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WO2013056096A1 - Polypeptide-opioid conjugates and uses thereof - Google Patents

Polypeptide-opioid conjugates and uses thereof Download PDF

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Publication number
WO2013056096A1
WO2013056096A1 PCT/US2012/060026 US2012060026W WO2013056096A1 WO 2013056096 A1 WO2013056096 A1 WO 2013056096A1 US 2012060026 W US2012060026 W US 2012060026W WO 2013056096 A1 WO2013056096 A1 WO 2013056096A1
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WIPO (PCT)
Prior art keywords
polypeptide
opioid
pain
angiopep
seq
Prior art date
Application number
PCT/US2012/060026
Other languages
French (fr)
Inventor
Michel Demeule
Sasmita Tripathy
Scott Bruce Duncan JARVIS
Christian Che
Gaoqiang Yang
Anthony Regina
Frank Porreca
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Angiochem Inc.
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Publication of WO2013056096A1 publication Critical patent/WO2013056096A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent

Definitions

  • the invention relates to opioid-polypeptides that are capable of crossing the blood-brain barrier and uses thereof.
  • Opioid analgesics are agonists of opioid receptors. Opioid receptors are present in high densities in the brain and spinal cord in the central nervous system (CNS) and in the gastrointestinal tract in the peripheral nervous system (PNS).
  • the analgesic effects of morphine are largely due to the action of the drug on receptors in the brain and spinal cord, while constipation is due to the action of opioids on receptors in the gastrointestinal tract.
  • opioid treatment is associated with a range of other side effects.
  • CNCP patients taking opioids 27% reported nausea, 9% reported vomiting, and 33% reported gastroesophageal reflux disease.
  • BBB blood-brain barrier
  • the brain is shielded against potentially toxic substances by the presence of two barrier systems: the BBB and the blood-cerebrospinal fluid barrier (BCSFB).
  • BBB is considered to be the major route for the uptake of serum ligands since its surface area is approximately 5000-fold greater than that of BCSFB.
  • the brain endothelium, which constitutes the BBB, represents the major obstacle for the use of potential drugs against many disorders of the CNS. As a general rule, only small lipophilic molecules may pass across the BBB, i.e., from circulating systemic blood to the brain.
  • New formulations of analgesic opioid drugs are needed to enhance delivery of active compounds across the BBB. Additionally, new formulations of analgesic opioid drugs are needed to deliver active compounds selectively to the brain and spinal cord (i.e., across the BBB). Summary of the Invention
  • the invention features a polypeptide-opioid conjugate, having (i): a polypeptide that is capable of crossing the blood-brain barrier; and (ii) one or more opioid moieties linked to the polypeptide by means of a linker; where each opioid moiety is linked to the polypeptide (e.g., the N- terminus, the C-terminus, or an amino acid side chain of the polypeptide).
  • the polypeptide-opioid conjugate features a polypeptide (a) substantially identical (e.g., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in a sequence selected from the group consisting of SEQ ID NO: 1-105, SEQ ID NO: 107-117, SEQ ID NO:120-138, and SEQ ID NO:166-178, or a fragment thereof.
  • the polypeptide contains between 6 and 21 amino acids.
  • the polypeptide-opioid conjugate features a polypeptide (a) substantially identical (e.g., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in Angiopep-2 (SEQ ID NO: 97), (3D)-Angiopep-2 (SEQ ID NO: 166), Angiopep-P6a (SEQ ID NO:174), Angiopep-descys- P6a (SEQ ID NO: 177), Angiopep-2-Cys (SEQ ID NO: 114), or (3D)-Angiopcp-2-Cys (SEQ ID NO: 178), or a pharmaceutically acceptable salt thereof.
  • Angiopep-2 SEQ ID NO: 97
  • 3D-Angiopep-2 SEQ ID NO: 166
  • the polypeptide-opioid conjugate features an opioid moiety selected from the group consisting of morphine, morphine-6-glucuronide, morphine-3-glucuronide, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, alazocine, alfentanil, bremazocine, buprenorphine, butorphanol, cyclazocine, dezocine, dihydrocodeine, diphenoxylate, diamorphine, fentanyl, levorphanol, meperidine, meptazinol, methadone, nalbuphine, pentazocine, propoxyphene, remifentanil, sufentanil, tramadol, naloxone, naltrexone, dipreno ⁇ hine, naloxonazine, met-enkephalin, leu-enkephalin, ⁇ -endorphin, dynorphin A,
  • the polypeptide-opioid conjugate features an opioid moiety selected from the group consisting of mo ⁇ hine, mo ⁇ hine-6-glucuronide, ⁇ 6-3- glucuronide, and
  • the polypeptide-opioid conjugate contains a linker selected from the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, adipyl, pimelyl, suberyl, azelayl, trans- ⁇ - hydromuconyl, 3,6,9-trioxaundecanedioyl, a poly(ethylene glycol) (PEG) linker (e.g., PEG 3 ),
  • PEG poly(ethylene glycol) linker
  • carboxybutylether carboxyethylether, a disulfide-containing linker, ethyl disulfide, ethyl-disulfide-ethyl, a cis-buten-mal linker, a hex-mal linker, a PEG 3 -mal linker, a triazole-containing linker, and methyl- triazolyl-propyl.
  • the opioid moiety of the polypeptide-opioid conjugate is linked to a lysine residue or a cysteine residue in the polypeptide or to the N-terminus of the polypeptide.
  • the polypeptide is linked to exactly one opioid moiety, and in other embodiments the polypeptide is linked to exactly three opioid moieties.
  • the polypeptide contains at least one D-amino acid. In related embodiments, the polypeptide contains exactly three D-amino acids.
  • the polypeptide opioid conjugate is selected from the group consisting of M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys, M6G-5 '-cis-buten-mal-Angiopep-2-Cys, M6G-4'- PEG 3 -mal-Angiopep-2-Cys, M6G-hex-mal-Angiopep-2-Cys, M6G-3-PEG 3 -mal-Angiopep-2-Cys, (M6G- amido-ethyl-disulfide-ethyl) 3 -Angiopep-2, (morphine-6- dimethylglutaryl) 3 -Angiopep-2, (morphine-3-glutaryl) 3 -Angiopep-2,
  • Angiopep-2 (morphine-3-suberyl) 3 -Angiopep-2, (morphine-3-succinyl) 3 -Angiopep-2, (morphine-3-PEG- 3 ) 3 -Angiopep-2, (mo hine-3-carbo ybutylether) 3 -Angio e -2, (mo ⁇ hine-6-succinyl) 3 -Angio ⁇ e ⁇ -2, mo ⁇ hine-3-PEG 3 -mal-Angiopep-2-Cys, and (M6G-5'-methyl-triazolyl-propyl) 3 -Angiopep-2.
  • the invention features a nanoparticle conjugated to a plurality of polypeptides having amino acid sequences (a) substantially identical (i.e., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1 , 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in a sequence selected from the group consisting of SEQ ID NO: 1 -105, SEQ ID NO: 107-117, SEQ ID NO: 120-138, and SEQ ID NO:l 66-178, or a fragment thereof, where the nanoparticle is bound to or contains a opioid moiety (e.g., ⁇ or M6G).
  • a opioid moiety e.g., ⁇ or M6G
  • the nanoparticle is conjugated to a plurality of polypeptides (a) substantially identical (e.g., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1 , 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1 , 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in Angiopep-2 (SEQ ID NO:97), (3D)-Angiopep-2 (SEQ ID NO: 166), Angiopep- P6a (SEQ ID NO: 174), Angiopep-descys-P6a (SEQ ID NO: 177), Angiopep-2-Cys (SEQ ID NO:l 14), or (3D)-Angiopep-2-Cys (SEQ ID NO: 178), or a pharmaceutically acceptable salt thereof, where the nanoparticle is bound to or contains an opioid moiety (e.g., ⁇ or M6G).
  • the invention features a method of treating pain in a subject by administering a composition containing a polypeptide-opioid conjugate or opioid-containing nanoparticle to the subject in an amount sufficient to treat the pain.
  • the pain is selected from the group consisting of postoperative pain, cancer pain, chronic pain, acute pain, somatic pain, neuropathic pain, visceral pain, inflammatory pain, migraine-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritic pain, skeletal pain, joint pain, gastrointestinal pain, muscle pain, angina pain, facial pain, pelvic pain, claudication, post traumatic pain, obstetric pain, labor pain, gynecological pain, and chemotherapy-induced pain.
  • the invention features a method of treating pain in a subject where said opioid conjugate is administered in an amount that reduces at least one opioid-induced side effect as compared to the same effective dosage of the corresponding unconjugated opioid.
  • the opioid-induced side effect is selected from the group consisting of constipation, respiratory depression, apnea, circulatory depression, respiratory arrest, shock, cardiac arrest, lightheadedness, dizziness, sedation, nausea, vomiting, sweating, dysphoria, euphoria, weakness, headache, agitation, tremor, uncoordinated muscle movements, seizure, alterations of mood, dreams, muscle rigidity, transient hallucinations and disorientation, visual disturbances, insomnia, increased cranial pressure, dry mouth, biliary tract spasm, laryngospasm, anorexia, diarrhea, cramps, taste alterations, flushing of the face, tachycardia, bradycardia, palpitation, faintness, syncope,
  • the side-effect is constipation.
  • the invention features a method of treating pain in a subject where the conjugate is administered at a lower equivalent dosage than would be required to treat the pain with a corresponding unconjugated opioid.
  • the opioid moiety is not codeine, dextropropoxyphene, diamorphine, dihydrocodeine, meptazinol, methadone, morphine, nalbuphine, or pentazocine.
  • the opioid moiety is not linked to the polypeptide with
  • the polypeptide-opioid conjugate is used to treat an opioid receptor- mediated condition including pain, diarrhea, cough, and dyspnea associated with acute left ventricular failure and pulmonary edema.
  • the invention features a pharmaceutically acceptable salt of any of the polypeptide-opioid conjugates described herein. Definitions
  • an amount sufficient is meant the amount of a compound of the invention required to treat pain in a clinically relevant manner.
  • a sufficient amount of a polypeptide-opioid conjugate used to practice the present invention varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the prescribers will decide the appropriate amount and dosage regimen. The appropriate amounts for any therapy can be determined from animal models, in vitro assays, and/or clinical studies.
  • conjugate is meant a compound having a polypeptide and a therapeutic agent (e.g., an opioid) linked to the polypeptide.
  • a therapeutic agent e.g., an opioid
  • polypeptide that is "capable of crossing the blood-brain barrier” is meant a polypeptide that is able to cross the BBB at a faster rate (e.g., a 5%, 10%, 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000% faster rate) than either a control substance or the unconjugated therapeutic agent.
  • a faster rate e.g., a 5%, 10%, 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000% faster rate
  • Ability to cross the BBB may be determined using any method known in the art (e.g., an in vitro model of the BBB or in situ brain perfusion as described in U.S. Patent No. 7,557,182).
  • the same effective dosage is meant the amount of a polypeptide-opioid conjugate of the invention required to achieve the same analgesic effect in a subject, as compared to the unconjugated opioid.
  • equivalent dosage is meant the amount of a polypeptide-opioid conjugate of the invention required to achieve the same molar amount of opioid in the conjugate of the invention, as compared to the unconjugated opioid.
  • fragment is meant a portion of a full-length amino acid or nucleic acid sequence (e.g., any sequence described herein). Fragments may include at least 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, or 50 amino acids or nucleic acids of the full length sequence. A fragment may retain at least one of the biological activities of the full length protein.
  • nanoparticle is meant a colloidal, polymeric, lipid, or elemental particle ranging in size from about 1 nm to about 1000 nm.
  • a polypeptide "linked to" a therapeutic agent By a polypeptide "linked to" a therapeutic agent is meant a covalent interaction between the polypeptide and the therapeutic agent.
  • the covalent interaction may be achieved through the use of a linker that forms covalent bonds with both the polypeptide and the therapeutic agent.
  • opioid-induced side effect is meant a non-analgesic condition induced by the treatment of a subject with an opioid.
  • Common opioid-induced side effects include constipation, respiratory depression, nausea, and vomiting.
  • substantially identical is meant a polypeptide or nucleic acid having at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or even 99% identity as compared to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, or 100) amino acids. It is to be understood herein that gaps may be found between the amino acids of sequences that are identical or similar to amino acids of the original polypeptide. The gaps may include no amino acids, one or more amino acids that are not identical or similar to the original polypeptide. Percent identity may be determined, for example, with an algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
  • subject is meant a human or non-human animal (e.g., a mammal).
  • therapeutic agent an agent that is capable of being used in the treatment or prophylactic treatment of a disease or condition or in the diagnosis of a disease or a condition.
  • treating pain in a subject is meant alleviating the pain experienced by the subject by administrating a polypeptide-opioid conjugate to the subject.
  • prophylactically treating pain in a subject is meant reducing the frequency of occurrence or severity of pain experienced by the subject by administering to the subject a polypeptide-opioid conjugate to the subject prior to the onset of pain.
  • a polypeptide-opioid conjugate may be administered to a subject before or during a surgical procedure to prophylactically treat pain.
  • Figure 1 is a scheme showing the synthesis of morphine-6-glucuronide (M6G) from morphine.
  • Figure 2 is a scheme showing the synthesis of M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys from M6G.
  • Figure 3 depicts polypeptide-opioid conjugates M6G-5'cis-buten-mal-Angiopep-2-Cys, M6G-3- hex-mal-Angiopep-2-Cys, M6G-4'-PEG 3 -mal-Angiopep-2-Cys, and M6G-3-PEG 3 -mal-Angiopep-2-Cys, where the polypeptide Angiopep-2-Cys is conjugated to a single opioid moiety through a cysteine residue in the polypeptide.
  • Figure 4 depicts the polypeptide-opioid conjugate (M6G-5 ' -amido-ethyl-disulfide-ethyl) 3 -
  • Angiopep-2 (ANG2010).
  • Figure 5 is a scheme showing the synthesis of (morphine-6-glutaryl) 3 -Angiopep-2 from morphine.
  • Figure 6 is a scheme showing the synthesis of (morphine-6-dimethylglutaryl) 3 -Angiopep-2 from morphine.
  • Figure 7 is a scheme showing the synthesis of ⁇ ⁇ 6-3 ⁇ 1 ⁇ 3 ⁇ 1)3- ⁇ 3 ⁇ 4 ⁇ ⁇ -2 from morphine.
  • Figure 8 is a scheme showing the synthesis of (mo ⁇ hine-3-dimethylglutaryl) 3 -Angio ep-2 from morphine.
  • Figure 9 is a scheme showing the synthesis of (morphine-3-suberyl) 3 -Angiopep-2 from morphine.
  • Figure 10 is a scheme showing the synthesis of (morphine-3-succinyl) 3 -Angiopep-2 from morphine.
  • Figure 11 is a scheme showing the synthesis of (morphine-3-PEG 3 ) 3 -Angiopep-2 from morphine.
  • Figure 12 is a scheme showing the synthesis of (mo ⁇ hine-3-carboxybutylether)3-Angiopep-2 from nunphine.
  • Figure 13 depicts polypeptide-opioid conjugates ( ⁇ 6-6-8 ⁇ 1) 3 -. ⁇ 6 ⁇ -2 and ⁇ ⁇ 6-3 -PEG 3 -mal-Angiopep-2-Cys .
  • Figure 14 is a scheme showing the synthesis of (M6G-5'-methyl-triazolyl-propyl) 3 -Angiopep-2.
  • Figures 15A-15C are graphs showing data for opioid-induced analgesia in the hot-plate test with
  • Figure 15A is a graph showing the effect of dose on hindpaw latency where the
  • FIG. 15B is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys with unconjugated M6G.
  • Figure 15C is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys and (M6G-5'-amido-ethyl-disulfide-ethyl) 3 -Angiopep-2 with a control and unconjugated M6G.
  • Figure 16 is a graph showing data for opioid-induced analgesia in the hot-plate test for M6G-5'- amido-ethyl-disulfide-Angiopep-2-Cys, M6G, and ⁇ .
  • Figure 17 is a graph showing data for opioid-induced analgesia in the hot-plate test using (morphine-3-PEG 3 )3-Angiopep-2 (which contains a cleavable linker), (morphine-carboxybutylether) 3 - Angiopep-2 (which contains a non-cleavable linker), and morphine.
  • Figures 18A-18C are graphs showing the effect of opioids on the body temperature of CD-I mice.
  • Figure 18A is a graph showing the effect of dose on body temperature where the polypeptide- opioid conjugate M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys is used at four different doses.
  • Figure 18B is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys with unconjugated M6G.
  • Figure 18C is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys and (M6G-5'-amido- ethyl-disulfide-ethyl)3-Angiopep-2 with a control and unconjugated M6G.
  • Figure 19 is a graph showing the brain perfusion and volume distribution of the polypeptide- opioid conjugates.
  • Figure 20 is a graph showing a time-course plot of the perfusion of the polypeptide-opioid conjugates into the brain.
  • Figures 21A-21C show data evaluating the binding of an Angiopep-2-M6G conjugate
  • FIG. 21 A shows the chemical structure and a schematic representation of ANG2010.
  • Figure 21 B is a graph showing competition binding of M6G and ANG2010 to Mu-opioid (MOPr).
  • Figure 21 C is a graph showing competition binding of M6G and ANG2010 to Delta-opiod (DOPr).
  • Figure 22 is a schematic showing the brain perfusion method that was used for evaluating brain uptake of ANG2010.
  • Figures 23A-23C show data evaluating brain uptake of ANG2010.
  • Figure 23A is a graph showing the in vivo brain uptake of the [ 125 I]-ANG2010 and [ 3 H]-morphine.
  • Figure 23B is a graph showing data from a brain capillary depletion assay to assess ANG2010 distribution in the brain compartments.
  • Figure 23C is a table showing initial brain transport rate (Kong) values for ANG2010, morphine, and M6G compared to that of other molecules.
  • Figures 24A-24B are graphs showing evaluation of the analgesic effect of ANG2010 using a hot plate model of pain.
  • Figure 24A is a graph showing increased paw flicking latency after intravenous (IV) bolus injection of ANG2010 compared to unconjugated M6G at an equimolar dose.
  • Figure 24B is a graph showing an increase in foot-licking latency for at least 3 hours after intravenous (IV) or sub- cutaneous (SC) administration of ANG2010.
  • Figures 25A-25B are graphs showing evaluation of the analgesic effect of ANG2010 using a rat tail flick model of pain.
  • Figure 25 A is a graph showing the analgesic effect of ANG2010 compared to that of unconjugated morphine and M6G in the rat tail flick model after IV bolus injection.
  • Figure 25B is a graph showing the results of Figure 25 A represented as the maximal possible effect (%) for the three drugs.
  • Figure 26 is a graph showing the analgesic effect of ANG2010 compared to that of unconjugated morphine and M6G in the rat tail flick model after SC bolus injection.
  • Figure 27 is a graph showing the effect of sub-cutaneous injection of ANG2010 on gastrointestinal transit.
  • the conjugates of the invention transport opioid receptor agonists to the brain and/or spinal cord.
  • the conjugates of the invention may either enhance the delivery of opioids that already cross the BBB (e.g., morphine) or allow for the delivery of opioids that do not cross the BBB or poorly cross the BBB (e.g., morphine-6-glucuronide).
  • Improved delivery of opioids to the brain and spinal cord can reduce opioid- associated side effects such as constipation and decrease the equivalent dosage required to effect analgesia in a subject.
  • Treatment of patients with the peptide-opioid conjugates of the invention may lead to fewer or reduced side effects compared with an equivalent dosage of the unconjugated opioid.
  • This advantage of the invention is due to the improved transport of the conjugate across the BBB.
  • the opioid receptors that regulate pain perception are located in the brain and spinal cord in the central nervous system (CNS) while the opioid receptors associated with certain opioid-induced side effects (e.g., constipation) are located in the peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • the patient may therefore experience reduced PNS-associated, opioid-induced side effects when treated with the polypeptide-opioid conjugate compared with the unconjugated opioid.
  • a common side effect of opioid treatment is constipation, which is a consequence of the dense distribution of opioid receptors in enteric neurons of the PNS.
  • the polypeptide of the polypeptide-opioid conjugates allow the opioid to be directed to the brain and/or spinal cord (and thereby away from enteric neurons of the PNS), patients treated with the conjugate may experience reduced levels of constipation.
  • any other PNS-associated side effect of opioid treatment e.g., intestinal motility, urinary retention, and pruritus
  • the polypeptide-opioid conjugates of the invention can effect analgesia in a subject using a dosage lower than the equivalent dosage of unconjugated opioid. Because the opioid (e.g., morphine or M6G) is delivered more efficiently to the brain and/or spinal cord, patients may receive lower systemic exposure to the opioid while receiving the same analgesic benefit.
  • opioid e.g., morphine or M6G
  • opioid moiety may be used in the polypeptide-opioid conjugates and methods of the invention.
  • opioid moieties are described below, and include morphine and morphine related compounds. Both morphine and morphine-6-glucuronide (M6G) act as agonists of opioid receptors, principally the ⁇ -opioid receptor. Opioid receptors are present in high densities throughout the central nervous system, including in the brainstem, the medial thalamus, the spinal cord, the limbic system, and the hypothalamus.
  • Agonist binding to the ⁇ -opioid receptor triggers a range of intracellular signals including inhibition of adenylate cyclase, reduced opening of voltage-gated calcium channels, and activation of potassium current, PKC, and PLC- ⁇ .
  • the analgesic effects of opioid receptor agonists have been partially attributed to the blocking of the release of GABA from tonically active neurons in the periaqueductal gray (PAG) matter in the brain.
  • PAG periaqueductal gray
  • monoamine receptors in the forebrain and spinal cord are deactivated, which affects sensory inputs from the spinal cord to higher centers.
  • Opioid receptors are also expressed in the spinal dorsal horn presynaptically in C-fibers and postsynaptically in second order neurons. Binding of opioid agonists at presynaptic C-fibers reduces opening of voltage-gated calcium channels that initiate neurotransmitter release. Binding of opioid agonists at the postsynaptic second order neuron enhances efflux of potassium and hyperpolarization. The reinforcing activity of opioid agonists at the presynaptic and postsynaptic sites attenuates activation of second order neurons (Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th Edition, p. 490-491, 2011).
  • Morphine is metabolized primarily to M6G and morphine-3-glucuronide (M3G).
  • M6G displays analgesic activity similar to morphine, while M3G is much less active.
  • the structures of morphine, M6G, and M3G are shown below.
  • the polypeptide-opioid conjugates may be prepared and used with any morphine congener or opioid.
  • Codeine, hydrocodone, hydromorphone, oxycodone, and oxymorphone, which are found in present day analgesic prescription drugs, are all congeners of morphine.
  • opioid receptor agonists e.g., alazocine, alfentanil, bremazocine, buprenorphine, butorphanol, cyclazocine, dezocine, dihydrocodeine, diphenoxylate, diamorphine, fentanyl, levorphanol, meperidine, meptazinol, methadone, nalbuphine, pentazocine, propoxyphene, remifentanil, sufentanil, and tramadol), opioid receptor antagonists (e.g., naloxone, naltrexone, diprenorphine, naloxonazine), and endogenous peptides (e.g., met-enkephalin, leu-enkephalin, ⁇ - endorphin, dynorphin A, dynorphin B, endomorphin-1, endomorphin-2, and nociceptin).
  • opioid receptor agonists e.g., al
  • the polypeptide-opioid conjugates of the invention can feature any of the polypeptides described herein, or a fragment or analog thereof.
  • the polypeptides described herein are capable of crossing the BBB.
  • the polypeptide of the polypeptide-opioid conjugate may have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to a polypeptide described herein.
  • the polypeptide may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or 15) substitutions relative to one of the sequences described herein.
  • the polypeptide may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15) additions and/or deletions of amino acids relative to one of the sequences described herein. Other modifications are described in greater detail below.
  • the polypeptide may be substantially identical to any of the sequences set forth in table 1 , or a fragment thereof.
  • the peptide vector has a sequence of Angiopep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97), Angiopep-3 (SEQ ID NO: 107), Angiopep-4a (SEQ ID NO:108), Angiopep-4b (SEQ ID NO: 109), Angiopep-5 (SEQ ED NO: l 10), Angiopep-6 (SEQ ID NO: 111), Angiopep-7 (SEQ ID NO: 112), Angiopep-2-Cys (SEQ ID NO: 114), or reversed Angiopep-2 (SEQ ID NO: 117).
  • the polypeptide may be of any length, for example, at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 35, 50, 75, 100, 200, or 500 amino acids, or any range between these numbers. In certain embodiments, the polypeptide is 10 to 50 amino acids in length.
  • the polypeptide may be produced by recombinant genetic technology or chemical synthesis.
  • Polypeptides Nos. 5, 67, 76, and 91 include the sequences of SEQ ID NOS:5, 67, 76, and 91, respectively, and are amidated at the C-terminus.
  • Polypeptides Nos. 107, 109, and 110 include the sequences of SEQ ID NOS:97, 109, and 110, respectively, and are acetylated at the N-terminus.
  • polypeptide may include a consensus sequence of
  • X3 is Asn or Gin
  • X4 is Asn or Gin
  • X5 is Phe, Tyr, or Trp.
  • the polypeptide may include a consensus sequence of
  • X3 is Asn or Gin
  • X4 is Asn or Gin
  • X5 is Phe, Tyr, or Trp
  • Zl is absent, Cys, Gly, Cys-Gly, Arg-Gly, Cys-Arg-Gly, Ser-Arg-Gly, Cys-Ser-Arg-Gly, Gly-Ser-Arg-Gly, Cys-Gly- Ser-Arg-Gly, Gly-Gly-Ser-Arg-Gly, Cys-Gly-Gly-Ser-Arg-Gly, Tyr- Gly-Gly-Ser-Arg-Gly, Cys-Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe- Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe-Tyr-Gly-Gly-Ser-Arg-G
  • Z2 is absent, Cys, Tyr, Tyr-Cys, Cys-Tyr, Thr-Glu-Glu-Tyr, or Thr-Glu-Glu-Tyr-Cys.
  • the consensus sequence of formulas (la) and (lb) include the amino acid sequence Lys-Arg-Asn- Asn-Phe-Lys (SEQ ID NO: 122) and conservative substitutions.
  • polypeptides include those having a consensus sequence of
  • XI is Lys or D-Lys; X2 is Arg or D-Arg;
  • X5 is Phe or D-Phe
  • X6 is Lys or D-Lys
  • XI , X2, X5, or X6 is a D-amino acid.
  • polypeptides include those having a consensus sequence of
  • XI is Lys or D-Lys
  • X2 is Arg or D-Arg
  • X5 is Phe or D-Phe
  • X6 is Lys or D-Lys
  • X7 is Tyr or D-Tyr
  • polypeptides may also contain a consensus sequence of
  • XI is Lys or D-Lys
  • X2 is Arg or D-Arg
  • X5 is Phe or D-Phe
  • X6 is Lys or D-Lys
  • X7 is Tyr or D-Tyr
  • Zl is absent, Cys, Gly, Cys-Gly, Arg-Gly, Cys-Arg-Gly, Ser-Arg-Gly, Cys-Ser-Arg-Gly, Gly-Ser-Arg-Gly, Cys-Gly-Ser-Arg-Gly, Gly-Gly-Ser-Arg-Gly, Cys-Gly-Gly-Ser-Arg-Gly, Tyr- Gly-Gly-Ser-Arg-Gly, Cys-Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Tyr-Gly-Gly- Ser-Arg-Gly, Cys-Phe- Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe-Tyr-Gly-Gly-Gly-Gly, Cys-Phe-Tyr-Gly-Gly-Ser-Arg-Gly,
  • Z2 is absent, Cys, Tyr, Tyr-Cys, Cys-Tyr, Thr-Glu-Glu-Tyr, or Thr-Glu-Glu-Tyr-Cys; where at least one of XI , X2, X5, X6, or X7 is a D-amino acid; and
  • polypeptide optionally includes one or more D-isomers of an amino acid recited in Zl or Z2.
  • the polypeptide may contain additions or deletions of amino acids to the consensus sequence of Lys-Arg-X3-X4-X5-Lys (formula la), where X3-X5 are as defined above; the consensus sequences of Xl-X2-Asn-Asn-X5-X6 and Xl -X2-Asn-Asn-X5-X6-X7 (formulas Ila and lib, respectively), where XI , X2, X5, X6, and X7 are as defined above; or the longer polypeptide of (3D)-Angiopep-2, as described herein.
  • deletions or additions can include any part of the consensus sequence of Lys-Arg-X3-X4- X5-Lys, Xl -X2-Asn-Asn-X5-X6, Xl -X2-Asn-Asn-X5-X6-X7, Lys-Arg-Asn-Asn-Phe-Lys, D-Lys-D- Arg-Asn-Asn-D-Phe-D-Lys, or D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-D-Tyr, or of the longer sequence 3D-Angiopep-2.
  • deletions or additions of 1, 2, 3, 4, or 5 amino acids may be made from the consensus sequence of the polypeptide.
  • the deletions or additions may be from 1 to 3 amino acids.
  • polypeptide of the invention can be (3D)-Angiopep-2 (SEQ ID NO: 1
  • the polypeptide may comprise a sequence shorter than Angiopep-2, shown in table 2.
  • PI, Pla, Plb, Pic, Pld, P2, P3, P4, P5, P5a, P5b, P5c, P6, P6a, P6b, P6, or decys-P6a is amidated at the C-terminus.
  • the polypeptide of the invention include additions and/or deletions of amino acids of the sequences of PI , Pl a, Plb, Pic, Pld, P2, P3, P4, P5, P5a, P5b, P5c, P6, P6a, P6b, P6, and decys-P6a.
  • the deletions or additions can include any part of these sequences.
  • deletions or additions of 1 , 2, 3, 4, or 5 amino acids may be made from these sequences of the polypeptide.
  • the deletions or additions may be from 1 to 3 amino acids.
  • the invention also features polypeptides having conservative substitutions of any of the above- described polypeptides.
  • Conservative substitutions and derivatives of amino acids and peptides are well known in the art and can be determined by any useful methods (e.g., by using a substitution matrix or any other method described herein).
  • a derivative of a polypeptide includes a polypeptide containing one or more conservative substitutions selected from the following groups or a subset of these groups: Ser, Thr, and Cys; Leu, He, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp (e.g., Phe and Tyr); and Gin, Asn, Glu, Asp, and His (e.g., Gin and Asn).
  • Conservative substitutions may also be determined by other methods, such as by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), and PAM substitution matrix (e.g., PAM 250 matrix).
  • BLAST Basic Local Alignment Search Tool
  • BLOSUM substitution matrix e.g., BLOSUM 62 matrix
  • PAM substitution matrix e.g., PAM 250 matrix
  • any useful substitutions, additions, and deletions can be made to the polypeptide that does not destroy significantly a desired biological activity (e.g., ability to cross the BBB or agonist activity).
  • the modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the consensus sequence or original polypeptide.
  • substitutions, additions, and deletions may have or may optimize a characteristic of the consensus sequence or polypeptide, such as charge (e.g., positive or negative charge), hydrophilicity, hydrophobicity, in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.
  • charge e.g., positive or negative charge
  • hydrophilicity e.g., from 1 to 3 amino acids
  • hydrophobicity e.g., in vivo stability
  • bioavailability toxicity
  • immunological activity e.g., immunological identity
  • conjugation properties e.g., conjugation properties.
  • positive charge can be promoted by deleting one or more amino acids (e.g., from 1 to 3 amino acids) that are not basic/positively charged (as described below based on common side chain properties) or less positively charged (e.g., as determined by pKa).
  • positive charge can be promoted by inserting one or more amino acids (e.g., from 1 to 3
  • substitutions or additions of D-amino acids can be made within the polypeptide.
  • Such substitutions or additions may provide peptides having increased resistance to cleavage by enzymes, where one of more amino acids for cleavage sites can be substituted with its D-isomer.
  • Exemplary enzymes include pepsin, trypsin, Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, glutamyl endopeptidase, LysC lysyl endopeptidase, LysN peptidyl-Lys metalloendopeptidase, proteinase K, and thermolysin; and exemplary cleavage sites for these enzymes are described herein.
  • In vivo stability may be optimized in any useful way.
  • stability in the presence of one or more digestive enzymes can be improved by substituting a naturally occurring L-amino acid for its D-isomer.
  • Exemplary digestive enzymes include pepsin and trypsin.
  • SI -SI ' indicates the cleavage site for a peptide Sn— S4-S3-S2-S1 -S1 '-S2'-S3'-S4'— Sm.
  • Cleavage by pepsin generally occurs when Phe, Tyr, Trp, or Leu is in position SI or SI '; or when Pro is in position S3 or S4.
  • Cleavage by trypsin generally occurs when Arg or Lys is in position S 1 ; when Pro is in position SI ', Lys is in position S I , and Trp is in position S2; when Pro is in position SI ', Arg is in position SI , and Met is in position S2; or when Pro is in position SI ' and Glu is in position S2.
  • exemplary cleavage sites include those for cleavage by Arg-C proteinase (e.g., Arg in position SI), Asp- N endopeptidase (e.g., Asp or Glu in position SI '), chymotrypsin (e.g., Trp, Tyr, or Phe in position SI for cleavage with high specificity; Leu, Met, or His in position SI for cleavage with low specificity), glutamyl endopeptidase (e.g., Glu at position SI), LysC lysyl endopeptidase (e.g., Lys at position SI), LysN peptidyl-Lys metalloendopeptidase (e.g., Lys at position S I '), proteinase K (e.g., an aliphatic or amino acid residue, such as Ala, Glu, Phe, He, Leu, Thr, Val, Trp, or Tyr, at position SI), and thermoly
  • Predictive models are also available for determining cleavage sites, such as PeptideCutter available on the ExPASy proteomics server.
  • Exemplary cleavage sites for polypeptides are C-terminal to positions 1 , 2, 3, 4, 14, 18, and 19 in Angiopep-2 (SEQ ID NO:97) for cleavage by pepsin and C-terminal to positions 8, 10, 1 1 , and 15 in Angiopep-2 (SEQ ID NO:97) for cleavage by trypsin.
  • exemplary cleavage sites in Angiopep-2 include C-terminal to positions 8 and 11 for cleavage by Arg-C proteinase; positions 16 and 17 for cleavage by Asp-N endopeptidase; positions 2, 3, 4, 14, and 19 for cleavage by chymotrypsin; positions 17 and 18 for cleavage by glutamyl endopeptidase; positions 10 and 15 for cleavage by LysC lysyl endopeptidase; positions 9 and 14 for cleavage by LysN peptidyl-Lys metalloendopeptidase; positions 1 , 2, 3, 4, 14, 16, and 1 for cleavage by proteinase K; and positions 1 , 2, and 13 for cleavage by thermolysin.
  • polypeptide of the invention also include polypeptides shorter than Angiopep-2 (SEQ ID NO:97) having one or more D-amino acid substitutions for one or more of positions 1 , 2, 3, 4, 8, 10, 1 1 , 13, 14, 15, 16, 17, 18, and 19 in Angiopep-2 (SEQ ID NO:97).
  • Substantial modifications in function or immunological identity are accomplished by selecting substitutions, additions, and deletions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (He), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), and Phenylalanine (Phe);
  • Cysteine Cysteine
  • Serine Serine
  • Threonine Thr
  • Trp Tryptophan
  • Tyrosine Tyrosine
  • Phe Phenylalanine
  • His His
  • the invention also features fragments of any of the above-described polypeptides (e.g., a functional fragment).
  • the fragments are efficiently transported across the BBB.
  • Truncations of the polypeptide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from either the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a combination thereof.
  • Other fragments include sequences where internal portions of the polypeptide are deleted.
  • Deletions of the polypeptide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from the internal portion of the polypeptide.
  • deletions may be 1, 2, 3, 4, or 5 amino acids from the consensus sequence of the polypeptide.
  • the polypeptide may be bound to an opioid moiety either directly (e.g., by a covalent bond) or through a linker.
  • Linkers include chemical linking agents (e.g., cleavable linkers and non-cleavable linkers), click-chemistry linkers, and peptides. Any of the linkers described below may be used in the polypeptide-opioid conjugates of the invention.
  • the linker is a chemical linking agent.
  • the polypeptide may be conjugated through sulfhydryl groups, amino groups (amines), or any appropriate reactive group.
  • the linker may comprise a flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms.
  • the flexible arm can be polyethylene glycol spacer, such as (PEG) n , where n is an integer between 1 and 20, or an amino acid, such as -NH- (CH 2 ) n -C(0)0-, where n is an integer between 2 and 10 (e.g., when n is 5).
  • Exemplary linkers include BS 3 ([bis(sulfosuccinimidyl)suberate]; BS 3 is a homobifunctional N- hydroxysuccinimide ester that targets accessible primary amines), NHS/EDC (N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS/EDC allows for the conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([ ⁇ - ⁇ -maleimidocaproic acid]hydrazide; sulfo-EMCS are heterobifunctional reactive groups (maleimide and NHS -ester) that are reactive toward sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazide is a useful reagent for linking carboxyl groups to primary amines), SATA (N-succinimidyl-S-acetylthioacetate; SATA is reactive towards
  • active carboxyl groups e.g., esters
  • Particular agents include N-hydroxysuccinimide (NHS), N-hydroxy- sulfosuccinimide (sulfo-NHS), maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS), maleimido propionic acid (MP A), maleimido hexanoic acid (MHA), and maleimido undecanoic acid (MUA).
  • NHS N-hydroxysuccinimide
  • sulfo-NHS N-hydroxy- sulfosuccinimide
  • MBS gamma-maleimido-butyryloxy succinimide ester
  • MP A maleimid
  • Primary amines are the principal targets for NHS esters. Accessible a-amine groups present on the N-termini of proteins and the ⁇ -amine of lysine react with NHS esters.
  • compounds of the invention can include a linker having a NHS ester conjugated to an N-terminal amino of a peptide or to an ⁇ -amine of lysine. An amide bond is formed when the NHS ester reacts with primary amines releasing N- hydroxysuccinimide.
  • succinimide containing reactive groups are herein referred to as succinimidyl groups.
  • the functional group on the protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as gamma-maleimide- butrylamide (GMBA or MP A).
  • GMBA gamma-maleimide- butrylamide
  • maleimide containing groups are referred to herein as maleimedo groups.
  • a conjugate of the invention can include a linker having a maleimido group conjugated to a sulfhydryl group of the polypeptide or of the therapeutic agent.
  • Amine-to-amine linkers include NHS esters and imidoesters.
  • exemplary NHS esters are DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS 3 (bis [sulfosuccinimidyl] suberate), TSAT (ira-succinimidyl aminotriacetate), variants of bis-succimmide ester-activated compounds that include a polyethylene glycol spacer, such as BS(PEG) n , where n is 1-20 (e.g., BS(PEG) 5 and BS(PEG) 9 ), DSP (Dithiobis[succinimidyl propionate]), DTSSP (3,3'-dithiobis[sulfosuccinimidylpropionate]), DST (disuccinimidyl tartarate), BSOCOES (bis[2-(succinimidooxycarbonyloxy)e
  • Imidoesters include DMA (dimethyl adipimidate*2 HCl), DMP (dimethyl pimelimidate'2 HCl), DMS (dimethyl suberimidate « 2 HCl), and DTBP (dimethyl S ⁇ '-dithiobispropionimidate ⁇ HCl).
  • Other amine- to-amine linkers include DFDNB (l ,5-difluoro-2,4-dinitrobenzene) and THPP (P-[tris(hydroxymethyl) phosphino] propionic acid (betaine)).
  • the linker may be a sulfhydryl-to-sulfhydryl linker.
  • Such linkers include maleimides and pyridyldithiols.
  • Exemplary maleimides include BMOE (bis-maleimidoethane), BMB (1 ,4- bismaleimidobutane), BMH (bismaleimidohexane), TMEA (irw[2-maleimidoethyl]amine), BM(PEG)2 1,8-bis-maleimidodiethyleneglycol) or BM(PEG) n , where n is 1 to 20 (e.g., 2 or 3), BMDB (1,4 bismaleimidyl-2,3-dihydroxybutane), and DTME (dithio-bismaleimidoethane).
  • BMOE bis-maleimidoethane
  • BMB bismaleimidobutane
  • BMH bismaleimidohexane
  • pyridyldithiols include DPDPB (l,4-di-[3'-(2'-pyridyldithio)-propionamido]butane).
  • Other sulfhydryl linkers include HBVS (1,6-hexane-bis-vinylsulfone).
  • the linker may be an amine-to-sulfhydryl linker, which includes NHS ester/maleimide compounds.
  • amine-to-sulfhydryl linkers can include ester linkers (e.g., any linker described herein containing an ester group).
  • AMAS N-(a- maleimidoacetoxy)succinimide ester
  • BMPS N-[ -maleimidopropyloxy]succinimide ester
  • GMBS ⁇ - [ ⁇ -maleimidobutyryloxyjsuccinimide ester
  • sulfo-GMBS N-[y-maleimidobutyryloxy]sulfosuccinimide ester
  • MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • sulfo-MBS m-maleimidobenzoyl-N- hydroxysulfosuccinimide ester
  • SMCC succinimidyl 4-[N-maleimidomethyl]cyclohexane-l- carboxylate
  • sulfo-SMCC Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate
  • the linker has the formula:
  • n is an integer between 1 and 15 (e.g., n is 2, 3, 4, 5, 6, or 1 1); and either Y is a thiol on the polypeptide and Z is a primary amine, alcohol, or phenol on the opioid moiety or Y is a thiol on the opioid moiety and Z is a primary amine, alcohol, or phenol on the polypeptide.
  • the linker is an amino-to-nonselective linker.
  • linkers include NHS ester/aryl azide and NHS ester/diazirine linkers.
  • NHS ester/aryl azide linkers include NHS- ASA (N-hydroxysuccinimidyl-4-azidosalicylic acid), ANB-NOS (N-5-azido-2- nitrobenzoyloxysuccinimide), sulfo-HSAB (N-hydroxysulfosuccinimidyl-4-azidobenzoate), sulfo-NHS- LC-ASA (sulfosuccinimidyl[4-azidosalicylamido]hexanoate), SANP H (N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate), sulfo-SANPAH (N-sulfosuccinimidyl-6-
  • NHS ester/diazirine linkers include SDA (succinimidyl 4,4'-azipentanoate), LC- SDA (succinimidyl 6-(4,4'-azipentanamido)hexanoate), SDAD (succinimidyl 2 -([4,4'- azipentanamido]ethyl)-l,3'-dithioproprionate), sulfo-SDA (sulfosuccinimidyl 4,4'-azipentanoate), sulfo- LC-SDA (sulfosuccinimidyl 6-(4,4'-azipentanamido)hexanoate), and sulfo-SDAD (sulfosuccinimidyl 2- ([4,4'-azipentanamido]ethyl)-l ,3 '-dithioproprionate).
  • Exemplary amine-to-carboxyl linkers include carbodiimide compounds (e.g., DCC (N,N- dicyclohexylcarbodimide) and EDC (l-ethyl-3-[3-dimethylaminopropyl]carbodiimide)).
  • Exemplary sulfhydryl-to-nonselective linkers include pyridyldithiol/aryl azide compounds (e.g., APDP ((N-[4-(p- azidosalicylamido)butyl] -3 ' -(2 ' -pyridyldithio)propionamide)) .
  • Exemplary sulfhydryl-to-carbohydrate linkers include maleimide/hydrazide compounds (e.g., BMPH (N-[P-maleimidopropionic acid]hydrazide), EMCH ([N-e-maleimidocaproic acid]hydrazide), MPBH 4-(4-N- maleimidophenyl)butyric acid hydrazide), and KMUH (N-[K-maleimidoundecanoic acid]hydrazide)) and pyridyldithiol/hydrazide compounds (e.g., PDPH (3-(2-pyridyldithio)propionyl hydrazide)).
  • maleimide/hydrazide compounds e.g., BMPH (N-[P-maleimidopropionic acid]hydrazide), EMCH ([N-e-maleimidocaproic acid]hydrazide), MPBH 4-(4-N-
  • Exemplary carbohydrate-to-nonselective linkers include hydrazide/aryl azide compounds (e.g., ABH (p-azidobenzoyl hydrazide)).
  • Exemplary hydroxyl-to-sulfhydryl linkers include isocyanate/maleimide compounds (e.g., (N-[p-maleimidophenyl]isocyanate)).
  • Exemplary amine-to-DNA linkers include NHS ester/psoralen compounds (e.g., SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate)).
  • Ri is H, Ci_ 6 alkyl, C 2- 6 alkenyl, C6-12 aryl or aralkyl or these coupled with a
  • R 2 is H, C 2 alkyl, C 6 , 2 aryl,
  • R 3 is ⁇ , ⁇ 0' ⁇ , ⁇ S' ⁇ , ⁇ 0 ' ⁇ , ⁇ S' ⁇ , H , H or another chemical structure which is able to delocalize the lone pair electrons of the adjacent nitrogen and R4 is a pendant reactive group capable of linking R 3 to a polypeptide or to a therapeutic agent.
  • the linker can be polyvalent or monovalent.
  • a monovalent linker has only one activated group available for forming a covalent bond.
  • the monovalent linker can include one or more functional groups that can be chemically modified by using a coupling agent, as described herein, to form a second activated group.
  • a coupling agent as described herein, to form a second activated group.
  • a terminal hydroxyl group of the linker can be activated by any number of coupling agents. Examples of coupling agents include N-hydroxysuccinimide,
  • a polyvalent linker (e.g., a multifunctional linker) has two or more activated groups.
  • the activated groups in the linker can be the same, as in a homopolyvalent linker, or different, as in a heteropolyvalent linker.
  • Heteropolyvalent linkers allow for conjugating a polypeptide and a transport vector with different functional groups. Examples of heteropolyvalent linkers include polyoxyethylene- bis(p-nitrophenyl carbonate), mal-PEG-DSPE, diisocyanate, succitiimidyl 4-hydrazinonicotinate acetone hydrazone.
  • homopolyvalent linkers with two activated groups include disuccinimidyl glutarate, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, bis(NHS)PEG 5 , bis(NHS)PEG(),
  • homopolyvalent linkers with three activated groups include tris-succinimidyl aminotriacetate, p-[tris(hydroxymethyl) phosphino] propionic acid, and tris[2-maleimidoethyl]amine.
  • heteropolyvalent linkers include those with an maleimide activated group and a succinimide activated group, such as N-[D-maleimidoacetoxy]succimmide ester, ⁇ -[ ⁇ - maleimidopropyloxy]-succinimide ester, N-[D -maleimidobutyryloxy]succinimide ester, m- maleimidoberizoyl-N-hydroxysuccimrnide ester, succinimidyl 4-[N-maleimidomethyl]cyclohexane-l- carboxylate, N-[D -maleimidocaproyloxy]succinimide ester, and succinimidyl 4-[p- maleimidophenyljbutyrate, including N-sulfosuccinimidyl derivatives; those with a PEG spacer molecule, such as succinimidyl-([N-maleimidopropionamido]-(ethyleneglycol) x
  • the linker is a trifunctional, tetrafunctional, or greater linking agent.
  • Exemplary trifunctional linkers include TMEA, THPP, TSAT, LC-TSAT (im-succinimidyl (6- aminocaproyl)aminotriacetate), ⁇ ' 5-succinimidyl-l ,3,5-benzenetricarboxylate, MDSI (maleimido-3,5- disuccinimidyl isophthalate), SDMB (succinimidyl-3,5-dimaleimidophenyl benzoate, Mal-4 ⁇ tetrakis- ⁇ 2>- maleinn ⁇ opropyl)pentaeiythritol, NHS-4 (te raA s-(N-succinimidylcarboxypropyl)pentaerythritol)).
  • TMEA has the structure:
  • TMEA through its maleimide groups, can react with sulfhydryl groups (e.g., through cysteine amino acid side chains).
  • THPP has the structure:
  • hydroxyl groups and carboxy group of THPP can react with primary or secondary amines.
  • the linker is formed by the reaction between a click-chemistry pair.
  • click-chemistry pair is meant a pair of reactive groups that participates in a modular reaction with high yield and a high thermodynamic gain, thus producing a click-chemistry linker.
  • one of the reactive groups is attached to the opioid moiety and the other reactive group is attached to the polypeptide.
  • Exemplary reactions and click-chemistry pairs include a Huisgen 1 ,3-dipolar cycloaddition reaction between an alkynyl group and an azido group to form a triazole-containing linker; a Diels-Alder reaction between a diene having a 4 ⁇ electron system (e.g., an optionally substituted 1,3 -unsaturated compound, such as optionally substituted 1,3 -butadiene, l-methoxy-3-trimethylsilyloxy-l,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) and a dienophile or heterodienophile having a 2 ⁇ electron system (e.g., an optionally substituted alkenyl group or an optionally substituted alkynyl group); a ring opening reaction with a nucleophile and a strained heterocyclyl electrophile; a splint ligation reaction with a phosphorothioate
  • the polypeptide is linked to the opioid moiety by means of a triazole-containing linker formed by the reaction between a alkynyl group and an azido group click-chemistry pair.
  • the azido group may be attached to the polypeptide and the alkynyl group may be attached to the opioid moiety.
  • the azido group may be attached to the opioid moiety and the alkynyl group may be attached to the polypeptide.
  • the reaction between an azido group and the alkynyl group is uncatalyzed, and in other embodiments the reaction is catalyzed by a copper(I) catalyst (e.g., copper(I) iodide), a copper(II) catalyst in the presence of a reducing agent (e.g., copper(II) sulfate or copper(II) acetate with sodium ascorbate), or a ruthenium- containing catalyst (e.g., Cp*RuCl(PPh 3 ) 2 or Cp*RuCl(COD)).
  • a copper(I) catalyst e.g., copper(I) iodide
  • a copper(II) catalyst in the presence of a reducing agent e.g., copper(II) sulfate or copper(II) acetate with sodium ascorbate
  • a ruthenium- containing catalyst e.g., Cp*RuCl(PPh 3
  • the linker includes at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids).
  • the linker is a single amino acid (e.g., any naturally occurring amino acid such as Cys).
  • a glycine-rich peptide such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] n (SEQ ID NO: 140) where n is 1 , 2, 3, 4, 5 or 6 is used, as described in U.S. Patent No. 7,271,149.
  • a serine-rich peptide linker is used, as described in U.S. Patent No. 5,525,491.
  • Serine rich peptide linkers include those of the formula [X-X-X-X-Gly] y (SEQ ID NO:141), where up to two of the X are Thr, and the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly (SEQ ID NO: 142), where y is greater than 1).
  • the linker is a single amino acid (e.g., any amino acid, such as Gly or Cys).
  • Amino acid linkers may be selected for flexibility (e.g., flexible or rigid) or may be selected on the basis of charge (e.g., positive, negative, or neutral).
  • Flexible linkers typically include those with Gly resides (e.g., [Gly-Gly-Gly-Gly-Ser] n where n is 1, 2, 3, 4, 5 or 6).
  • Other linkers include rigid linkers (e.g., PAPAP (SEQ ID NO: 143) and (PT) n P (SEQ ID NO: 144), where n is 2, 3, 4, 5, 6, or 7) and ⁇ x- helical linkers (e.g., A(EAAAK) n A (SEQ ID NO: 145), where n is 1 , 2, 3, 4, or 5).
  • linkers are succinyl, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys.
  • linker When the linker is succinyl, one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the other carboxyl group thereof may, for example, form an ester bond with a hydroxyl group of opioid moiety.
  • linker When the linker is Lys, Glu, or Asp, the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the amino group thereof may, for example, form an amide bond with a carboxyl group of the opioid moiety.
  • Lys is used as the linker, a further linker may be inserted between the ⁇ -amino group of Lys and the opioid moiety.
  • the further linker is succinyl acid, which can form an amide bond with the ⁇ - amino group of Lys and an ester bond with a hydroxyl group present in the opioid moiety.
  • the further linker is Glu or Asp (e.g., which forms an amide bond with the ⁇ -amino group of Lys and an ester or amide bond with a hydroxyl or amino group present in the opioid moiety).
  • the peptide linker is a branched polypeptide.
  • Exemplary branched peptide linkers are described in U.S. Patent No. 6,759,509.
  • linkers described herein may be modified.
  • the linkers can include a spacer molecule.
  • the spacer molecule within linker can be of any suitable molecule.
  • spacer molecules include aliphatic carbon groups (e.g., C 2 -C 20 alkyl groups), cleavable heteroatomic carbon groups (e.g., C -C 20 alkyl groups with dithio groups), and hydrophilic polymer groups.
  • hydrophilic polymer groups include poly(ethylene glycol) (PEG), polyvinylpyrrolidone,
  • polyvinylmethylether polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,
  • hydroxyethylcellulose polyethyleneglycol, polyaspartamide, and a hydrophilic peptide sequence.
  • the hydrophilic polymer is PEG, such as a PEG chain having a molecular weight between 500-10,000 Da (e.g., between 1,000-5,000 Da such as 2,000 Da). Methoxy or ethoxy-capped analogues of PEG can also be used. These are commercially available in sizes ranging between 120-
  • lipid-tether conjugates for use in liposomes is described, for example, in U.S. Patent No. 5,395,619, hereby incorporated by reference.
  • Other spacer molecules include polynucleotides (e.g., DNA or RNA), polysaccharides such as dextran or xanthan, cellulose derivatives (e.g., carboxymethyl cellulose), polystyrene, polyvinyl alcohol, poly methylacrylic acid, and poly(NIPAM).
  • Synthetic reaction schemes for activating PEG with coupling agents are set forth in U.S. Pat. Nos.
  • PEG for example, can be conjugated to a polypeptide of the invention by any means known in the art.
  • the PEG molecule is derivatized with a linker, which is then reacted with the protein to form a conjugate.
  • Suitable linkers include aldehydes, tresyl or tosyl linkers,
  • the conjugates of the invention include a linker selected from the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, adipyl, pimelyl, suberyl, azelayl, trans- ⁇ - hydromuconyl, 3,6,9-trioxaundecanedioyl (PEG 3 ), carboxybutylether, and carboxyethylether (depicted in table 4).
  • a linker selected from the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, adipyl, pimelyl, suberyl, azelayl, trans- ⁇ - hydromuconyl, 3,6,9-trioxaundecanedioyl (PEG 3 ), carboxybutylether, and carboxyethylether (depicted in table 4).
  • Additional linkers include those containing a disulfide bond, such as the ethyl disulfide linker formed when pyridyldithioethylamine is coupled with a carboxylic acid of an opioid moiety and then allowed to react with a cysteine-containing polypeptide.
  • Other exemplary linkers of invention may be formed by the reaction between the sulfur of a cysteine group in a polypeptide and a maleimido group attached to the opioid moiety (e.g., cis-buten-mal, hex-mal, and PEG 3 -mal depicted in table 4).
  • Still other exemplary linkers of the invention may be formed by the reaction between a click-chemistry pair, where one reactive group is attached to the polypeptide and the other reactive group is attached to the opioid moiety.
  • An exemplary embodiment of a click-chemistry linker is methyl-triazolyl-propyl, as depicted in table 4. In each of the linkers depicted in table 4, one terminus of the linker is attached to a functional group in the opioid moiety and the other terminus is attached to a functional group in the polypeptide.
  • the polypeptide Angiopep-2 is conjugated to three opioid moieties by means of a linker.
  • the opioid moiety is a phenanthrene opioid receptor agonist or the opioid moiety is morphine or M6G.
  • ⁇ contains two hydroxyl groups, a phenol at the 3-position and a secondary alcohol at the 6-position.
  • the polypeptide Angiopep-2 may be linked through its N-terminus and lysine residues at positions 10 and 15 by means of a linker.
  • Such a structure is depicted in formula Ilia.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • Related polypeptide-opioid conjugates of the invention are described by formula Ilia where the morphine moiety is replaced with a different
  • the polypeptide Angiopep-2 is conjugated to three morphine molecules through the C-3 hydroxyl group of the morphine. These embodiments are depicted in formula Illb.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • Related polypeptide-opioid conjugates of the invention are described by formula Illb where the morphine moiety is replaced with a different phenanthrene-based opioid agonist having a free C-3 hydroxyl group (e.g., hydromorphone).
  • the polypeptide Angiopep-2 is conjugated to three M6G molecules through the 5' carboxylic acid of M6G through ester linkages. These embodiments are depicted in formula IIIc.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2 is conjugated to three M6G molecules through the 5' carboxylic acid of M6G through amide linkages. These embodiments are depicted in formula Illd.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2 is conjugated to three M6G molecules through the 2' hydroxyl group of M6G. These embodiments are depicted in formula Ille.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers
  • the polypeptide Angiopep-2 is conjugated to three M6G molecules through the C-3 hydroxyl of M6G. These embodiments are depicted in formula Illf.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2 is conjugated to three M3G molecules through the C-6 hydroxyl of M3G. These embodiments are depicted in formula Illg.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2 -Cys is conjugated to one M6G molecule through the 5' carboxylic acid of M6G via an ester linkage.
  • Exemplary embodiments are depicted in formula IIHi.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein. —
  • the polypeptide Angiopep-2-Cys is conjugated to one M6G molecule through the 5' carboxylic acid of M6G via an amide linkage.
  • exemplary embodiments are depicted in formula IIIi.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2-Cys is conjugated to one M6G molecule through the 4' alcohol group of M6G.
  • Exemplary embodiments are depicted in formula Illj .
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2-Cys is conjugated to one M6G molecule through the C-3 hydroxyl group of M6G.
  • Exemplary embodiments are depicted in formula Illk.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2-Cys is conjugated to one morphine molecule through the C-3 hydroxyl group of morphine.
  • Exemplary embodiments are depicted in formula IIIl.
  • the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • the polypeptide Angiopep-2-Cys is conjugated to one morphine molecule through the C-6 hydroxyl group of morphine.
  • Exemplary embodiments are depicted in formula Illm.
  • the Ln KE moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
  • polypeptide-opioid conjugate is described by any one of formulas Illa-IIIm, where residues 8, 10, and 11 are D-amino acids.
  • polypeptides of the invention include those listed in table 5.
  • Polypeptide-opioid conjugates of the invention may be prepared by linking one or more opioid moieties to side chains (e.g., lysine or cysteine side chains) of residues in any of the polypeptides listed in table 5.
  • side chains e.g., lysine or cysteine side chains
  • the sulfur atom of the cysteine residue may be incorporated into a disulfide moiety of a linker.
  • the N- terminus and/or C-terminus of a polypeptide shown in table 5 may be modified with an opioid moiety.
  • the N-terminus of a polypeptide is linked to an opioid moiety and two amino acid side chains of the polypeptide are linked to opioid moieties.
  • the C- terminus may be either an amide or a carboxylic acid.
  • the present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a polypeptide-opioid conjugate of the invention.
  • the composition can be formulated for use in a variety of drug delivery systems.
  • One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation.
  • Suitable formulations for use in the present invention are found in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985.
  • Langer Science 249:1527-1533, 1990.
  • the pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration.
  • compositions for parenteral administration that include the above mentioned agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like.
  • an acceptable carrier preferably an aqueous carrier
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • the invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like.
  • this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.
  • compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.
  • the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules.
  • the composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
  • compositions containing an effective amount of the polypeptide-opioid conjugate can be administered for prophylactic or therapeutic treatments.
  • Compositions of the invention can be administered to the subject (e.g., a human) in an amount sufficient to delay, reduce, or prevent the onset of pain.
  • compositions are administered to a subject (e.g., a human) already experiencing pain in an amount sufficient to alleviate or partially alleviate the pain.
  • Amounts effective for this use may depend on the severity of the pain and the weight and general state of the subject, but generally range from about 0.1-3,000 mg of an equivalent dose of the opioid (e.g., morphine) per dose per subject.
  • Typical morphine sulfate dosages for pain are 5-30 mg (e.g., 5, 10, 15, 20, 25, 30 mg), orally, every 3 to 4 hours; 10-600 mg (e.g., 10, 20, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 mg), extended release, daily; 4-15 mg (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg), intravenously, every 3 to 4 hours; and 0.8-440 mg/hour (e.g., 0.8, 1, 2, 3, 5, 8, 10, 15, 20, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 440), continuous intravenous infusion.
  • the opioid e.g., morphine
  • the polypeptide-opioid conjugate of the invention is administered (e.g., intravenously or orally) at a lower dose than the equivalent typical dose of morphine or morphine sulfate (e.g., less than or equal to about 90%, 85%, 80%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the equivalent dose of morphine or morphine sulfate).
  • morphine or morphine sulfate e.g., less than or equal to about 90%, 85%, 80%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the
  • Suitable regimes for administration are typified by an initial administration followed by repeated doses at one or more hourly or daily intervals by a subsequent administration.
  • the total effective amount of an opioid present in the compositions of the invention can be administered to a mammal as a single dose, orally, as a bolus, or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time (e.g., a dose every 1-2, 4-6, 8-12, 14-16, or 18-24 hours).
  • continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are used.
  • the therapeutically effective amount of the opioid moiety present within the compositions of the invention and used in the methods of this invention applied to mammals can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because the polypeptide-opioid conjugates exhibit an enhanced ability to cross the BBB, the dosage of the compounds of the invention can be lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dosage required for a therapeutic effect of the unconjugated agent.
  • the polypeptide-opioid conjugates of the invention are administered to a subject (e.g. a mammal, such as a human) in an effective amount, which is an amount that produces analgesia in a treated subject.
  • Therapeutically effective amounts can also be determined empirically by those of skill in the art.
  • the subject may also receive an polypeptide-opioid conjugate in the range of about 0.1-3,000 mg equivalent dose, as compared to unconjugated opioid, per dose one or more times per day (e.g., 2, 3, 4, 5, 6, or 6 or more times per day).
  • an polypeptide-opioid conjugate in the range of about 0.1-3,000 mg equivalent dose, as compared to unconjugated opioid, per dose one or more times per day (e.g., 2, 3, 4, 5, 6, or 6 or more times per day).
  • Single or multiple administrations of the polypeptide-opioid conjugates of the invention, including an effective amount, can be carried out with dose levels and pattern being selected by the treating physician.
  • the dose and administration schedule can be determined and adjusted based on the severity of the pain being treated, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.
  • polypeptide-opioid conjugates of the invention may be administered in combination therapies with other agents.
  • the conjugates and other agent or agents may be administered sequentially or concurrently to an individual.
  • pharmaceutical compositions according to the present invention may be comprised of a combination of a conjugate of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.
  • Nanoparticles coated with a polypeptide of the invention may be used to deliver an opioid (e.g., mo hme or M6G) to a subject.
  • an opioid e.g., mo hme or M6G
  • a "nanoparticle” is a colloidal, polymeric, lipid, or elemental particle ranging in size from about 1 nm to about 1000 nm.
  • Nanoparticles can be made up of silica, carbohydrate, lipid, or polymer molecules.
  • Opioid molecules can be embedded or encapsulated in the nanoparticle matrix or may be adsorbed onto its surface.
  • the nanoparticle may be made up of a biodegradable polymer such as poly(butylcyanoacrylate) (PBCA).
  • PBCA poly(butylcyanoacrylate)
  • elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic and/or a polypeptide of the invention.
  • OA oleic acid
  • an opioid e.g., morphine or M6G
  • Other nanoparticles are made of silica, and include those described, for example, in Burns et al., Nano Lett. 9:442-448, 2009.
  • Nanoparticles can be formed, in part, from any useful polymer.
  • polymers include biodegradable polymers, such as poly(butyl cyanoacrylate), poly(lactide), poly(glycolide), poly-D - caprolactone, poly(butylene succinate), poly(ethylene succinate), and poly(p-dioxanone);
  • a certain percentage e.g., 0.1 %, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%
  • a polypeptide e.g., Angiopep-2
  • Polymeric nanoparticles can be produced by any useful process.
  • the solvent evaporation method the polymer and opioid are dissolved in a solvent to form a nanoemulsion and the solvent is evaporated.
  • Appropriate solvent systems and surfactants can be used to obtain either oil-in-water or water-in-oil nanoemulsions.
  • This method can optionally include filtration, centrifugation, sonication, or lyophilization.
  • a solution of the polymer and an opioid is formed in a first solvent. Then, the solution is added to a second solvent that is miscible with the first solvent but does not solubilize the polymer. During phase separation, nanoparticles are formed spontaneously.
  • the monomer is dispersed into an aqueous solution to form micelles.
  • Initiator radicals e.g. hydroxyl ions
  • Additional methods include dialysis, ionic gelation, interfacial polymerization, and solvent casting with porogens.
  • the polymer is a cyanoacrylate copolymer containing a hydrophilic polymer group: poly(aminopoly(ethyleneglycol) cyanoacrylate-co-hexadecyl cyanoacrylate), which is synthesized as described in Stella et al., J. Pharm. Sci. 89: 1452-1464 (2000).
  • the cyanoacrylate polymer, or a certain fraction of the polymer may be conjugated to any polypeptide described herein using any conjugation method known in the art.
  • the polymer and an opioid are added to an organic solvent, and the mixture is emulsified by adding an aqueous solution.
  • the monomer is added dropwise to an acidic aqueous solution.
  • the mixture is stirred to promote polymerization and then neutralized.
  • the nanoparticles are then filtered, centrifuged, sonicated, and washed.
  • the monomer of butyl cyanoacrylate monomer is provided and the aqueous solution also includes dextran in a dilute aqueous solution of hydrochloric acid.
  • the poly(butyl cyanoacrylate) nanoparticles are lyophilized and then resuspended in saline. An opioid is added to the saline solution with the nanoparticles under constant stirring.
  • the opioid is added to during the polymerization process.
  • the nanoparticles are coated with a polypeptide of the invention and optionally coated with a surfactant, such as polysorbate 80.
  • a surfactant such as polysorbate 80.
  • Nanoparticles include solid lipid nanoparticles (SLN).
  • SSN approaches are described, for example, in Kreuter, Ch. 24, In V. P. Torchilin (ed), Nanoparticles as Drug Carriers pp. 527-548, Imperial College Press, 2006).
  • lipid molecules for solid lipid nanoparticles include stearic acid and modified stearic acid, such as stearic acid-PEG 2000; soybean lechitin; and emulsifying wax.
  • Solid lipid nanoparticles can optionally include other components, including surfactants, such as Epicuron® 200, poloxamer 188 (Pluronic® F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts, such as taurocholate sodium.
  • surfactants such as Epicuron® 200, poloxamer 188 (Pluronic® F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts, such as taurocholate sodium.
  • Opioids can be introduced into solid lipid nanoparticles by a number of methods known in the art.
  • SLNs include stearic acid, Epicuron 2000 (surfactant), and taurocholate sodium loaded with an opioid.
  • SLNs include stearic acid, soybean lecithin, and poloxamer 188.
  • SLNs can also be made from polyoxyl 2-stearyl ether (Brij 72), or a mixture of emulsifying wax and polyoxyl 20-stearyl ether (Brij 78) (see, e.g., Koziara et al., Pharm Res 20: 1772-1778, 2003).
  • a microemulsion is formed by adding a surfactant (e.g.
  • Emulsifying wax is a waxy solid that is prepared from cetostearyl alcohol and contains a polyoxyethylene derivative of a fatty acid ester of sorbitan. Nanoparticles are formed by cooling the mixture while stirring.
  • the opioid can be introduced by adding the opioid to the heated mixture containing the emulsifying wax in water.
  • Nanoparticles can also include nanometer-sized micelles.
  • Micelles can be formed from any polymers described herein.
  • Exemplary polymers for forming micelles include block copolymers, such as poly(ethylene glycol) and poly(8-caprolactone).
  • PEO-b-PCL block copolymer is synthesized via controlled ring-opening polymerization of ⁇ -caprolactone by using an a-methoxy- ⁇ - hydroxy-poly(ethylene glycol) as a macroinitiator.
  • the PEO-b-PCL block copolymers are dissolved in an organic solvent (e.g., tetrahydrofuran) and then deionized water is added to form a micellar solution. The organic solvent is evaporated to obtain nanometer-sized micelles.
  • an organic solvent e.g., tetrahydrofuran
  • the properties of the nanoparticle are altered by coating with a surfactant.
  • a surfactant Any biocompatible surfactant may be used, for example, polysorbate surfactants, such as polysorbate 20, 40, 60, and 80 (Tween 80); Epicuron® 200; poloxamer surfactants, such as 188
  • Nanoparticles can be optionally coated with a surfactant.
  • Nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethyleneglycol) or poly(propyleneglycol)).
  • the surface of the nanoparticle can be modified by covalently attaching hydrophilic polymer groups.
  • nanoparticles can be formed by using polymers that contain hydrophilic polymer groups, such as poly[methoxy poly (ethyleneglycol) cyanoacrylate-co-hexadecyl cyanoacrylate]. Nanoparticles can be optionally cross-linked, which can be particularly use for protein-based nanoparticles.
  • a polypeptide of the invention may be covalently linked to the polymer or lipid used to form a nanoparticle before or after the formation of the nanoparticle.
  • Angiopep-2 may be covalently linked to the polymer or lipid used to form a nanoparticle before or after the formation of the nanoparticle.
  • the polypeptide is linked to the nanoparticle through a spacer moiety (e.g., a poly(ethylene glycol)- or poly(lactic acid)-containing spacer).
  • the spacer precursor may by attached to any polymer or lipid of the nanoparticle and may include a reactive group (e.g., an NHS ester, aldehyde, or maleimide) that reacts selectively with an amino or a thiol group of the polypeptide, thereby linking the polypeptide to the nanoparticles.
  • Opioids can be introduced to nanoparticles by any useful method. Opioids can be incorporated into the nanoparticle at, during, or after the formation of the nanoparticle.
  • the opioid is added to the solvent with the polymer or monomer before the formation of the nanoparticles.
  • the opioid is incorporated into pre-formed nanoparticles by adsorption.
  • the opioid is covalently bound to the nanoparticle.
  • the opioid can be physically adsorbed to the surface of the nanoparticle with the optional step of further coating the nanoparticle with a surfactant.
  • surfactants include polysorbate 80 (Tween 80). Further examples of this approach are described in Kreuter, Nanoparticular Carriers for Drug Delivery to the Brain, Chapter 24, in Torchilin (ed.), Nanoparticulates as Drug Carriers (2006), Imperial College Press.
  • the invention features any of the compositions or methods for the transport of therapeutic agents
  • Morphine- -glucuronide was synthesized by the route shown in Figure 1. Morphine sulfate (320 mg) was acylated with acetic anhydride and saturated aqueous sodium bicarbonate to provide 3-O-acetylmorphine (250 mg). The phenol group of the product was glucuronated with acetobromo-D- glucuronic acid methyl ester. The glucuronidation reaction was allowed to go to completion, i.e., the reaction was not stopped until the 3-O-acetylmorphine was fully consumed. The product (490 mg) was used directly in a hydrolysis reaction with sodium hydroxide in methanol and water to provide M6G in 56% yield (200 mg). B. Preparation of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys
  • M6G (25 mg) was coupled with pyridyldithioethylamine using benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and diisopropylethylamine (DIEA) in DMF to provide the amide product in 100% conversion by mass.
  • the product was not isolated, but was treated in situ with 1.3 equiv of Angiopep-2 -Cys and DBEA to provide the final product, M6G-5'-Amido- Ethyl-Disulfide-Angiopep-2-Cys in 48% yield (76 mg) ( Figure 2).
  • TBSC1 tert-butyldimethylsilyl chloride
  • the secondary alcohol of the product was functionalized with glutaric anhydride to yield a carboxylic acid.
  • the carboxylic acid was treated with the coupling agent 0-(benzotriazol-l -yl)-N,N,N',N- tetramethyluronium tetrafluoroborate (TBTU) in DMF and then Angiopep-2 in DMF to yield a conjugate that was deprotected with tetra- «-butylammonium fluoride (TBAF) to yield (Morphine-6-Glutaryl) 3 - Angiopep-2 ( Figure 5).
  • TBTU 0-(benzotriazol-l -yl)-N,N,N',N- tetramethyluronium tetrafluoroborate
  • Morphine was protected at the C-3 and C-6 positions with TBSC1.
  • the bis-TBS compound was treated with TBAF to selectively remove the phenolic TBS group.
  • the phenol of the product was functionalized with glutaric anhydride to yield a carboxylic acid.
  • the carboxylic acid was treated with the coupling agent TBTU and DIEA, and then Angiopep-2 in DMSO to yield a conjugate that was deprotected with trifluoroacetic acid in water to provide (Moiphine-6-Glutaryl) 3 -Angiopep-2 ( Figure 7).
  • the phenol group of morphine was coupled with suberic acid using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide » HCl (EDC).
  • EDC 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide » HCl
  • the product carboxylic acid was treated with TBTU in DMF and then Angiopep-2 in DMF to provide (Morphine-3-Suberyl) 3 -Angiopep-2 ( Figure 9).
  • the phenol group of morphine was coupled with 3,6,9-trioxaundecanedioic acid in DMF with PyBOP and DIEA to provide the product carboxylic acid in 51% yield.
  • the carboxylic acid was treated with TBTU and DIEA in DMF then Angiopep-2 in DMSO/DMF to provide (Morphine-3-PEG 3 ) 3 - Angiopep 2 in 40% yield ( Figure 11).
  • Morphine was protected at the C-3 and C-6 positions with TBSC1 and imidazole.
  • the bis-TBS compound was treated with TBAF to selectively remove the phenolic TBS group.
  • the phenol of the product was functionalized with methyl 5-bromopentanoate and sodium methoxide to yield a terminal ester product.
  • the ester was saponified with lithium hydroxide.
  • the carboxylic acid product was treated with TBTU and DIEA then Angiopep-2 in DMSO to yield a conjugate that was deprotected with trifluoroacetic acid in water to yield a trifluoroacetate salt that was subsequently converted into the hydrochloride salt of (Morphine-Carboxybutylefher) 3 -Angiopep-2 ( Figure 12).
  • Example 8 Polypeptide-Opioid Conjugates Induce Analgesia in the Hot-PIate Test
  • Angiopep-2-Cys over unconjugated M6G and morphine, and indicate that a cleavable linker is preferred to a non-cleavable linker.
  • CD-I mice were treated intravenously with four different dosages of M6G-5'-Amido-Efhyl- Disulfide-Angiopep-2-Cys (3.8 mg/kg, 9.5 mg/kg, 1 mg/kg, and 38 mg/kg). After a set period of time (15 min, 30 min, 60 min, 90 min, or 120 min), mice were placed on an electrically heated surface maintained at 55 °C. The mice were constrained to the hot plate using a transparent barrier and monitored. The time until flicking of the hindpaw was measured and recorded as hindpaw latency.
  • Figure 1 A depicts hindpaw latency in terms of post-injection time for the four different doses of M6G- 5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys.
  • Untreated animals exhibited a hindpaw latency time of, approximately, between 5 and 10 seconds, while treated mice showed higher hindpaw latency times. The greatest increase in hindpaw latency was observed at the highest dose.
  • the data indicate that M6G-5'- Amido-Ethyl-Disulfide-Angiopep-2-Cys induces an analgesic effect with respect to thermal nociception, and that this effect increases with increasing dose over the range of 3.8 mg/kg to 38 mg/kg.
  • mice were treated intravenously with either M6G-5'-Amido-Ethyl-Disulfide- Angiopep-2 - Cys at 9.5 mg/kg or with M6G at 1.5 mg/kg. After a set period of time (15 min, 60 min, 120 min, 180 min, or 240 min), mice were placed on an electrically heated surface maintained at 55 °C. The mice were constrained to the hot plate using a transparent barrier and monitored. The time until flicking of the hindpaw was measured and recorded as hindpaw latency.
  • Figure 15B depicts hindpaw latency in terms of post-injection time for the two compounds.
  • mice receiving M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2- Cys exhibited hindpaw latency times of greater than 20 seconds, while mice receiving M6G exhibited lower hindpaw latency times.
  • the data indicate that conjugating M6G to a polypeptide capable of crossing the BBB increases the analgesic effects of M6G with respect to thermal nociception.
  • CD-I mice were treated intravenously with M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys at
  • mice 25 mg/kg, (M6G-5'-Amido-Ethyl-Disulfide-Ethyl) 3 -Angiopep-2 at 6.7 mg/kg, M6G at 3.2 mg/kg, or saline.
  • mice were placed on an electrically heated surface maintained at 55 °C.
  • Figure 15C depicts hindpaw latency in terms of post- injection time for the four treatments. The mice were constrained to the hot plate using a transparent barrier and monitored. The time until flicking of the hindpaw was measured and recorded as hindpaw latency.
  • Figure 16 illustrates hot-plate assay data (obtained by the procedure of Example 8B) showing that M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys (at 9.5 mg/kg) is more active than both unconjugated M6G (at 1.5 mg/kg) and morphine (at 1.25 mg/kg) and fully active for at least 3-4 hours.
  • Figure 17 illustrates hot-plate data (obtained by the procedure of Example 8 A) comparing a polypeptide-opioid conjugate having a cleavable linker with a conjugate having a non-cleavable linker ((mo hine-carbo ybutylether)3-Angiopep-2).
  • the data shows that the conjugate with the cleavable linker provided morphine-like analgesia while the conjugate with the non-cleavable linker did not provide analgesia.
  • FIG. 21 A The chemical structure and schematic representation of Angiopep-2-M6G conjugate (ANG2010) are shown in Figure 21 A. Competition binding of M6G and ANG2010 to Mu-opioid (MOPr) ( Figure 2 IB) or Delta-opioid (DOPr) ( Figure 21 C) receptors in rat brain membranes was evaluated. Brain membranes were incubated with either 1 nM [ 3 H]-DAMGO or 1 nM [ 3 ⁇ ]- ⁇ 1 ⁇ 1 ⁇ II and increasing concentrations (10 " to 10 "5 M) of unlabeled M6G or unlabeled ANG2010 for 60 minutes at 37 °C. Binding inhibition results were analyzed using GraphPad Prism software. Each curve represents the mean of 6 measurements. Data are expressed as the mean Ki ⁇ SEM.
  • Example 12 Brain uptake of ANG2010
  • Example 13 Evaluation of analgesic effect in a hot plate model
  • the hot plate model was used to evaluate the analgesic effect of ANG2010.
  • mice were placed onto a hot metal plate maintained at 54°C and foot-licking response was measured after dosing. Latency to a hind limb response (lick, shake, or jump) was recorded, with a maximum time on the hot plate of 30 seconds. A statistically significant increase from baseline pain-threshold measurement was interpreted as induction of analgesia.
  • ANG2010 significantly increased the paw flicking latency after intravenous (IV) bolus injection compared to an equimolar dose of unconjugated M6G (Figure 24A). In addition, ANG2010, after IV or sub-cutaneous (SC) administration, significantly increased the foot licking latency for at least 3hrs ( Figure 24B).
  • Example 14 Evaluation of analgesic effect in a rat tail flick mouse model
  • the rat tail flick model was also used to evaluate the analgesic effect of ANG2010. Pain threshold was measured before (baseline) and after drug administration, using a standard hot-water tail- flick assay. The dependent variable was the latency (in seconds) for the rat to flick its tail from the hot- water bath. The water was maintained at 53°C in a constant-temperature water bath. The distal first 5 cm of the rat's tail was immersed in the bath, and the time required for the rat to remove its tail was measured. A statistically significant increase from baseline pain-threshold measurement was interpreted as induction of analgesia.
  • rV or sub-cutaneous (SC) administration of ANG2010 significantly increased the latency to tail withdrawal for at least 3hours ( Figures 25A and 25B, and Figure 26).
  • Example 15 Evaluation of sub-cutaneous injection of ANG2010 on gastro-intestinal transit

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Abstract

The invention features polypeptide-opioid conjugates capable of crossing the blood-brain barrier. The polypeptide-opioid conjugates can be used to treat a condition that is modulated by opioid receptors such as pain (e.g., postoperative pain, cancer pain, acute pain, and chronic pain).

Description

POLYPEPTIDE-OPIOID CONJUGATES AND USES THEREOF
Background of the Invention
The invention relates to opioid-polypeptides that are capable of crossing the blood-brain barrier and uses thereof.
Approximately 70% of patients with advanced cancer and 65% of patients dying from non- malignant diseases experience pain (Colvin et al., BMJ332: 1081-1083,' 2006). For many years, the opioid morphine has been considered to be the most important drug for the treatment of acute and chronic pain (van Dorp et al., Anesth. Analg. 102: 1789-1797, 2006). However, a variety of side effects are associated with the use of morphine and other opioids in pain management. Constipation is one of the most prevalent side effects of opioid treatment. A recent study found that 47% of chronic non-cancer pain (CNCP) patients taking opioids reported suffering from constipation (Tuteja et al.,
Neurogastroenterol. Motil. 22: 424-430, 2010).
Opioid analgesics are agonists of opioid receptors. Opioid receptors are present in high densities in the brain and spinal cord in the central nervous system (CNS) and in the gastrointestinal tract in the peripheral nervous system (PNS). The analgesic effects of morphine are largely due to the action of the drug on receptors in the brain and spinal cord, while constipation is due to the action of opioids on receptors in the gastrointestinal tract. In addition to constipation, opioid treatment is associated with a range of other side effects. Among CNCP patients taking opioids, 27% reported nausea, 9% reported vomiting, and 33% reported gastroesophageal reflux disease.
Because pain perception is modulated by opioid receptors in the brain and spinal cord, therapeutics that act on such receptors must cross the blood-brain barrier (BBB). The BBB is considered a major obstacle for the potential use of drugs for treating disorders of the CNS. The global market for CNS drugs was $68 billion in 2006, which was roughly half that of the global market for cardiovascular drugs, even though in the United States nearly twice as many people suffer from CNS disorders as from cardiovascular diseases. The reason for this imbalance is, in part, that more than 98% of all potential CNS drugs do not cross the BBB.
The brain is shielded against potentially toxic substances by the presence of two barrier systems: the BBB and the blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered to be the major route for the uptake of serum ligands since its surface area is approximately 5000-fold greater than that of BCSFB. The brain endothelium, which constitutes the BBB, represents the major obstacle for the use of potential drugs against many disorders of the CNS. As a general rule, only small lipophilic molecules may pass across the BBB, i.e., from circulating systemic blood to the brain.
New formulations of analgesic opioid drugs are needed to enhance delivery of active compounds across the BBB. Additionally, new formulations of analgesic opioid drugs are needed to deliver active compounds selectively to the brain and spinal cord (i.e., across the BBB). Summary of the Invention
In one aspect, the invention features a polypeptide-opioid conjugate, having (i): a polypeptide that is capable of crossing the blood-brain barrier; and (ii) one or more opioid moieties linked to the polypeptide by means of a linker; where each opioid moiety is linked to the polypeptide (e.g., the N- terminus, the C-terminus, or an amino acid side chain of the polypeptide).
In some embodiments, the polypeptide-opioid conjugate features a polypeptide (a) substantially identical (e.g., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in a sequence selected from the group consisting of SEQ ID NO: 1-105, SEQ ID NO: 107-117, SEQ ID NO:120-138, and SEQ ID NO:166-178, or a fragment thereof. In related embodiments, the polypeptide contains between 6 and 21 amino acids. In particular embodiments, the polypeptide-opioid conjugate features a polypeptide (a) substantially identical (e.g., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in Angiopep-2 (SEQ ID NO: 97), (3D)-Angiopep-2 (SEQ ID NO: 166), Angiopep-P6a (SEQ ID NO:174), Angiopep-descys- P6a (SEQ ID NO: 177), Angiopep-2-Cys (SEQ ID NO: 114), or (3D)-Angiopcp-2-Cys (SEQ ID NO: 178), or a pharmaceutically acceptable salt thereof.
In related embodiments, the polypeptide-opioid conjugate features an opioid moiety selected from the group consisting of morphine, morphine-6-glucuronide, morphine-3-glucuronide, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, alazocine, alfentanil, bremazocine, buprenorphine, butorphanol, cyclazocine, dezocine, dihydrocodeine, diphenoxylate, diamorphine, fentanyl, levorphanol, meperidine, meptazinol, methadone, nalbuphine, pentazocine, propoxyphene, remifentanil, sufentanil, tramadol, naloxone, naltrexone, diprenoφhine, naloxonazine, met-enkephalin, leu-enkephalin, β-endorphin, dynorphin A, dynorphin B, α-ηεο-ε^οφΐιίη, endonu^hin-l ,
εηάοπιοφ1πη-2, and nociceptin. In particular embodiments, the polypeptide-opioid conjugate features an opioid moiety selected from the group consisting of moφhine, moφhine-6-glucuronide, ηιοφηίη6-3- glucuronide, and
Figure imgf000003_0001
In related embodiments, the polypeptide-opioid conjugate contains a linker selected from the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, adipyl, pimelyl, suberyl, azelayl, trans-β- hydromuconyl, 3,6,9-trioxaundecanedioyl, a poly(ethylene glycol) (PEG) linker (e.g., PEG3),
carboxybutylether, carboxyethylether, a disulfide-containing linker, ethyl disulfide, ethyl-disulfide-ethyl, a cis-buten-mal linker, a hex-mal linker, a PEG3-mal linker, a triazole-containing linker, and methyl- triazolyl-propyl.
In particular embodiments, the opioid moiety of the polypeptide-opioid conjugate is linked to a lysine residue or a cysteine residue in the polypeptide or to the N-terminus of the polypeptide. In some embodiments, the polypeptide is linked to exactly one opioid moiety, and in other embodiments the polypeptide is linked to exactly three opioid moieties. In some embodiments, the polypeptide contains at least one D-amino acid. In related embodiments, the polypeptide contains exactly three D-amino acids. In specific embodiments, the polypeptide opioid conjugate is selected from the group consisting of M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys, M6G-5 '-cis-buten-mal-Angiopep-2-Cys, M6G-4'- PEG3-mal-Angiopep-2-Cys, M6G-hex-mal-Angiopep-2-Cys, M6G-3-PEG3-mal-Angiopep-2-Cys, (M6G- amido-ethyl-disulfide-ethyl)3-Angiopep-2,
Figure imgf000004_0001
(morphine-6- dimethylglutaryl)3-Angiopep-2, (morphine-3-glutaryl)3-Angiopep-2,
Figure imgf000004_0002
Angiopep-2, (morphine-3-suberyl)3-Angiopep-2, (morphine-3-succinyl)3-Angiopep-2, (morphine-3-PEG- 3)3-Angiopep-2, (mo hine-3-carbo ybutylether)3-Angio e -2, (moφhine-6-succinyl)3-Angioρeρ-2, moφhine-3-PEG3-mal-Angiopep-2-Cys, and (M6G-5'-methyl-triazolyl-propyl)3-Angiopep-2.
In another aspect, the invention features a nanoparticle conjugated to a plurality of polypeptides having amino acid sequences (a) substantially identical (i.e., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1 , 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in a sequence selected from the group consisting of SEQ ID NO: 1 -105, SEQ ID NO: 107-117, SEQ ID NO: 120-138, and SEQ ID NO:l 66-178, or a fragment thereof, where the nanoparticle is bound to or contains a opioid moiety (e.g., ηιοφΐιίηε or M6G). In particular embodiments, the nanoparticle is conjugated to a plurality of polypeptides (a) substantially identical (e.g., at least 70%, 80%, 85%, or 90% identical) to, or (b) having 0, 1 , 2, 3, 4, 5, 6, or 7 substitutions, deletions, or insertions (e.g., 0-1 , 0-2, 0-3, 0-4, 0-5, 0-6, or 0-7) in Angiopep-2 (SEQ ID NO:97), (3D)-Angiopep-2 (SEQ ID NO: 166), Angiopep- P6a (SEQ ID NO: 174), Angiopep-descys-P6a (SEQ ID NO: 177), Angiopep-2-Cys (SEQ ID NO:l 14), or (3D)-Angiopep-2-Cys (SEQ ID NO: 178), or a pharmaceutically acceptable salt thereof, where the nanoparticle is bound to or contains an opioid moiety (e.g., ιηοφηϊηβ or M6G).
In another aspect, the invention features a method of treating pain in a subject by administering a composition containing a polypeptide-opioid conjugate or opioid-containing nanoparticle to the subject in an amount sufficient to treat the pain. In particular embodiments, the pain is selected from the group consisting of postoperative pain, cancer pain, chronic pain, acute pain, somatic pain, neuropathic pain, visceral pain, inflammatory pain, migraine-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritic pain, skeletal pain, joint pain, gastrointestinal pain, muscle pain, angina pain, facial pain, pelvic pain, claudication, post traumatic pain, obstetric pain, labor pain, gynecological pain, and chemotherapy-induced pain.
In related embodiments, the invention features a method of treating pain in a subject where said opioid conjugate is administered in an amount that reduces at least one opioid-induced side effect as compared to the same effective dosage of the corresponding unconjugated opioid. In particular embodiments, the opioid-induced side effect is selected from the group consisting of constipation, respiratory depression, apnea, circulatory depression, respiratory arrest, shock, cardiac arrest, lightheadedness, dizziness, sedation, nausea, vomiting, sweating, dysphoria, euphoria, weakness, headache, agitation, tremor, uncoordinated muscle movements, seizure, alterations of mood, dreams, muscle rigidity, transient hallucinations and disorientation, visual disturbances, insomnia, increased cranial pressure, dry mouth, biliary tract spasm, laryngospasm, anorexia, diarrhea, cramps, taste alterations, flushing of the face, tachycardia, bradycardia, palpitation, faintness, syncope, hypotension, hypertension, urine retention or hesitance, reduced libido and/or potency, pruritus, skin rashes, edema, diaphoresis, antidiuretic effects, paresthesia, muscle tremor, and blurred vision. In preferred
embodiments, the side-effect is constipation.
In related embodiments, the invention features a method of treating pain in a subject where the conjugate is administered at a lower equivalent dosage than would be required to treat the pain with a corresponding unconjugated opioid.
In certain embodiments of the invention, the opioid moiety is not codeine, dextropropoxyphene, diamorphine, dihydrocodeine, meptazinol, methadone, morphine, nalbuphine, or pentazocine. In particular embodiments, the opioid moiety is not linked to the polypeptide with
bis(sulfosuccinimidyl)suberate (BS3), N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl) (NHS/EDC), N-epsilon-maleimidocaproyl-oxysulfosuccinimide ester (Sulfo-EMCS), N-succinimidyl S- acetylthioacetate (SATA), or succinic anhydride.
In some embodiments, the polypeptide-opioid conjugate is used to treat an opioid receptor- mediated condition including pain, diarrhea, cough, and dyspnea associated with acute left ventricular failure and pulmonary edema.
In certain embodiments, the invention features a pharmaceutically acceptable salt of any of the polypeptide-opioid conjugates described herein. Definitions
By "an amount sufficient" is meant the amount of a compound of the invention required to treat pain in a clinically relevant manner. A sufficient amount of a polypeptide-opioid conjugate used to practice the present invention varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the prescribers will decide the appropriate amount and dosage regimen. The appropriate amounts for any therapy can be determined from animal models, in vitro assays, and/or clinical studies.
By "conjugate" is meant a compound having a polypeptide and a therapeutic agent (e.g., an opioid) linked to the polypeptide.
By a polypeptide that is "capable of crossing the blood-brain barrier" is meant a polypeptide that is able to cross the BBB at a faster rate (e.g., a 5%, 10%, 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000% faster rate) than either a control substance or the unconjugated therapeutic agent. Ability to cross the BBB may be determined using any method known in the art (e.g., an in vitro model of the BBB or in situ brain perfusion as described in U.S. Patent No. 7,557,182).
By "the same effective dosage" is meant the amount of a polypeptide-opioid conjugate of the invention required to achieve the same analgesic effect in a subject, as compared to the unconjugated opioid. By "equivalent dosage" is meant the amount of a polypeptide-opioid conjugate of the invention required to achieve the same molar amount of opioid in the conjugate of the invention, as compared to the unconjugated opioid.
By "fragment" is meant a portion of a full-length amino acid or nucleic acid sequence (e.g., any sequence described herein). Fragments may include at least 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, or 50 amino acids or nucleic acids of the full length sequence. A fragment may retain at least one of the biological activities of the full length protein.
By "nanoparticle" is meant a colloidal, polymeric, lipid, or elemental particle ranging in size from about 1 nm to about 1000 nm.
By a polypeptide "linked to" a therapeutic agent is meant a covalent interaction between the polypeptide and the therapeutic agent. The covalent interaction may be achieved through the use of a linker that forms covalent bonds with both the polypeptide and the therapeutic agent.
By "opioid-induced side effect" is meant a non-analgesic condition induced by the treatment of a subject with an opioid. Common opioid-induced side effects include constipation, respiratory depression, nausea, and vomiting.
By "substantially identical" is meant a polypeptide or nucleic acid having at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or even 99% identity as compared to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, or 100) amino acids. It is to be understood herein that gaps may be found between the amino acids of sequences that are identical or similar to amino acids of the original polypeptide. The gaps may include no amino acids, one or more amino acids that are not identical or similar to the original polypeptide. Percent identity may be determined, for example, with an algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
By "subject" is meant a human or non-human animal (e.g., a mammal).
By "therapeutic agent" is meant an agent that is capable of being used in the treatment or prophylactic treatment of a disease or condition or in the diagnosis of a disease or a condition.
By "treating" pain in a subject is meant alleviating the pain experienced by the subject by administrating a polypeptide-opioid conjugate to the subject.
By "prophylactically treating" pain in a subject is meant reducing the frequency of occurrence or severity of pain experienced by the subject by administering to the subject a polypeptide-opioid conjugate to the subject prior to the onset of pain. For example, a polypeptide-opioid conjugate may be administered to a subject before or during a surgical procedure to prophylactically treat pain.
Recitation of an amino acid residue refers to a naturally occurring L-amino acid, unless otherwise specified.
Other features and advantages of the invention will be apparent from the following Detailed Description and the claims. Brief Description of the Drawings
Figure 1 is a scheme showing the synthesis of morphine-6-glucuronide (M6G) from morphine. Figure 2 is a scheme showing the synthesis of M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys from M6G.
Figure 3 depicts polypeptide-opioid conjugates M6G-5'cis-buten-mal-Angiopep-2-Cys, M6G-3- hex-mal-Angiopep-2-Cys, M6G-4'-PEG3-mal-Angiopep-2-Cys, and M6G-3-PEG3-mal-Angiopep-2-Cys, where the polypeptide Angiopep-2-Cys is conjugated to a single opioid moiety through a cysteine residue in the polypeptide.
Figure 4 depicts the polypeptide-opioid conjugate (M6G-5 ' -amido-ethyl-disulfide-ethyl)3-
Angiopep-2 (ANG2010).
Figure 5 is a scheme showing the synthesis of (morphine-6-glutaryl)3-Angiopep-2 from morphine.
Figure 6 is a scheme showing the synthesis of (morphine-6-dimethylglutaryl)3-Angiopep-2 from morphine.
Figure 7 is a scheme showing the synthesis of ^ο ηώ6-3^1υί3^1)3-Ατ¾ίο ε -2 from morphine.
Figure 8 is a scheme showing the synthesis of (moφhine-3-dimethylglutaryl)3-Angio ep-2 from morphine.
Figure 9 is a scheme showing the synthesis of (morphine-3-suberyl)3-Angiopep-2 from morphine.
Figure 10 is a scheme showing the synthesis of (morphine-3-succinyl)3-Angiopep-2 from morphine.
Figure 11 is a scheme showing the synthesis of (morphine-3-PEG3)3-Angiopep-2 from morphine. Figure 12 is a scheme showing the synthesis of (moφhine-3-carboxybutylether)3-Angiopep-2 from nunphine.
Figure 13 depicts polypeptide-opioid conjugates (πιοφηίη6-6-8^οί^1)3-.Α^ίορ6ρ-2 and ηιοφ ίη6-3 -PEG3-mal-Angiopep-2-Cys .
Figure 14 is a scheme showing the synthesis of (M6G-5'-methyl-triazolyl-propyl)3-Angiopep-2. Figures 15A-15C are graphs showing data for opioid-induced analgesia in the hot-plate test with
CD-I mice. Figure 15A is a graph showing the effect of dose on hindpaw latency where the
polylpeptide-opioid conjugate M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys is used at four different doses. Figure 15B is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys with unconjugated M6G. Figure 15C is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys and (M6G-5'-amido-ethyl-disulfide-ethyl)3-Angiopep-2 with a control and unconjugated M6G.
Figure 16 is a graph showing data for opioid-induced analgesia in the hot-plate test for M6G-5'- amido-ethyl-disulfide-Angiopep-2-Cys, M6G, and ηιοφΐήηβ. Figure 17 is a graph showing data for opioid-induced analgesia in the hot-plate test using (morphine-3-PEG3)3-Angiopep-2 (which contains a cleavable linker), (morphine-carboxybutylether)3- Angiopep-2 (which contains a non-cleavable linker), and morphine.
Figures 18A-18C are graphs showing the effect of opioids on the body temperature of CD-I mice. Figure 18A is a graph showing the effect of dose on body temperature where the polypeptide- opioid conjugate M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys is used at four different doses. Figure 18B is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys with unconjugated M6G. Figure 18C is a graph comparing M6G-5'-amido-ethyl-disulfide-Angiopep-2-Cys and (M6G-5'-amido- ethyl-disulfide-ethyl)3-Angiopep-2 with a control and unconjugated M6G.
Figure 19 is a graph showing the brain perfusion and volume distribution of the polypeptide- opioid conjugates.
Figure 20 is a graph showing a time-course plot of the perfusion of the polypeptide-opioid conjugates into the brain.
Figures 21A-21C show data evaluating the binding of an Angiopep-2-M6G conjugate
(ANG2010) to receptors in rat brain membranes. Figure 21 A shows the chemical structure and a schematic representation of ANG2010. Figure 21 B is a graph showing competition binding of M6G and ANG2010 to Mu-opioid (MOPr). Figure 21 C is a graph showing competition binding of M6G and ANG2010 to Delta-opiod (DOPr).
Figure 22 is a schematic showing the brain perfusion method that was used for evaluating brain uptake of ANG2010.
Figures 23A-23C show data evaluating brain uptake of ANG2010. Figure 23A is a graph showing the in vivo brain uptake of the [125I]-ANG2010 and [3H]-morphine. Figure 23B is a graph showing data from a brain capillary depletion assay to assess ANG2010 distribution in the brain compartments. Figure 23C is a table showing initial brain transport rate (K„) values for ANG2010, morphine, and M6G compared to that of other molecules.
Figures 24A-24B are graphs showing evaluation of the analgesic effect of ANG2010 using a hot plate model of pain. Figure 24A is a graph showing increased paw flicking latency after intravenous (IV) bolus injection of ANG2010 compared to unconjugated M6G at an equimolar dose. Figure 24B is a graph showing an increase in foot-licking latency for at least 3 hours after intravenous (IV) or sub- cutaneous (SC) administration of ANG2010.
Figures 25A-25B are graphs showing evaluation of the analgesic effect of ANG2010 using a rat tail flick model of pain. Figure 25 A is a graph showing the analgesic effect of ANG2010 compared to that of unconjugated morphine and M6G in the rat tail flick model after IV bolus injection. Figure 25B is a graph showing the results of Figure 25 A represented as the maximal possible effect (%) for the three drugs.
Figure 26 is a graph showing the analgesic effect of ANG2010 compared to that of unconjugated morphine and M6G in the rat tail flick model after SC bolus injection. Figure 27 is a graph showing the effect of sub-cutaneous injection of ANG2010 on gastrointestinal transit.
Detailed Description
We have developed polypeptide-opioid conjugates that are capable of crossing the BBB. The conjugates of the invention transport opioid receptor agonists to the brain and/or spinal cord. The conjugates of the invention may either enhance the delivery of opioids that already cross the BBB (e.g., morphine) or allow for the delivery of opioids that do not cross the BBB or poorly cross the BBB (e.g., morphine-6-glucuronide). Improved delivery of opioids to the brain and spinal cord can reduce opioid- associated side effects such as constipation and decrease the equivalent dosage required to effect analgesia in a subject.
Reduced Side Effects
Treatment of patients with the peptide-opioid conjugates of the invention may lead to fewer or reduced side effects compared with an equivalent dosage of the unconjugated opioid. This advantage of the invention is due to the improved transport of the conjugate across the BBB. The opioid receptors that regulate pain perception are located in the brain and spinal cord in the central nervous system (CNS) while the opioid receptors associated with certain opioid-induced side effects (e.g., constipation) are located in the peripheral nervous system (PNS). When a polypeptide-opioid conjugate of the invention is administered to a patient, a lower percentage of opioid reaches receptors in the PNS and a higher percentage of opioid reaches receptors in the CNS, compared with an equivalent dosage of the unconjugated opioid. The patient may therefore experience reduced PNS-associated, opioid-induced side effects when treated with the polypeptide-opioid conjugate compared with the unconjugated opioid. A common side effect of opioid treatment is constipation, which is a consequence of the dense distribution of opioid receptors in enteric neurons of the PNS. Because the polypeptide of the polypeptide-opioid conjugates allow the opioid to be directed to the brain and/or spinal cord (and thereby away from enteric neurons of the PNS), patients treated with the conjugate may experience reduced levels of constipation. Likewise, any other PNS-associated side effect of opioid treatment (e.g., intestinal motility, urinary retention, and pruritus) can be reduced through use of the polypeptide-opioid conjugates.
Decreased Equivalent Dosage
The polypeptide-opioid conjugates of the invention can effect analgesia in a subject using a dosage lower than the equivalent dosage of unconjugated opioid. Because the opioid (e.g., morphine or M6G) is delivered more efficiently to the brain and/or spinal cord, patients may receive lower systemic exposure to the opioid while receiving the same analgesic benefit. Opioid Moieties
Any opioid moiety may be used in the polypeptide-opioid conjugates and methods of the invention. Exemplary opioid moieties are described below, and include morphine and morphine related compounds. Both morphine and morphine-6-glucuronide (M6G) act as agonists of opioid receptors, principally the μ-opioid receptor. Opioid receptors are present in high densities throughout the central nervous system, including in the brainstem, the medial thalamus, the spinal cord, the limbic system, and the hypothalamus. Agonist binding to the μ-opioid receptor triggers a range of intracellular signals including inhibition of adenylate cyclase, reduced opening of voltage-gated calcium channels, and activation of potassium current, PKC, and PLC-β. The analgesic effects of opioid receptor agonists have been partially attributed to the blocking of the release of GABA from tonically active neurons in the periaqueductal gray (PAG) matter in the brain. In the absence of GABA release, monoamine receptors in the forebrain and spinal cord are deactivated, which affects sensory inputs from the spinal cord to higher centers. Opioid receptors are also expressed in the spinal dorsal horn presynaptically in C-fibers and postsynaptically in second order neurons. Binding of opioid agonists at presynaptic C-fibers reduces opening of voltage-gated calcium channels that initiate neurotransmitter release. Binding of opioid agonists at the postsynaptic second order neuron enhances efflux of potassium and hyperpolarization. The reinforcing activity of opioid agonists at the presynaptic and postsynaptic sites attenuates activation of second order neurons (Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th Edition, p. 490-491, 2011).
Morphine is metabolized primarily to M6G and morphine-3-glucuronide (M3G). M6G displays analgesic activity similar to morphine, while M3G is much less active. The structures of morphine, M6G, and M3G are shown below.
Figure imgf000010_0001
morphine M6G M3G
The polypeptide-opioid conjugates may be prepared and used with any morphine congener or opioid. Codeine, hydrocodone, hydromorphone, oxycodone, and oxymorphone, which are found in present day analgesic prescription drugs, are all congeners of morphine. A broad range of other opioids may be used as well, including opioid receptor agonists (e.g., alazocine, alfentanil, bremazocine, buprenorphine, butorphanol, cyclazocine, dezocine, dihydrocodeine, diphenoxylate, diamorphine, fentanyl, levorphanol, meperidine, meptazinol, methadone, nalbuphine, pentazocine, propoxyphene, remifentanil, sufentanil, and tramadol), opioid receptor antagonists (e.g., naloxone, naltrexone, diprenorphine, naloxonazine), and endogenous peptides (e.g., met-enkephalin, leu-enkephalin, β- endorphin, dynorphin A, dynorphin B,
Figure imgf000011_0001
endomorphin-1, endomorphin-2, and nociceptin). Preferably, the opioid is linked to the linker or polypeptide through a functional group on the opioid (e.g., an alcohol, phenol, amine, or carboxylic acid).
Polypeptides
The polypeptide-opioid conjugates of the invention can feature any of the polypeptides described herein, or a fragment or analog thereof. The polypeptides described herein are capable of crossing the BBB. In certain embodiments, the polypeptide of the polypeptide-opioid conjugate may have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to a polypeptide described herein. The polypeptide may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or 15) substitutions relative to one of the sequences described herein. The polypeptide may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15) additions and/or deletions of amino acids relative to one of the sequences described herein. Other modifications are described in greater detail below.
The polypeptide may be substantially identical to any of the sequences set forth in table 1 , or a fragment thereof. In certain embodiments, the peptide vector has a sequence of Angiopep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97), Angiopep-3 (SEQ ID NO: 107), Angiopep-4a (SEQ ID NO:108), Angiopep-4b (SEQ ID NO: 109), Angiopep-5 (SEQ ED NO: l 10), Angiopep-6 (SEQ ID NO: 111), Angiopep-7 (SEQ ID NO: 112), Angiopep-2-Cys (SEQ ID NO: 114), or reversed Angiopep-2 (SEQ ID NO: 117). The polypeptide may be of any length, for example, at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 35, 50, 75, 100, 200, or 500 amino acids, or any range between these numbers. In certain embodiments, the polypeptide is 10 to 50 amino acids in length. The polypeptide may be produced by recombinant genetic technology or chemical synthesis.
Table 1 : Exemplary Polypeptides of the Invention
SEQ BD NO:
1 T F V Y G G C R A K R N N F K s A E D
2 T F Q Y G G C M G N G N N F V T E K E
3 P F F Y G G C G G N R N N F D T E E Y
4 s F Y Y G G C L G N K N N Y L R E E E
5 T F F Y G G C R A K R N N F K R A K Y
6 T F F Y G G C R G K R N N F K R A K Y
7 T F F Y G G C R A K K N N Y K R A K Y
8 T F F Y G G C R G K K N N F K R A K Y
9 T F Q Y G G C R A K R N N F K R A K Y
10 T F Q Y G G C R G K K N N F K R A K Y
11 T F F Y G G C L G K R N N F K R A K Y
12 T F F Y G G s L G K R N N F K R A K Y P F F Y G G C G G K K N N F K R A K Y
T F F Y G G C R G K G N N Y K R A K Y
P F F Y G G C R G K R N N F L R A K Y
T F F Y G G C R G K R N N F K R E K Y
P F F Y G G C R A K K N N F K R A K E
T F F Y G G C R G K R N N F K R A K D
T F F Y G G C R A K R N N F D R A K Y
T F F Y G G C R G K K N N F K R A E Y
P F F Y G G C G A N R N N F K R A K Y
T F F Y G G C G G K K N N F K T A K Y
T F F Y G G C R G N R N N F L R A K Y
T F F Y G G C R G N R N N F K T A K Y
T F F Y G G s R G N R N N F K T A K Y
T F F Y G G c L G N G N N F K R A K Y
T F F Y G G c L G N R N N F L R A K Y
T F F Y G G c L G N R N N F K T A K Y
T F F Y G G c R G N G N N F K S A K Y
T F F Y G G c R G K K N N F D R E K Y
T F F Y G G c R G K R N N F L R E K E
T F F Y G G c R G K G N N F D R A K Y
T F F Y G G s R G K G N N F D R A K Y
T F F Y G G c R G N G N N F V T A K Y
P F F Y G G c G G K G N N Y V T A Y
T F F Y G G c L G K G N N F L T A K Y s F F Y G G c L G N K N N F L T A K Y
T F F Y G G c G G N K N N F V R E K Y
T F F Y G G c M G N N N F V R E K Y
T F F Y G G s M G N K N N F V R E K Y
P F F Y G G c L G N R N N Y V R E K Y
T F F Y G G c L G N R N N F V R E K Y
T F F Y G G c L G N K N N Y V R E K Y
T F F Y G G c G G N G N N F L T A K Y
T F F Y G G c R G N R N N F L T A E Y
T F F Y G G c R G N G N N F K s A E Y
P F F Y G G c L G N K N N F K T A E Y
T F F Y G G c R G N R N N F K T E E Y
T F F Y G G c R G K R N N F K T E E D
P F F Y G G c G G N G N N F V R E K Y s F F Y G G c M G N G N N F V R E K Y
P F F Y G G c G G N G N N F L R E K Y
T F F Y G G c L G N G N N F V R E K Y s F F Y G G c L G N G N N Y L R E K Y
T F F Y G G s L G N G N N F V R E K Y
T F F Y G G c R G N G N N F V T A E Y
T F F Y G G c L G K G N N F V S A E Y
T F F Y G G c L G N R N N F D R A E Y
T F F Y G G c L G N R N N F L R E E Y
T F F Y G G c L G N K N N Y L R E E Y
P F F Y G G c G G N R N N Y L R E E Y
P F F Y G G s G G N R N N Y L R E E Y
M R P D F C L E P P Y T G P C V A R I
A R I I R Y F Y N A K A G L C Q T F V Y G G C R A K R N N Y K S A E D c M R T C G
P D F c L E P P Y T G P C V A R I I R Y F Y
T F F Y G G C R G K R N N F K T E E Y
K F F Y G G C R G K R N N F K T E E Y
Τ F Y Y G G C R G K R N N Y K T E E Y
τ F F Y G G S R G K R N N F K T E E Y
C Τ F F Y G c C R G K R N N F K T E E Y τ F F Y G G c R G K R N N F K T E E Y c
C Τ F F Y G s C R G K R N N F K T E E Y τ F F Y G G s R G K R N N F K T E E Y c
Ρ F F Y G G c R G K R N N F K T E E Y τ F F Y G G c R G K R N N F K T K E Y τ F F Y G G K R G K R N N F K T E E Y τ F F Y G G c R G K R N N F K T K R Y τ F F Y G G K R G K R N N F K T A E Y τ F F Y G G K R G K R N N F K T A G Y τ F F Y G G K R G K R N N F K R E K Y τ F F Y G G K R G K R N N F K R A K Y τ F F Y G G c L G N R N N F K T E E Y τ F F Y G C G R G K R N N F K T E E Y τ F F Y G G R C G K R N N F K T E E Y τ F F Y G G C L G N G N N F D T E E E τ F Q Y G G c R G K R N N F K T E E Y
Υ Ν K E F G T F N T K G C E R G Y R F
R F K Y G G c L G N M N N F E T L E E
R F K Y G G c L G N K N N F L R L K Y
R F K Y G G c L G N K N N Y L R L K Y
Κ Τ K R K R K K Q R V K I A Y E E I F K N Y
Κ Τ K R K R K K Q R V K I A Y
R G G R L S Y S R R F s T S T G R
R R L S Y S R R R F
R Q I K I W F Q N R R M K W K K
Τ F F Y G G S R G K R N N F K T E E Y
Μ R P D F C L E P P Y T G P C V A R I
I R Y F Y N A K A G L c Q T F V Y G G
C R A K R N N F K S A E D c M R T C G G A
Τ F F Y G G C R G K R N N F K T K E Y
R F K Y G G C L G N K N N Y L R L K Y
Τ F F Y G G c R A K R N N F K R A K Y
Ν A A G L c Q T F V Y G G C L A K R N N F
Ε S A E D C M R T C G G A
Υ G G C R A K R N N F K S A E D C M R T C G
G A
G L C Q T F V Y G G C R A K R N N F K s A E
L C Q T F V Y G G C E A K R N N F K S A
Τ F F Y G G s R G K R N N F K T E E Y
R F F Y G G s R G K R N N F K T E E Y 109 R F F Y G G S R G K R N N F K T E E Y
1 10 R F F Y G G S R G K R N N F R T E E Y
111 T F F Y G G s R G K R N N F R T E E Y
112 T F F Y G G s R G R R N N F R T E E Y
113 c T F F Y G G S R G K R N N F K T E E Y
114 T F F Y G G s R G K R N N F K T E E Y c
115 c T F F Y G G S R G R R N N F R T E E Y
116 T F F Y G G S R G R R N N F R T E E Y c
117 Y E E T K F N N R K G R S G G Y F F T
Polypeptides Nos. 5, 67, 76, and 91 include the sequences of SEQ ID NOS:5, 67, 76, and 91, respectively, and are amidated at the C-terminus.
Polypeptides Nos. 107, 109, and 110 include the sequences of SEQ ID NOS:97, 109, and 110, respectively, and are acetylated at the N-terminus.
In other embodiments, the polypeptide may include a consensus sequence of
Lys-Arg-X3-X4-X5-Lys (SEQ ID NO:120) (formula la), where
X3 is Asn or Gin;
X4 is Asn or Gin; and
X5 is Phe, Tyr, or Trp.
The polypeptide may include a consensus sequence of
Zl -Lys-Arg-X3-X4-X5-Lys-Z2 (SEQ ID NO: 121) (formula lb), where
X3 is Asn or Gin;
X4 is Asn or Gin;
X5 is Phe, Tyr, or Trp;
Zl is absent, Cys, Gly, Cys-Gly, Arg-Gly, Cys-Arg-Gly, Ser-Arg-Gly, Cys-Ser-Arg-Gly, Gly-Ser-Arg-Gly, Cys-Gly- Ser-Arg-Gly, Gly-Gly-Ser-Arg-Gly, Cys-Gly-Gly-Ser-Arg-Gly, Tyr- Gly-Gly-Ser-Arg-Gly, Cys-Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe- Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe-Phe-Tyr-Gly-Gly-Ser- Arg-Gly, Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, or Cys-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg- Gly; and
Z2 is absent, Cys, Tyr, Tyr-Cys, Cys-Tyr, Thr-Glu-Glu-Tyr, or Thr-Glu-Glu-Tyr-Cys. The consensus sequence of formulas (la) and (lb) include the amino acid sequence Lys-Arg-Asn- Asn-Phe-Lys (SEQ ID NO: 122) and conservative substitutions.
Other polypeptides include those having a consensus sequence of
X 1 -X2-Asn-Asn-X5-X6 (SEQ ID NO: 124) (formula Ila), where
XI is Lys or D-Lys; X2 is Arg or D-Arg;
X5 is Phe or D-Phe; and
X6 is Lys or D-Lys; and
where at least one of XI , X2, X5, or X6 is a D-amino acid.
Yet other polypeptides include those having a consensus sequence of
Xl-X2-Asn-Asn-X5-X6-X7 (SEQ ID NO: 125) (formula lib), where
XI is Lys or D-Lys;
X2 is Arg or D-Arg;
X5 is Phe or D-Phe;
X6 is Lys or D-Lys;
X7 is Tyr or D-Tyr; and
where at least one of XI , X2, X5, X6, or X7 is a D-amino acid.
The polypeptides may also contain a consensus sequence of
Zl-Xl -X2-Asn-Asn-X5-X6-X7-Z2 (SEQ ID NO: 126) (formula lie), where
XI is Lys or D-Lys;
X2 is Arg or D-Arg;
X5 is Phe or D-Phe;
X6 is Lys or D-Lys;
X7 is Tyr or D-Tyr;
Zl is absent, Cys, Gly, Cys-Gly, Arg-Gly, Cys-Arg-Gly, Ser-Arg-Gly, Cys-Ser-Arg-Gly, Gly-Ser-Arg-Gly, Cys-Gly-Ser-Arg-Gly, Gly-Gly-Ser-Arg-Gly, Cys-Gly-Gly-Ser-Arg-Gly, Tyr- Gly-Gly-Ser-Arg-Gly, Cys-Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Tyr-Gly-Gly- Ser-Arg-Gly, Cys-Phe- Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe-Phe-Tyr-Gly-Gly-Ser-
Arg-Gly, Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, or Cys-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg- Gly; and
Z2 is absent, Cys, Tyr, Tyr-Cys, Cys-Tyr, Thr-Glu-Glu-Tyr, or Thr-Glu-Glu-Tyr-Cys; where at least one of XI , X2, X5, X6, or X7 is a D-amino acid; and
where the polypeptide optionally includes one or more D-isomers of an amino acid recited in Zl or Z2.
The polypeptide may contain additions or deletions of amino acids to the consensus sequence of Lys-Arg-X3-X4-X5-Lys (formula la), where X3-X5 are as defined above; the consensus sequences of Xl-X2-Asn-Asn-X5-X6 and Xl -X2-Asn-Asn-X5-X6-X7 (formulas Ila and lib, respectively), where XI , X2, X5, X6, and X7 are as defined above; or the longer polypeptide of (3D)-Angiopep-2, as described herein. The deletions or additions can include any part of the consensus sequence of Lys-Arg-X3-X4- X5-Lys, Xl -X2-Asn-Asn-X5-X6, Xl -X2-Asn-Asn-X5-X6-X7, Lys-Arg-Asn-Asn-Phe-Lys, D-Lys-D- Arg-Asn-Asn-D-Phe-D-Lys, or D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-D-Tyr, or of the longer sequence 3D-Angiopep-2. In some embodiments, deletions or additions of 1, 2, 3, 4, or 5 amino acids may be made from the consensus sequence of the polypeptide. In particular embodiments, the deletions or additions may be from 1 to 3 amino acids.
In other embodiments, the polypeptide of the invention can be (3D)-Angiopep-2 (SEQ ID
NO: 166) or (3D)-Angiopep-2-Cys (SEQ ID NO: 177).
SEQ ID NO.
166 T F F Y G G S ^ G ^' ^ N N F K T E E Y 177 T F F Y G G S G N N F K T E E Y C
In other embodiments, the polypeptide may comprise a sequence shorter than Angiopep-2, shown in table 2.
Table 2: Exemplary Polypeptides with Sequences Shorter Than Angiopep-2
SEQ ID NO.
127 F Y G G S R G K R N N F K T E E Y C (PI)
D- D-
167 F Y G G S R G N D-F K T E E Y C (Pla)
K R
168 F Y G G s D- D- D-
R G N ND-F T E E Y C (Plb) K R K
D- D- D-
169 F Y G G s D-
R G N ND-F T E E
K R K Y c (Pic)
D- D- D- D- D-
170 D-F G G G N ND-F T ED-E
Y s D- R K R K Y c (Pld)
128 G G s R G K R N N F K T E E Y c (P2)
129 s R G K R N N F K T E E Y c (P3)
130 G K R N N F K T E E Y c (P4)
K R N N F K T E E Y
131 c (P5)
D- D-
171 N ND-F K T E E Y
K R c (P5a)
D- D- D-
172 N ND-F T E E Y
K R K c (P5b)
D- D- D- D-
173 N ND-F T E E
K R K Y c (P5c)
132 K R N N F K Y C (P6)
D- D-
174 N ND-F K Y C (P6a)
K R D- D- D-
175 N N D-F Y C (P6b)
K R K
D- D- D- 176 N N D-F (P6c)
K R K
D- D- (decys 178 N D-F K
K R P6a)
In some embodiments, PI, Pla, Plb, Pic, Pld, P2, P3, P4, P5, P5a, P5b, P5c, P6, P6a, P6b, P6, or decys-P6a is amidated at the C-terminus. The polypeptide of the invention include additions and/or deletions of amino acids of the sequences of PI , Pl a, Plb, Pic, Pld, P2, P3, P4, P5, P5a, P5b, P5c, P6, P6a, P6b, P6, and decys-P6a. The deletions or additions can include any part of these sequences. In some embodiments, deletions or additions of 1 , 2, 3, 4, or 5 amino acids may be made from these sequences of the polypeptide. In particular embodiments, the deletions or additions may be from 1 to 3 amino acids.
The invention also features polypeptides having conservative substitutions of any of the above- described polypeptides. Conservative substitutions and derivatives of amino acids and peptides are well known in the art and can be determined by any useful methods (e.g., by using a substitution matrix or any other method described herein). A derivative of a polypeptide includes a polypeptide containing one or more conservative substitutions selected from the following groups or a subset of these groups: Ser, Thr, and Cys; Leu, He, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp (e.g., Phe and Tyr); and Gin, Asn, Glu, Asp, and His (e.g., Gin and Asn). Conservative substitutions may also be determined by other methods, such as by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), and PAM substitution matrix (e.g., PAM 250 matrix).
Any useful substitutions, additions, and deletions can be made to the polypeptide that does not destroy significantly a desired biological activity (e.g., ability to cross the BBB or agonist activity). The modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the consensus sequence or original polypeptide.
Furthermore, substitutions, additions, and deletions may have or may optimize a characteristic of the consensus sequence or polypeptide, such as charge (e.g., positive or negative charge), hydrophilicity, hydrophobicity, in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties. For example, positive charge can be promoted by deleting one or more amino acids (e.g., from 1 to 3 amino acids) that are not basic/positively charged (as described below based on common side chain properties) or less positively charged (e.g., as determined by pKa). In another example, positive charge can be promoted by inserting one or more amino acids (e.g., from 1 to 3 amino acids) that are basic/positively charged or more positively charged (e.g., as determined by pKa).
In particular, substitutions or additions of D-amino acids can be made within the polypeptide. Such substitutions or additions may provide peptides having increased resistance to cleavage by enzymes, where one of more amino acids for cleavage sites can be substituted with its D-isomer. Exemplary enzymes include pepsin, trypsin, Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, glutamyl endopeptidase, LysC lysyl endopeptidase, LysN peptidyl-Lys metalloendopeptidase, proteinase K, and thermolysin; and exemplary cleavage sites for these enzymes are described herein.
In vivo stability may be optimized in any useful way. For example, stability in the presence of one or more digestive enzymes can be improved by substituting a naturally occurring L-amino acid for its D-isomer. Exemplary digestive enzymes include pepsin and trypsin. Using the subsite nomenclature for cleavage sites, SI -SI ' indicates the cleavage site for a peptide Sn— S4-S3-S2-S1 -S1 '-S2'-S3'-S4'— Sm. Cleavage by pepsin generally occurs when Phe, Tyr, Trp, or Leu is in position SI or SI '; or when Pro is in position S3 or S4. Cleavage by trypsin generally occurs when Arg or Lys is in position S 1 ; when Pro is in position SI ', Lys is in position S I , and Trp is in position S2; when Pro is in position SI ', Arg is in position SI , and Met is in position S2; or when Pro is in position SI ' and Glu is in position S2. Other exemplary cleavage sites include those for cleavage by Arg-C proteinase (e.g., Arg in position SI), Asp- N endopeptidase (e.g., Asp or Glu in position SI '), chymotrypsin (e.g., Trp, Tyr, or Phe in position SI for cleavage with high specificity; Leu, Met, or His in position SI for cleavage with low specificity), glutamyl endopeptidase (e.g., Glu at position SI), LysC lysyl endopeptidase (e.g., Lys at position SI), LysN peptidyl-Lys metalloendopeptidase (e.g., Lys at position S I '), proteinase K (e.g., an aliphatic or amino acid residue, such as Ala, Glu, Phe, He, Leu, Thr, Val, Trp, or Tyr, at position SI), and thermolysin (e.g., a bulky or an amino acid residue, such as He, Leu, Val, Ala, Met, or Phe, at position SI ').
Predictive models are also available for determining cleavage sites, such as PeptideCutter available on the ExPASy proteomics server. Exemplary cleavage sites for polypeptides are C-terminal to positions 1 , 2, 3, 4, 14, 18, and 19 in Angiopep-2 (SEQ ID NO:97) for cleavage by pepsin and C-terminal to positions 8, 10, 1 1 , and 15 in Angiopep-2 (SEQ ID NO:97) for cleavage by trypsin. Other exemplary cleavage sites in Angiopep-2 (SEQ ID NO:97) include C-terminal to positions 8 and 11 for cleavage by Arg-C proteinase; positions 16 and 17 for cleavage by Asp-N endopeptidase; positions 2, 3, 4, 14, and 19 for cleavage by chymotrypsin; positions 17 and 18 for cleavage by glutamyl endopeptidase; positions 10 and 15 for cleavage by LysC lysyl endopeptidase; positions 9 and 14 for cleavage by LysN peptidyl-Lys metalloendopeptidase; positions 1 , 2, 3, 4, 14, 16, and 1 for cleavage by proteinase K; and positions 1 , 2, and 13 for cleavage by thermolysin. Accordingly, the polypeptide of the invention also include polypeptides shorter than Angiopep-2 (SEQ ID NO:97) having one or more D-amino acid substitutions for one or more of positions 1 , 2, 3, 4, 8, 10, 1 1 , 13, 14, 15, 16, 17, 18, and 19 in Angiopep-2 (SEQ ID NO:97).
Substantial modifications in function or immunological identity are accomplished by selecting substitutions, additions, and deletions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (He), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), and Phenylalanine (Phe);
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), and Threonine (Thr);
(3) acidic/negatively charged: Aspartic acid (Asp) and Glutamic acid (Glu);
(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), and Arginine (Arg);
(5) residues that influence chain orientation: Glycine (Gly) and Proline (Pro);
(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), and Histidine (His);
(7) polar: Ser, Thr, Asn, Gin;
(8) basic positively charged: Arg, Lys, His; and
(9) charged: Asp, Glu, Arg, Lys, His.
Other amino acid substitutions are listed in table 3.
Table 3: Amino Acid Substitutions
Figure imgf000019_0001
The invention also features fragments of any of the above-described polypeptides (e.g., a functional fragment). In certain embodiments, the fragments are efficiently transported across the BBB. Truncations of the polypeptide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from either the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a combination thereof. Other fragments include sequences where internal portions of the polypeptide are deleted. Deletions of the polypeptide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from the internal portion of the polypeptide. In some embodiments, deletions may be 1, 2, 3, 4, or 5 amino acids from the consensus sequence of the polypeptide. Linkers
The polypeptide may be bound to an opioid moiety either directly (e.g., by a covalent bond) or through a linker. Linkers include chemical linking agents (e.g., cleavable linkers and non-cleavable linkers), click-chemistry linkers, and peptides. Any of the linkers described below may be used in the polypeptide-opioid conjugates of the invention.
Chemical linking agents
In some embodiments, the linker is a chemical linking agent. The polypeptide may be conjugated through sulfhydryl groups, amino groups (amines), or any appropriate reactive group.
Homomultifunctional and heteromultifunctional linkers (conjugation agents, including bifunctional and trifunctional agents) are available from many commercial sources. Sites available for linking may be found on the polypeptide or therapeutic agents described herein. The linker may comprise a flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. The flexible arm can be polyethylene glycol spacer, such as (PEG)n, where n is an integer between 1 and 20, or an amino acid, such as -NH- (CH2)n-C(0)0-, where n is an integer between 2 and 10 (e.g., when n is 5).
Exemplary linkers include BS3 ([bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N- hydroxysuccinimide ester that targets accessible primary amines), NHS/EDC (N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS/EDC allows for the conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([Ν-ε-maleimidocaproic acid]hydrazide; sulfo-EMCS are heterobifunctional reactive groups (maleimide and NHS -ester) that are reactive toward sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazide is a useful reagent for linking carboxyl groups to primary amines), SATA (N-succinimidyl-S-acetylthioacetate; SATA is reactive towards amines and adds protected sulfhydryls groups), and BMOE (bis-maleimidoethane).
To form covalent bonds, one can use as a chemically reactive group a wide variety of active carboxyl groups (e.g., esters) where the hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide. Particular agents include N-hydroxysuccinimide (NHS), N-hydroxy- sulfosuccinimide (sulfo-NHS), maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS), maleimido propionic acid (MP A), maleimido hexanoic acid (MHA), and maleimido undecanoic acid (MUA).
Primary amines are the principal targets for NHS esters. Accessible a-amine groups present on the N-termini of proteins and the ε-amine of lysine react with NHS esters. Thus, compounds of the invention can include a linker having a NHS ester conjugated to an N-terminal amino of a peptide or to an ε-amine of lysine. An amide bond is formed when the NHS ester reacts with primary amines releasing N- hydroxysuccinimide. These succinimide containing reactive groups are herein referred to as succinimidyl groups. In certain embodiments of the invention, the functional group on the protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as gamma-maleimide- butrylamide (GMBA or MP A). Such maleimide containing groups are referred to herein as maleimedo groups.
The maleimido group is most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls (e.g., thiol groups on proteins such as serum albumin) is approximately 1000-fold faster than with amines. Thus, a stable thioether linkage between the maleimido group and the sulfhydryl can be selectively formed. Accordingly, a conjugate of the invention can include a linker having a maleimido group conjugated to a sulfhydryl group of the polypeptide or of the therapeutic agent.
Amine-to-amine linkers include NHS esters and imidoesters. Exemplary NHS esters are DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis [sulfosuccinimidyl] suberate), TSAT (ira-succinimidyl aminotriacetate), variants of bis-succimmide ester-activated compounds that include a polyethylene glycol spacer, such as BS(PEG)n, where n is 1-20 (e.g., BS(PEG)5 and BS(PEG)9), DSP (Dithiobis[succinimidyl propionate]), DTSSP (3,3'-dithiobis[sulfosuccinimidylpropionate]), DST (disuccinimidyl tartarate), BSOCOES (bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), EGS (ethylene glycol bisfsuccinimidylsuccinate]), and sulfo-EGS (ethylene glycol bis[sulfosuccinimidylsuccinate]). Imidoesters include DMA (dimethyl adipimidate*2 HCl), DMP (dimethyl pimelimidate'2 HCl), DMS (dimethyl suberimidate«2 HCl), and DTBP (dimethyl S^'-dithiobispropionimidate^ HCl). Other amine- to-amine linkers include DFDNB (l ,5-difluoro-2,4-dinitrobenzene) and THPP (P-[tris(hydroxymethyl) phosphino] propionic acid (betaine)).
The linker may be a sulfhydryl-to-sulfhydryl linker. Such linkers include maleimides and pyridyldithiols. Exemplary maleimides include BMOE (bis-maleimidoethane), BMB (1 ,4- bismaleimidobutane), BMH (bismaleimidohexane), TMEA (irw[2-maleimidoethyl]amine), BM(PEG)2 1,8-bis-maleimidodiethyleneglycol) or BM(PEG)n, where n is 1 to 20 (e.g., 2 or 3), BMDB (1,4 bismaleimidyl-2,3-dihydroxybutane), and DTME (dithio-bismaleimidoethane). Exemplary
pyridyldithiols include DPDPB (l,4-di-[3'-(2'-pyridyldithio)-propionamido]butane). Other sulfhydryl linkers include HBVS (1,6-hexane-bis-vinylsulfone).
The linker may be an amine-to-sulfhydryl linker, which includes NHS ester/maleimide compounds. Such amine-to-sulfhydryl linkers can include ester linkers (e.g., any linker described herein containing an ester group). Examples of these compounds are AMAS (N-(a- maleimidoacetoxy)succinimide ester), BMPS (N-[ -maleimidopropyloxy]succinimide ester), GMBS (Ν- [γ-maleimidobutyryloxyjsuccinimide ester), sulfo-GMBS (N-[y-maleimidobutyryloxy]sulfosuccinimide ester), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), sulfo-MBS (m-maleimidobenzoyl-N- hydroxysulfosuccinimide ester), SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-l- carboxylate), sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate), EMCS ([Ν-ε-maleimidocaproyloxyJsuccinimide ester), Sulfo-EMCS ([Ν-ε- maleimidocaproyloxyjsulfosuccinimide ester), SMPB (succinimidyl 4-[ ?-maleimidophenyl]butyrate), sulfo-SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate), SMPH (succinimidyl-6-[p- maleimidopropionamido]hexanoate), LC-SMCC (succinimidyl-4-[N-maleimidomethyl]cyclohexane-l - carboxy-[6-amidocaproate]), sulfo-KMUS (N-[K-maleirnidoundecanoyloxy]sulfosuccinimide ester), SM(PEG)n (succirumidyl-([N-maleimidopropionamido-polyethyleneglycol) ester), where n is 1 to 30 (e.g., 2, 4, 6, 8, 12, or 24), SPDP (7V-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP
(succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'- [2-pyridyldithio]-propionamido)hexanoate), SMPT (4-succmimidyloxycarbonyl-a-methyl-a-[2- pyridyldithio]toluene), Sulfo-LC-SMPT (4-sulfosuccinimidyl-6-[a-methyl-a-(2- pyridyldithio)toluamido]hexanoate), SIA (N-succinimidyl iodoacetate), SBAP (succinimidyl 3- [bromoacetamido]propionate), SLAB (N-succinimidyl[4-iodoacetyl]aminobenzoate), and sulfo-SIAB (N- sulfosuccinimidyl [4-iodoacetyl] aminobenzoate) .
In some embodiments, the linker has the formula:
Figure imgf000022_0001
where n is an integer between 1 and 15 (e.g., n is 2, 3, 4, 5, 6, or 1 1); and either Y is a thiol on the polypeptide and Z is a primary amine, alcohol, or phenol on the opioid moiety or Y is a thiol on the opioid moiety and Z is a primary amine, alcohol, or phenol on the polypeptide.
In other embodiments, the linker is an amino-to-nonselective linker. Examples of such linkers include NHS ester/aryl azide and NHS ester/diazirine linkers. NHS ester/aryl azide linkers include NHS- ASA (N-hydroxysuccinimidyl-4-azidosalicylic acid), ANB-NOS (N-5-azido-2- nitrobenzoyloxysuccinimide), sulfo-HSAB (N-hydroxysulfosuccinimidyl-4-azidobenzoate), sulfo-NHS- LC-ASA (sulfosuccinimidyl[4-azidosalicylamido]hexanoate), SANP H (N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate), sulfo-SANPAH (N-sulfosuccinimidyl-6-(4' -azido-2'- nitrophenylamino)hexanoate), sulfo-SFAD (sulfosuccinimidyl-(perfluoroazidobenzamido)-ethyl-l ,3'- dithioproprionate), sulfo-SAND (sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-l ,3'- proprionate), and sulfo-SAED (sulfosuccinimidyl 2-[7-amino-4-methylcoumarin-3-acetamido]ethyl- 1 ,3'dithiopropionate). NHS ester/diazirine linkers include SDA (succinimidyl 4,4'-azipentanoate), LC- SDA (succinimidyl 6-(4,4'-azipentanamido)hexanoate), SDAD (succinimidyl 2 -([4,4'- azipentanamido]ethyl)-l,3'-dithioproprionate), sulfo-SDA (sulfosuccinimidyl 4,4'-azipentanoate), sulfo- LC-SDA (sulfosuccinimidyl 6-(4,4'-azipentanamido)hexanoate), and sulfo-SDAD (sulfosuccinimidyl 2- ([4,4'-azipentanamido]ethyl)-l ,3 '-dithioproprionate).
Exemplary amine-to-carboxyl linkers include carbodiimide compounds (e.g., DCC (N,N- dicyclohexylcarbodimide) and EDC (l-ethyl-3-[3-dimethylaminopropyl]carbodiimide)). Exemplary sulfhydryl-to-nonselective linkers include pyridyldithiol/aryl azide compounds (e.g., APDP ((N-[4-(p- azidosalicylamido)butyl] -3 ' -(2 ' -pyridyldithio)propionamide)) . Exemplary sulfhydryl-to-carbohydrate linkers include maleimide/hydrazide compounds (e.g., BMPH (N-[P-maleimidopropionic acid]hydrazide), EMCH ([N-e-maleimidocaproic acid]hydrazide), MPBH 4-(4-N- maleimidophenyl)butyric acid hydrazide), and KMUH (N-[K-maleimidoundecanoic acid]hydrazide)) and pyridyldithiol/hydrazide compounds (e.g., PDPH (3-(2-pyridyldithio)propionyl hydrazide)). Exemplary carbohydrate-to-nonselective linkers include hydrazide/aryl azide compounds (e.g., ABH (p-azidobenzoyl hydrazide)). Exemplary hydroxyl-to-sulfhydryl linkers include isocyanate/maleimide compounds (e.g., (N-[p-maleimidophenyl]isocyanate)). Exemplary amine-to-DNA linkers include NHS ester/psoralen compounds (e.g., SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate)).
Linkers are also described in U.S. Patent No. 4,680,338 having the formula Y=C=N-Q-A-C(0)- Z, where Q is a homoaromatic or heteroaromatic ring system; A is a single bond or an unsubstituted or substituted divalent Ci .30 bridging group, Y is O or S; and Z is CI, Br, I, N3 , N-succinimidyloxy, imidazolyl, 1-benzotriazolyloxy, OAr where Ar is an electron-deficient activating aryl group, or OC(0)R where R is -A-Q-N=C=Y or C4 -20 tertiary-alkyl.
Linkers are also described in U.S. Patent No. 5,306,809, which describes linkers having the
formula
Figure imgf000023_0001
Ri is H, Ci_6 alkyl, C2-6 alkenyl, C6-12 aryl or aralkyl or these coupled with a
R'
divalent organic -0-, -S-, or where R' is Ci_6 alkyl, linking moiety; R2 is H, C 2 alkyl, C6 , 2 aryl,
O s or C6.i2 aralkyl, R3 is ^^ , ^0'^, ^S'^, ^0'^, ^S'^, H , H or another chemical structure which is able to delocalize the lone pair electrons of the adjacent nitrogen and R4 is a pendant reactive group capable of linking R3 to a polypeptide or to a therapeutic agent.
The linker can be polyvalent or monovalent. A monovalent linker has only one activated group available for forming a covalent bond. However, the monovalent linker can include one or more functional groups that can be chemically modified by using a coupling agent, as described herein, to form a second activated group. For example, a terminal hydroxyl group of the linker can be activated by any number of coupling agents. Examples of coupling agents include N-hydroxysuccinimide,
ethylchloroformate, dicyclohexylcarbodiimide, and trifluoromethanesulfonyl chloride. See, e.g. U.S. Patent Nos. 5,395,619 and 6,316,024.
A polyvalent linker (e.g., a multifunctional linker) has two or more activated groups. The activated groups in the linker can be the same, as in a homopolyvalent linker, or different, as in a heteropolyvalent linker. Heteropolyvalent linkers allow for conjugating a polypeptide and a transport vector with different functional groups. Examples of heteropolyvalent linkers include polyoxyethylene- bis(p-nitrophenyl carbonate), mal-PEG-DSPE, diisocyanate, succitiimidyl 4-hydrazinonicotinate acetone hydrazone.
Examples of homopolyvalent linkers with two activated groups include disuccinimidyl glutarate, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, bis(NHS)PEG5, bis(NHS)PEG(),
dithiobis(succinimidyl propionate), 3,3 r-dithiobis(sulfosuccinimidylpropionate), disuccinimidyl tartrate, bis[2-(succinimido oxycarbonyloxy)ethyl]sulfone, ethylene glycol bisfsuccinimidylsuccinate]), ethylene glycol bis[sulfosuccinimidylsuccinate]), dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate, l,5-difluoro-2,4-dinitrobenzene, bis- maleimidoethane, 1 ,4-bismaleimidobutane, bismaleimidohexane, 1 ,8-bis-maleimidodiethyleneglycol, 1, 11-bis-maleimido-triethyleneglycol, l ,4-di-[3'-(2'-pyridyldithio)-propionamido]butane, 1 ,6-hexane-bis- vinylsulfone, and bis-[b-(4-azidosalicylamido)ethyl]disulfide.
Examples of homopolyvalent linkers with three activated groups include tris-succinimidyl aminotriacetate, p-[tris(hydroxymethyl) phosphino] propionic acid, and tris[2-maleimidoethyl]amine.
Examples of heteropolyvalent linkers include those with an maleimide activated group and a succinimide activated group, such as N-[D-maleimidoacetoxy]succimmide ester, Ν-[β- maleimidopropyloxy]-succinimide ester, N-[D -maleimidobutyryloxy]succinimide ester, m- maleimidoberizoyl-N-hydroxysuccimrnide ester, succinimidyl 4-[N-maleimidomethyl]cyclohexane-l- carboxylate, N-[D -maleimidocaproyloxy]succinimide ester, and succinimidyl 4-[p- maleimidophenyljbutyrate, including N-sulfosuccinimidyl derivatives; those with a PEG spacer molecule, such as succinimidyl-([N-maleimidopropionamido]-(ethyleneglycol)x)ester, wherein x is from 2 to 24; those with a pyridylditliio activated group and a succinimide activated group, such as N-succinimidyl-3- (2-pyridyldithio)propionate, succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate, 4- succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene, and 4-sulfosuccinimidyl-6-rnethyl-a-(2- pyridyldithio)toluamido]hexanoate); those with a haloacetyl activated group and a succinimide activated group, such as N-succinimidyl iodoacetate and N-succinimidyl[4-iodoacetyl]aminobenzoate; those with an aryl azide activated group and a succinimide activated group, such as N-hydroxysuccinimidyl-4- azidosalicylic acid, sulfosuccinimidyl[4-azidosalicylamido]-hexanoate, and N-succinimidyl-6-(4'-azido- 2'-nitrophenylamino) hexanoate; those with an diazirine activated group and a succinimide activated group, such as succinimidyl 4,4'-azipentanoate and succinimidyl 6-(4,4'-azipentanamido)hexanoate; Ν- [4-(p-azidosalicylamido) butyl]-3 '-(2'-pyridyldithio)propionamide; N-[ -maleimidopropionic acid] hydrazide; N-(e-maleirnidocaproic acid) hydrazide; 4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride; (N-[K-maleimidoundecanoic acid]-hydrazide); 3-(2-pyridyldithio)propionyl hydrazide; p- azidobenzoyl hydrazide; and N-[p-maleimidophenyl]isocyanate.
In other embodiments, the linker is a trifunctional, tetrafunctional, or greater linking agent.
Exemplary trifunctional linkers include TMEA, THPP, TSAT, LC-TSAT (im-succinimidyl (6- aminocaproyl)aminotriacetate), ίπ'5-succinimidyl-l ,3,5-benzenetricarboxylate, MDSI (maleimido-3,5- disuccinimidyl isophthalate), SDMB (succinimidyl-3,5-dimaleimidophenyl benzoate, Mal-4 {tetrakis-{2>- maleinn^opropyl)pentaeiythritol, NHS-4 (te raA s-(N-succinimidylcarboxypropyl)pentaerythritol)).
TMEA has the structure:
Figure imgf000025_0001
TMEA, through its maleimide groups, can react with sulfhydryl groups (e.g., through cysteine amino acid side chains).
THPP has the structure:
Figure imgf000025_0002
The hydroxyl groups and carboxy group of THPP can react with primary or secondary amines.
Click-chemistry linkers
In particular embodiments, the linker is formed by the reaction between a click-chemistry pair. By click-chemistry pair is meant a pair of reactive groups that participates in a modular reaction with high yield and a high thermodynamic gain, thus producing a click-chemistry linker. In this embodiment, one of the reactive groups is attached to the opioid moiety and the other reactive group is attached to the polypeptide. Exemplary reactions and click-chemistry pairs include a Huisgen 1 ,3-dipolar cycloaddition reaction between an alkynyl group and an azido group to form a triazole-containing linker; a Diels-Alder reaction between a diene having a 4π electron system (e.g., an optionally substituted 1,3 -unsaturated compound, such as optionally substituted 1,3 -butadiene, l-methoxy-3-trimethylsilyloxy-l,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) and a dienophile or heterodienophile having a 2π electron system (e.g., an optionally substituted alkenyl group or an optionally substituted alkynyl group); a ring opening reaction with a nucleophile and a strained heterocyclyl electrophile; a splint ligation reaction with a phosphorothioate group and an iodo group; and a reductive amination reaction with an aldehyde group and an amino group (Kolb et al., Angew. Chem. Int. Ed., 40:2004-2021 (2001); Van der Eycken et al., QSAR Comb. Sci., 26:1115-1326 (2007)).
In particular embodiments of the invention, the polypeptide is linked to the opioid moiety by means of a triazole-containing linker formed by the reaction between a alkynyl group and an azido group click-chemistry pair. In such cases, the azido group may be attached to the polypeptide and the alkynyl group may be attached to the opioid moiety. Alternatively, the azido group may be attached to the opioid moiety and the alkynyl group may be attached to the polypeptide. In certain embodiments, the reaction between an azido group and the alkynyl group is uncatalyzed, and in other embodiments the reaction is catalyzed by a copper(I) catalyst (e.g., copper(I) iodide), a copper(II) catalyst in the presence of a reducing agent (e.g., copper(II) sulfate or copper(II) acetate with sodium ascorbate), or a ruthenium- containing catalyst (e.g., Cp*RuCl(PPh3)2 or Cp*RuCl(COD)). Amino acid and peptide linkers
In other embodiments, the linker includes at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino acid such as Cys). In other embodiments, a glycine-rich peptide such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n (SEQ ID NO: 140) where n is 1 , 2, 3, 4, 5 or 6 is used, as described in U.S. Patent No. 7,271,149. In other embodiments, a serine-rich peptide linker is used, as described in U.S. Patent No. 5,525,491. Serine rich peptide linkers include those of the formula [X-X-X-X-Gly]y (SEQ ID NO:141), where up to two of the X are Thr, and the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly (SEQ ID NO: 142), where y is greater than 1). In some cases, the linker is a single amino acid (e.g., any amino acid, such as Gly or Cys).
Amino acid linkers may be selected for flexibility (e.g., flexible or rigid) or may be selected on the basis of charge (e.g., positive, negative, or neutral). Flexible linkers typically include those with Gly resides (e.g., [Gly-Gly-Gly-Gly-Ser]n where n is 1, 2, 3, 4, 5 or 6). Other linkers include rigid linkers (e.g., PAPAP (SEQ ID NO: 143) and (PT)nP (SEQ ID NO: 144), where n is 2, 3, 4, 5, 6, or 7) and <x- helical linkers (e.g., A(EAAAK)nA (SEQ ID NO: 145), where n is 1 , 2, 3, 4, or 5).
Examples of suitable linkers are succinyl, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys.
When the linker is succinyl, one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the other carboxyl group thereof may, for example, form an ester bond with a hydroxyl group of opioid moiety. When the linker is Lys, Glu, or Asp, the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the amino group thereof may, for example, form an amide bond with a carboxyl group of the opioid moiety. When Lys is used as the linker, a further linker may be inserted between the ε-amino group of Lys and the opioid moiety. In one particular embodiment, the further linker is succinyl acid, which can form an amide bond with the ε- amino group of Lys and an ester bond with a hydroxyl group present in the opioid moiety. In one embodiment, the further linker is Glu or Asp (e.g., which forms an amide bond with the ε-amino group of Lys and an ester or amide bond with a hydroxyl or amino group present in the opioid moiety).
In other embodiments, the peptide linker is a branched polypeptide. Exemplary branched peptide linkers are described in U.S. Patent No. 6,759,509.
Modifications to linkers
Any of the linkers described herein (e.g., chemical linking agents, click-chemistry linkers, or amino acid and peptide linkers) may be modified. For example, the linkers can include a spacer molecule. The spacer molecule within linker can be of any suitable molecule. Examples of spacer molecules include aliphatic carbon groups (e.g., C2-C20 alkyl groups), cleavable heteroatomic carbon groups (e.g., C -C20 alkyl groups with dithio groups), and hydrophilic polymer groups. Examples of hydrophilic polymer groups include poly(ethylene glycol) (PEG), polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose,
hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, and a hydrophilic peptide sequence.
In one example, the hydrophilic polymer is PEG, such as a PEG chain having a molecular weight between 500-10,000 Da (e.g., between 1,000-5,000 Da such as 2,000 Da). Methoxy or ethoxy-capped analogues of PEG can also be used. These are commercially available in sizes ranging between 120-
20,000 Da. Preparation of lipid-tether conjugates for use in liposomes is described, for example, in U.S. Patent No. 5,395,619, hereby incorporated by reference. Other spacer molecules include polynucleotides (e.g., DNA or RNA), polysaccharides such as dextran or xanthan, cellulose derivatives (e.g., carboxymethyl cellulose), polystyrene, polyvinyl alcohol, poly methylacrylic acid, and poly(NIPAM). Synthetic reaction schemes for activating PEG with coupling agents are set forth in U.S. Pat. Nos.
5,631,018, 5,527,528, and 5,395,619. Synthetic reaction schemes for linkers with PEG spacer molecules are set forth in U.S. Pat. Nos. 6,828,401 and 7,217,845. Further examples of PEG linkers are set forth in U.S. Pat. No. 6,294,697.
PEG, for example, can be conjugated to a polypeptide of the invention by any means known in the art. In certain embodiments, the PEG molecule is derivatized with a linker, which is then reacted with the protein to form a conjugate. Suitable linkers include aldehydes, tresyl or tosyl linkers,
dichlorotriazine or chlorotriazine, epoxide, carboxylates such as succinimidyl succinate, carbonates such as a p-nitrophenyl carbonate, benzotriazolyl carbonate, 2,3,5-trichlorophenyl carbonate, and PEG- succinimidyl carbonate, or reactive thiols such as pyridyldisufide, maleimide, vinylsulfone, and iodo acetamide. Conjugation can take place at amino groups (e.g., the N-terminal amino group or amino groups within the lysine side chain), or at thiol hydroxyl, or amide groups, depending on the linker used. See, e.g., Veronese et al., DrugDiscov. Today 10: 1451-1458, 2005.
Exemplary Conjugates of the Invention
In certain embodiments, the conjugates of the invention include a linker selected from the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, adipyl, pimelyl, suberyl, azelayl, trans-β- hydromuconyl, 3,6,9-trioxaundecanedioyl (PEG3), carboxybutylether, and carboxyethylether (depicted in table 4). Additional linkers include those containing a disulfide bond, such as the ethyl disulfide linker formed when pyridyldithioethylamine is coupled with a carboxylic acid of an opioid moiety and then allowed to react with a cysteine-containing polypeptide. Other exemplary linkers of invention may be formed by the reaction between the sulfur of a cysteine group in a polypeptide and a maleimido group attached to the opioid moiety (e.g., cis-buten-mal, hex-mal, and PEG3-mal depicted in table 4). Still other exemplary linkers of the invention may be formed by the reaction between a click-chemistry pair, where one reactive group is attached to the polypeptide and the other reactive group is attached to the opioid moiety. An exemplary embodiment of a click-chemistry linker is methyl-triazolyl-propyl, as depicted in table 4. In each of the linkers depicted in table 4, one terminus of the linker is attached to a functional group in the opioid moiety and the other terminus is attached to a functional group in the polypeptide.
Table 4: Exemplary Linkers of the Invention
Figure imgf000028_0001
Figure imgf000029_0001
In certain embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three opioid moieties by means of a linker. In particular embodiments, the opioid moiety is a phenanthrene opioid receptor agonist or the opioid moiety is morphine or M6G. Μοφΐιίηε contains two hydroxyl groups, a phenol at the 3-position and a secondary alcohol at the 6-position. The polypeptide Angiopep-2 may be linked through its N-terminus and lysine residues at positions 10 and 15 by means of a linker. Such a structure is depicted in formula Ilia. In formula Ilia, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein. Related polypeptide-opioid conjugates of the invention are described by formula Ilia where the morphine moiety is replaced with a different
Figure imgf000029_0002
In related embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three morphine molecules through the C-3 hydroxyl group of the morphine. These embodiments are depicted in formula Illb. In formula Illb, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein. Related polypeptide-opioid conjugates of the invention are described by formula Illb where the morphine moiety is replaced with a different phenanthrene-based opioid agonist having a free C-3 hydroxyl group (e.g., hydromorphone).
Figure imgf000030_0001
In related embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three M6G molecules through the 5' carboxylic acid of M6G through ester linkages. These embodiments are depicted in formula IIIc. In formula IIIc, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000030_0002
In related embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three M6G molecules through the 5' carboxylic acid of M6G through amide linkages. These embodiments are depicted in formula Illd. In formula Illd, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000031_0001
(formula Hid)
In related embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three M6G molecules through the 2' hydroxyl group of M6G. These embodiments are depicted in formula Ille. In formula Ille, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers
Figure imgf000031_0002
Ille)
In related embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three M6G molecules through the C-3 hydroxyl of M6G. These embodiments are depicted in formula Illf. In formula Illf, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000032_0001
In related embodiments of the invention, the polypeptide Angiopep-2 is conjugated to three M3G molecules through the C-6 hydroxyl of M3G. These embodiments are depicted in formula Illg. In formula Illg, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000032_0002
a Illg)
In related embodiments of the invention, the polypeptide Angiopep-2 -Cys is conjugated to one M6G molecule through the 5' carboxylic acid of M6G via an ester linkage. Exemplary embodiments are depicted in formula IIHi. In formula HQi, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein. —
Figure imgf000033_0001
(formula Illh)
In related embodiments of the invention, the polypeptide Angiopep-2-Cys is conjugated to one M6G molecule through the 5' carboxylic acid of M6G via an amide linkage. Exemplary embodiments are depicted in formula IIIi. In formula IIIi, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000033_0002
In related embodiments of the invention, the polypeptide Angiopep-2-Cys is conjugated to one M6G molecule through the 4' alcohol group of M6G. Exemplary embodiments are depicted in formula Illj . In formula Illj, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000033_0003
In related embodiments of the invention, the polypeptide Angiopep-2-Cys is conjugated to one M6G molecule through the C-3 hydroxyl group of M6G. Exemplary embodiments are depicted in formula Illk. In formula Illk, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000034_0001
(formula Illk)
In related embodiments of the invention, the polypeptide Angiopep-2-Cys is conjugated to one morphine molecule through the C-3 hydroxyl group of morphine. Exemplary embodiments are depicted in formula IIIl. In formula IIIl, the LINKER moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000034_0002
In related embodiments of the invention, the polypeptide Angiopep-2-Cys is conjugated to one morphine molecule through the C-6 hydroxyl group of morphine. Exemplary embodiments are depicted in formula Illm. In formula Illm, the Ln KE moiety may be any of the groups appearing in table 4 or any of the linkers described herein.
Figure imgf000034_0003
In further embodiments, the polypeptide-opioid conjugate is described by any one of formulas Illa-IIIm, where residues 8, 10, and 11 are D-amino acids. In certain embodiments, polypeptides of the invention include those listed in table 5.
Polypeptide-opioid conjugates of the invention may be prepared by linking one or more opioid moieties to side chains (e.g., lysine or cysteine side chains) of residues in any of the polypeptides listed in table 5. Where a cysteine residue is present (e.g., in Angiopep-2-Cys, (3D)-Angiopep-2-Cys, or Angiopep-P6a), the sulfur atom of the cysteine residue may be incorporated into a disulfide moiety of a linker. The N- terminus and/or C-terminus of a polypeptide shown in table 5 may be modified with an opioid moiety. In some embodiments, the N-terminus of a polypeptide is linked to an opioid moiety and two amino acid side chains of the polypeptide are linked to opioid moieties. In any of the sequences of table 5, the C- terminus may be either an amide or a carboxylic acid.
Table 5: Exem lar Pol e tides of the Invention
Figure imgf000035_0001
Administration and Dosage
The present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a polypeptide-opioid conjugate of the invention. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).
The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides compositions for parenteral administration that include the above mentioned agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. The invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.
These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
The compositions containing an effective amount of the polypeptide-opioid conjugate can be administered for prophylactic or therapeutic treatments. Compositions of the invention can be administered to the subject (e.g., a human) in an amount sufficient to delay, reduce, or prevent the onset of pain. In therapeutic applications, compositions are administered to a subject (e.g., a human) already experiencing pain in an amount sufficient to alleviate or partially alleviate the pain.
Amounts effective for this use may depend on the severity of the pain and the weight and general state of the subject, but generally range from about 0.1-3,000 mg of an equivalent dose of the opioid (e.g., morphine) per dose per subject. Typical morphine sulfate dosages for pain are 5-30 mg (e.g., 5, 10, 15, 20, 25, 30 mg), orally, every 3 to 4 hours; 10-600 mg (e.g., 10, 20, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 mg), extended release, daily; 4-15 mg (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg), intravenously, every 3 to 4 hours; and 0.8-440 mg/hour (e.g., 0.8, 1, 2, 3, 5, 8, 10, 15, 20, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 440), continuous intravenous infusion. In particular embodiments of the invention, the polypeptide-opioid conjugate of the invention is administered (e.g., intravenously or orally) at a lower dose than the equivalent typical dose of morphine or morphine sulfate (e.g., less than or equal to about 90%, 85%, 80%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the equivalent dose of morphine or morphine sulfate).
Suitable regimes for administration are typified by an initial administration followed by repeated doses at one or more hourly or daily intervals by a subsequent administration. The total effective amount of an opioid present in the compositions of the invention can be administered to a mammal as a single dose, orally, as a bolus, or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time (e.g., a dose every 1-2, 4-6, 8-12, 14-16, or 18-24 hours). Alternatively, continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are used.
The therapeutically effective amount of the opioid moiety present within the compositions of the invention and used in the methods of this invention applied to mammals (e.g., humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because the polypeptide-opioid conjugates exhibit an enhanced ability to cross the BBB, the dosage of the compounds of the invention can be lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dosage required for a therapeutic effect of the unconjugated agent. The polypeptide-opioid conjugates of the invention are administered to a subject (e.g. a mammal, such as a human) in an effective amount, which is an amount that produces analgesia in a treated subject.
Therapeutically effective amounts can also be determined empirically by those of skill in the art.
The subject may also receive an polypeptide-opioid conjugate in the range of about 0.1-3,000 mg equivalent dose, as compared to unconjugated opioid, per dose one or more times per day (e.g., 2, 3, 4, 5, 6, or 6 or more times per day).
Single or multiple administrations of the polypeptide-opioid conjugates of the invention, including an effective amount, can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the pain being treated, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.
The polypeptide-opioid conjugates of the invention may be administered in combination therapies with other agents. The conjugates and other agent or agents may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention may be comprised of a combination of a conjugate of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.
Delivery of Opioids Using Nanoparticles
Nanoparticles coated with a polypeptide of the invention (e.g., Angiopep-2) may be used to deliver an opioid (e.g., mo hme or M6G) to a subject. As used herein, a "nanoparticle" is a colloidal, polymeric, lipid, or elemental particle ranging in size from about 1 nm to about 1000 nm. Nanoparticles can be made up of silica, carbohydrate, lipid, or polymer molecules. Opioid molecules can be embedded or encapsulated in the nanoparticle matrix or may be adsorbed onto its surface. In one example, the nanoparticle may be made up of a biodegradable polymer such as poly(butylcyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic and/or a polypeptide of the invention. In this approach, an opioid (e.g., morphine or M6G) is loaded into the nanoparticle. For related approaches, see Jain et al., Mol. Pharm. 2: 1 4-205, 2005. Other nanoparticles are made of silica, and include those described, for example, in Burns et al., Nano Lett. 9:442-448, 2009.
Nanoparticles can be formed, in part, from any useful polymer. Examples of polymers include biodegradable polymers, such as poly(butyl cyanoacrylate), poly(lactide), poly(glycolide), poly-D - caprolactone, poly(butylene succinate), poly(ethylene succinate), and poly(p-dioxanone);
poly(ethyleneglycol); poly-2-hydroxyethylmethacry.late (poly(HEMA)); copolymers, such as poly(lactide-co-glycolide), poly(lactide)-poly(ethyleneglycol), poly(poly(ethyleneglycol)cyanoacrylate- co-hexadecylcyanoacrylate, and poly[HEMA-co-methacrylic acid]; proteins, such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides, such as amylopectin, a-amylose, and chitosan. Any of the polymers may be bonded to or linked to a polypeptide of the invention. In some embodiments, a certain percentage (e.g., 0.1 %, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the polymer moieties used to form a nanoparticle is conjugated to a polypeptide (e.g., Angiopep-2) of the invention.
Polymeric nanoparticles can be produced by any useful process. Using the solvent evaporation method, the polymer and opioid are dissolved in a solvent to form a nanoemulsion and the solvent is evaporated. Appropriate solvent systems and surfactants can be used to obtain either oil-in-water or water-in-oil nanoemulsions. This method can optionally include filtration, centrifugation, sonication, or lyophilization. Using the nanoprecipitation method, a solution of the polymer and an opioid is formed in a first solvent. Then, the solution is added to a second solvent that is miscible with the first solvent but does not solubilize the polymer. During phase separation, nanoparticles are formed spontaneously.
Using the emulsion polymerization method, the monomer is dispersed into an aqueous solution to form micelles. Initiator radicals (e.g. hydroxyl ions) in the aqueous solution initiate anionic polymerization of the monomers. Additional methods include dialysis, ionic gelation, interfacial polymerization, and solvent casting with porogens.
In an example of the solvent evaporation method, the polymer is a cyanoacrylate copolymer containing a hydrophilic polymer group: poly(aminopoly(ethyleneglycol) cyanoacrylate-co-hexadecyl cyanoacrylate), which is synthesized as described in Stella et al., J. Pharm. Sci. 89: 1452-1464 (2000). The cyanoacrylate polymer, or a certain fraction of the polymer, may be conjugated to any polypeptide described herein using any conjugation method known in the art. The polymer and an opioid are added to an organic solvent, and the mixture is emulsified by adding an aqueous solution. Then, the organic solvent is evaporated under reduced pressure and the resultant nanoparticles are washed and lyophilized. Examples related to this approach are described in Li et al., Int. J. Pharm. 259:93-101 (2003); and Yu et al., Int. J. Pharm. 288:361-368 (2005).
In an example of the emulsion polymerization method, the monomer is added dropwise to an acidic aqueous solution. The mixture is stirred to promote polymerization and then neutralized. The nanoparticles are then filtered, centrifuged, sonicated, and washed. In one particular example of this method, the monomer of butyl cyanoacrylate monomer is provided and the aqueous solution also includes dextran in a dilute aqueous solution of hydrochloric acid. To introduce the opioid, the poly(butyl cyanoacrylate) nanoparticles are lyophilized and then resuspended in saline. An opioid is added to the saline solution with the nanoparticles under constant stirring. Alternatively, the opioid is added to during the polymerization process. The nanoparticles are coated with a polypeptide of the invention and optionally coated with a surfactant, such as polysorbate 80. Further examples related to this approach are described in Kreuter et al., Brain Res. 674: 171-174 (1995); Kreuter et al, Pharm. Res. 20:409-416 (2003); and Steiniger et al, Int. J. Cancer 109:759-767 (2004).
Other nanoparticles include solid lipid nanoparticles (SLN). SLN approaches are described, for example, in Kreuter, Ch. 24, In V. P. Torchilin (ed), Nanoparticles as Drug Carriers pp. 527-548, Imperial College Press, 2006). Examples of lipid molecules for solid lipid nanoparticles include stearic acid and modified stearic acid, such as stearic acid-PEG 2000; soybean lechitin; and emulsifying wax. Solid lipid nanoparticles can optionally include other components, including surfactants, such as Epicuron® 200, poloxamer 188 (Pluronic® F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts, such as taurocholate sodium. Opioids can be introduced into solid lipid nanoparticles by a number of methods known in the art.
In one example, SLNs include stearic acid, Epicuron 2000 (surfactant), and taurocholate sodium loaded with an opioid. In another example, SLNs include stearic acid, soybean lecithin, and poloxamer 188. SLNs can also be made from polyoxyl 2-stearyl ether (Brij 72), or a mixture of emulsifying wax and polyoxyl 20-stearyl ether (Brij 78) (see, e.g., Koziara et al., Pharm Res 20: 1772-1778, 2003). In one example of making solid lipid nanoparticles, a microemulsion is formed by adding a surfactant (e.g. Brij 78 or Tween 80) to a mixture of emulsifying wax in water at 50°C to 55°C. Emulsifying wax is a waxy solid that is prepared from cetostearyl alcohol and contains a polyoxyethylene derivative of a fatty acid ester of sorbitan. Nanoparticles are formed by cooling the mixture while stirring. The opioid can be introduced by adding the opioid to the heated mixture containing the emulsifying wax in water.
Examples related to this approach are described in Koziara et al., Pharm. Res. 20: 1772-1778 (2003).
Nanoparticles can also include nanometer-sized micelles. Micelles can be formed from any polymers described herein. Exemplary polymers for forming micelles include block copolymers, such as poly(ethylene glycol) and poly(8-caprolactone). In one particular example, PEO-b-PCL block copolymer is synthesized via controlled ring-opening polymerization of ε-caprolactone by using an a-methoxy-ω- hydroxy-poly(ethylene glycol) as a macroinitiator. To form micelles, the PEO-b-PCL block copolymers are dissolved in an organic solvent (e.g., tetrahydrofuran) and then deionized water is added to form a micellar solution. The organic solvent is evaporated to obtain nanometer-sized micelles.
In certain embodiments, the properties of the nanoparticle are altered by coating with a surfactant. Any biocompatible surfactant may be used, for example, polysorbate surfactants, such as polysorbate 20, 40, 60, and 80 (Tween 80); Epicuron® 200; poloxamer surfactants, such as 188
(Pluronic® F68) poloxamer 908 and 1508; and Brij surfactants, such as Brij 72 and Brij 78. In other embodiments, the surfactant is covalently attached to the nanoparticle, as is described in PCT Publication No. WO 2008/085556. Such an approach may reduce toxicity by preventing the surfactant from leeching out of the nanoparticle. Nanoparticles can be optionally coated with a surfactant.
Nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethyleneglycol) or poly(propyleneglycol)). The surface of the nanoparticle can be modified by covalently attaching hydrophilic polymer groups. Alternatively, nanoparticles can be formed by using polymers that contain hydrophilic polymer groups, such as poly[methoxy poly (ethyleneglycol) cyanoacrylate-co-hexadecyl cyanoacrylate]. Nanoparticles can be optionally cross-linked, which can be particularly use for protein-based nanoparticles.
A polypeptide of the invention (e.g., Angiopep-2) may be covalently linked to the polymer or lipid used to form a nanoparticle before or after the formation of the nanoparticle. In certain
embodiments, the polypeptide is linked to the nanoparticle through a spacer moiety (e.g., a poly(ethylene glycol)- or poly(lactic acid)-containing spacer). The spacer precursor may by attached to any polymer or lipid of the nanoparticle and may include a reactive group (e.g., an NHS ester, aldehyde, or maleimide) that reacts selectively with an amino or a thiol group of the polypeptide, thereby linking the polypeptide to the nanoparticles.
Opioids can be introduced to nanoparticles by any useful method. Opioids can be incorporated into the nanoparticle at, during, or after the formation of the nanoparticle. In one example, the opioid is added to the solvent with the polymer or monomer before the formation of the nanoparticles. In another example, the opioid is incorporated into pre-formed nanoparticles by adsorption. In yet another example, the opioid is covalently bound to the nanoparticle. The opioid can be physically adsorbed to the surface of the nanoparticle with the optional step of further coating the nanoparticle with a surfactant. Examples of surfactants include polysorbate 80 (Tween 80). Further examples of this approach are described in Kreuter, Nanoparticular Carriers for Drug Delivery to the Brain, Chapter 24, in Torchilin (ed.), Nanoparticulates as Drug Carriers (2006), Imperial College Press.
The invention features any of the compositions or methods for the transport of therapeutic agents
(e.g., morphine or M6G) described in WO 2011/041897, which is hereby incorporated by references.
Examples
Example 1: Synthesis of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys
A. Synthesis of M6G
Morphine- -glucuronide (M6G) was synthesized by the route shown in Figure 1. Morphine sulfate (320 mg) was acylated with acetic anhydride and saturated aqueous sodium bicarbonate to provide 3-O-acetylmorphine (250 mg). The phenol group of the product was glucuronated with acetobromo-D- glucuronic acid methyl ester. The glucuronidation reaction was allowed to go to completion, i.e., the reaction was not stopped until the 3-O-acetylmorphine was fully consumed. The product (490 mg) was used directly in a hydrolysis reaction with sodium hydroxide in methanol and water to provide M6G in 56% yield (200 mg). B. Preparation of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys
M6G (25 mg) was coupled with pyridyldithioethylamine using benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and diisopropylethylamine (DIEA) in DMF to provide the amide product in 100% conversion by mass. The product was not isolated, but was treated in situ with 1.3 equiv of Angiopep-2 -Cys and DBEA to provide the final product, M6G-5'-Amido- Ethyl-Disulfide-Angiopep-2-Cys in 48% yield (76 mg) (Figure 2).
Example 2: Synthesis of (Morphine-6-Gutaryl)3- Angiopep-2 and (Morphine-6-Dimethylglutaryl)3- Angiopep-2
Morphine was protected at the C-3 phenol position with tert-butyldimethylsilyl chloride (TBSC1).
The secondary alcohol of the product was functionalized with glutaric anhydride to yield a carboxylic acid. The carboxylic acid was treated with the coupling agent 0-(benzotriazol-l -yl)-N,N,N',N- tetramethyluronium tetrafluoroborate (TBTU) in DMF and then Angiopep-2 in DMF to yield a conjugate that was deprotected with tetra-«-butylammonium fluoride (TBAF) to yield (Morphine-6-Glutaryl)3- Angiopep-2 (Figure 5).
(Μοφηώ6-6-θ™6^^Μί3^1)3-Α^ΐορερ-2 was prepared by the same route when 3,3- dimethylglutaric anhydride was used in place of glutaric anhydride (Figure 6).
Example 3: Synthesis of (Morphine-3-Glutaryl)3- Angiopep-2 and (Morphine-3-Dimethylglutaryl)3- Angiopep-2
Morphine was protected at the C-3 and C-6 positions with TBSC1. The bis-TBS compound was treated with TBAF to selectively remove the phenolic TBS group. The phenol of the product was functionalized with glutaric anhydride to yield a carboxylic acid. The carboxylic acid was treated with the coupling agent TBTU and DIEA, and then Angiopep-2 in DMSO to yield a conjugate that was deprotected with trifluoroacetic acid in water to provide (Moiphine-6-Glutaryl)3-Angiopep-2 (Figure 7).
(Μοφηώε-3-0^6^¾1υί3^1)3-Α^ίορερ-2 was prepared by the same route when 3,3- dimethylglutaric anhydride was used in place of glutaric anhydride (Figure 8).
Example 4: Synthesis of (Morphine-3-Suberyl)3- Angiopep-2
The phenol group of morphine was coupled with suberic acid using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide»HCl (EDC). The product carboxylic acid was treated with TBTU in DMF and then Angiopep-2 in DMF to provide (Morphine-3-Suberyl)3-Angiopep-2 (Figure 9).
Example 5: Synthesis of (Morphine-3-Succinyl)3- Angiopep-2
The phenol group of morphine was coupled with succinic anhydride and DIEA in DMF. The product carboxylic acid was treated with TBTU and DIEA in DMF and then Angiopep-2 in DMF to provide (Μοφηίη6-3-8υοοί^1)3-Α¾ϊορερ-2 (Figure 10). Example 6: Synthesis of (Morphine-3-PEG3)3- Angiopep-2
The phenol group of morphine was coupled with 3,6,9-trioxaundecanedioic acid in DMF with PyBOP and DIEA to provide the product carboxylic acid in 51% yield. The carboxylic acid was treated with TBTU and DIEA in DMF then Angiopep-2 in DMSO/DMF to provide (Morphine-3-PEG3)3- Angiopep 2 in 40% yield (Figure 11).
Example 7: Synthesis of (Morphine-Carboxybutylether)3-Angiopep-2
Morphine was protected at the C-3 and C-6 positions with TBSC1 and imidazole. The bis-TBS compound was treated with TBAF to selectively remove the phenolic TBS group. The phenol of the product was functionalized with methyl 5-bromopentanoate and sodium methoxide to yield a terminal ester product. The ester was saponified with lithium hydroxide. The carboxylic acid product was treated with TBTU and DIEA then Angiopep-2 in DMSO to yield a conjugate that was deprotected with trifluoroacetic acid in water to yield a trifluoroacetate salt that was subsequently converted into the hydrochloride salt of (Morphine-Carboxybutylefher)3-Angiopep-2 (Figure 12).
Example 8: Polypeptide-Opioid Conjugates Induce Analgesia in the Hot-PIate Test
Several polypeptide-opioid conjugates of the invention were tested for analgesic activity in mice using the hot-plate assay. The data shows improved analgesic activity with increasing dose of M6G-5'- Amido-Ethyl-Disulfide-Angiopep-2-Cys, improved activity of M6G-5 ' -Amido-Efhyl-Disulfide-
Angiopep-2-Cys over unconjugated M6G and morphine, and indicate that a cleavable linker is preferred to a non-cleavable linker.
A. Comparison of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys at four dosages
CD-I mice were treated intravenously with four different dosages of M6G-5'-Amido-Efhyl- Disulfide-Angiopep-2-Cys (3.8 mg/kg, 9.5 mg/kg, 1 mg/kg, and 38 mg/kg). After a set period of time (15 min, 30 min, 60 min, 90 min, or 120 min), mice were placed on an electrically heated surface maintained at 55 °C. The mice were constrained to the hot plate using a transparent barrier and monitored. The time until flicking of the hindpaw was measured and recorded as hindpaw latency. Figure 1 A depicts hindpaw latency in terms of post-injection time for the four different doses of M6G- 5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys. Untreated animals exhibited a hindpaw latency time of, approximately, between 5 and 10 seconds, while treated mice showed higher hindpaw latency times. The greatest increase in hindpaw latency was observed at the highest dose. The data indicate that M6G-5'- Amido-Ethyl-Disulfide-Angiopep-2-Cys induces an analgesic effect with respect to thermal nociception, and that this effect increases with increasing dose over the range of 3.8 mg/kg to 38 mg/kg.
B. Comparison of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys with unconjugated M6G
CD-I mice were treated intravenously with either M6G-5'-Amido-Ethyl-Disulfide- Angiopep-2 - Cys at 9.5 mg/kg or with M6G at 1.5 mg/kg. After a set period of time (15 min, 60 min, 120 min, 180 min, or 240 min), mice were placed on an electrically heated surface maintained at 55 °C. The mice were constrained to the hot plate using a transparent barrier and monitored. The time until flicking of the hindpaw was measured and recorded as hindpaw latency. Figure 15B depicts hindpaw latency in terms of post-injection time for the two compounds. Mice receiving M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2- Cys exhibited hindpaw latency times of greater than 20 seconds, while mice receiving M6G exhibited lower hindpaw latency times. The data indicate that conjugating M6G to a polypeptide capable of crossing the BBB increases the analgesic effects of M6G with respect to thermal nociception.
C. Comparison of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys and (M6G-5'-Amido- Ethyl-Disulfide-Ethyl)3-Angiopep-2 with a saline control and M6G
CD-I mice were treated intravenously with M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys at
25 mg/kg, (M6G-5'-Amido-Ethyl-Disulfide-Ethyl)3-Angiopep-2 at 6.7 mg/kg, M6G at 3.2 mg/kg, or saline. After a set period of time (15 min, 60 min, 120 min, 180 min, or 240 min), mice were placed on an electrically heated surface maintained at 55 °C. Figure 15C depicts hindpaw latency in terms of post- injection time for the four treatments. The mice were constrained to the hot plate using a transparent barrier and monitored. The time until flicking of the hindpaw was measured and recorded as hindpaw latency. The data indicate that both M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys and (M6G-5'- Amido-Ethyl-Disulfide-Ethyl)3-Angiopep-2 exhibit greater analgesic activity than unconjugated M6G or the saline control.
D. Comparison of M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys with M6G and morphine
Figure 16 illustrates hot-plate assay data (obtained by the procedure of Example 8B) showing that M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys (at 9.5 mg/kg) is more active than both unconjugated M6G (at 1.5 mg/kg) and morphine (at 1.25 mg/kg) and fully active for at least 3-4 hours.
E. Comparison of a conjugate having a cleavable linker with a conjugate having a non- cleavable linker
Figure 17 illustrates hot-plate data (obtained by the procedure of Example 8 A) comparing a polypeptide-opioid conjugate having a cleavable linker
Figure imgf000043_0001
with a conjugate having a non-cleavable linker ((mo hine-carbo ybutylether)3-Angiopep-2). The data shows that the conjugate with the cleavable linker provided morphine-like analgesia while the conjugate with the non-cleavable linker did not provide analgesia.
Observations made from the hot-plate test data are shown in tables 6 and 7. Comparison of Polypeptide-Opioid Conjugal
with Morphine in the Hot-Plate Test
Figure imgf000044_0001
Example 9: Effect of M6G and Polypeptide-Opioid Conjugates on Body Temperature in Mice
We then examined the effect of polypeptide-opioid conjugates of the invention on the body temperature of mice. Mice receiving the same intravenous doses of compounds described in Examples 8A-8C were monitored by measurement of body temperature. The recorded data is depicted in Figures 18A-18C. Mice receiving M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys at a dose of 38 mg/kg or (M6G-5'-Amido-Ethyl-Disulfide-Ethyl)3-Angiopep-2 at a dose of 6.7 mg/kg exhibited a slight decrease in body temperature (Figures 18 A and 18C). No significant difference was seen in body temperature when M6G-5'-Amido-Ethyl-Disulfide-Angiopep-2-Cys at 1.5 mg/kg was compared with unconjugated M6G (Figure 18B).
Observations made from the hot-plate test data are shown in tables 8 and 9. Table 8: Comparison of Polypeptide-Opioid Conjugates
with Morphine in Body Temperature Measurements
Figure imgf000045_0001
Example 10: Brain Perfusion and Volume Distribution of Polypeptide-Opioid Conjugates
The ability of the polypeptide-opioid conjugates to cross the BBB in situ was also measured. This assay can be performed as described in PCT Publication WO 2006/086870, which is herein inco orated by reference. Figure 19 show transcytosis of the polypeptide-opioid conjugates of the invention into the brain parenchyma at two minutes. The corresponding data is shown in table 10. A time-course experiment demonstrating uptake of the polypeptide-opioid conjugates into the brain at a 50 nM concentration over 4 minutes is shown in Figure 20 and the corresponding data is presented in table 11. Table 10: Brain Perfusion of Morphine Conjugates
Figure imgf000046_0001
Example 11: Receptor binding of Angiopep-2-M6G conjugate (ANG2010)
The chemical structure and schematic representation of Angiopep-2-M6G conjugate (ANG2010) are shown in Figure 21 A. Competition binding of M6G and ANG2010 to Mu-opioid (MOPr) (Figure 2 IB) or Delta-opioid (DOPr) (Figure 21 C) receptors in rat brain membranes was evaluated. Brain membranes were incubated with either 1 nM [3H]-DAMGO or 1 nM [3Η]-Οε1ΐθφ1ιϊη II and increasing concentrations (10" to 10"5 M) of unlabeled M6G or unlabeled ANG2010 for 60 minutes at 37 °C. Binding inhibition results were analyzed using GraphPad Prism software. Each curve represents the mean of 6 measurements. Data are expressed as the mean Ki ± SEM.
Example 12: Brain uptake of ANG2010
In vivo brain uptake of the [12 I]-ANG2010 and [3Η]-ηιοφηϊηβ was measured by in situ brain perfusion, as shown in Figure 22. ANG2010 was transported into the brain more efficiently than moφhme (Figure 23 A). Brain capillary depletion was performed to assess the ANG2010 distribution in the brain compartments (Figure 23B). Initial brain transport rate (¾„) values for ANG2010, ηιοφηίηβ, and M6G compared to that of other molecules showed that ANG2010 had the highest brain transport rate (Figure 23C).
Example 13: Evaluation of analgesic effect in a hot plate model
The hot plate model was used to evaluate the analgesic effect of ANG2010. In this model, mice were placed onto a hot metal plate maintained at 54°C and foot-licking response was measured after dosing. Latency to a hind limb response (lick, shake, or jump) was recorded, with a maximum time on the hot plate of 30 seconds. A statistically significant increase from baseline pain-threshold measurement was interpreted as induction of analgesia.
ANG2010 significantly increased the paw flicking latency after intravenous (IV) bolus injection compared to an equimolar dose of unconjugated M6G (Figure 24A). In addition, ANG2010, after IV or sub-cutaneous (SC) administration, significantly increased the foot licking latency for at least 3hrs (Figure 24B). Example 14: Evaluation of analgesic effect in a rat tail flick mouse model
The rat tail flick model was also used to evaluate the analgesic effect of ANG2010. Pain threshold was measured before (baseline) and after drug administration, using a standard hot-water tail- flick assay. The dependent variable was the latency (in seconds) for the rat to flick its tail from the hot- water bath. The water was maintained at 53°C in a constant-temperature water bath. The distal first 5 cm of the rat's tail was immersed in the bath, and the time required for the rat to remove its tail was measured. A statistically significant increase from baseline pain-threshold measurement was interpreted as induction of analgesia.
In the first experiment, the analgesic effect of ANG2010 compared to that of unconjugated morphine and M6G was evaluated in the rat tail flick model after IV bolus injection (equivalent to 3 mg/kg of morphine sulfate). The results are shown in Figure 25 A.
The results were then represented as the maximal possible effect (%) for the three drugs (Figure 25B).
In the second experiment, analgesic effect of ANG2010 compared to that of unconjugated morphine, and M6G was evaluated in the rat tail flick model after SC bolus injection (equivalent to 3 mg/kg of morphine sulfate) (n = 10 rats/group).
rV or sub-cutaneous (SC) administration of ANG2010 significantly increased the latency to tail withdrawal for at least 3hours (Figures 25A and 25B, and Figure 26).
Example 15: Evaluation of sub-cutaneous injection of ANG2010 on gastro-intestinal transit
The effect of sub-cutaneous injection of ANG2010 on gastro-intestinal transit (n = 10 rats/group) was evaluated. The data shown in Figure 27 demonstrate that gastro-intestinal transit was not significantly affected by sub-cutaneous administration of ANG2010 as compared to M6G or morphine. Other Embodiments
All publications, patents, and patent applications including U.S. Provisional Application No. 61/546,851, filed 13 October 2011, mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
Other embodiments are within the claims.

Claims

1. A polypeptide-opioid conjugate, comprising:
(i) a polypeptide that is capable of crossing the blood-brain barrier;
(ii) one or more opioid moieties linked to the polypeptide by means of a linker;
wherein each of said opioid moieties is linked to said polypeptide.
2. The polypeptide-opioid conjugate of claim 1 , wherein each of said opioid moieties is linked to the N-terminus, C-terminus, or an amino acid side chain of said polypeptide.
3. The polypeptide-opioid conjugate of claim 1 , wherein said polypeptide has an amino acid sequence identity of at least 70% compared to a sequence selected from the group consisting of SEQ ID NO: 1-105, SEQ ID NO:107-117, SEQ ID NO:120-138, and SEQ ID NO: 166-178, or a fragment thereof.
4. The polypeptide-opioid conjugate of claim 3, wherein said sequence identity is at least
90%.
5. The polypeptide-opioid conjugate of claim 1 , wherein said polypeptide contains between 6 and 21 amino acids.
6. The polypeptide-opioid conjugate of claim 1 , wherein said polypeptide comprises an amino acid sequence at least 70% identical to Angiopep-2 (SEQ ID NO: 97), (3D)-Angiopep-2 (SEQ ID NO: 166), Angiopep-P6a (SEQ ID NO: 174), Angiopep-descys-P6a (SEQ ID NO: 177), Angiopep-2-Cys (SEQ ID NO: 114), or (3D)-Angiopep-2-Cys (SEQ ID NO:178).
7. The polypeptide-opioid conjugate of claim 6, wherein said sequence identity is at least
90%.
8. The polypeptide-opioid conjugate of any one of claims 1-7, wherein said opioid moieties are selected from the group consisting of morphine, morphine-6-glucuronide, morphine-3-glucuronide, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, alazocine, alfentanil, bremazocine, buprenorphme, butorphanol, cyclazocine, dezocine, dihydrocodeine, diphenoxylate, diamorphine, fentanyl, levorphanol, meperidine, meptazinol, methadone, nalbuphine, pentazocine, propoxyphene, remifentanil, sufentanil, tramadol, naloxone, naltrexone, diprenorphine, naloxonazine, met-enkephalin, leu-enkephalin, β-endorph n, dynorphin A, dynorphin B, a-neo-endorphin, endomorphin-1,
endomorphin-2, and nociceptin.
9. The polypeptide-opioid conjugate of claim 8, wherein said opioid moieties are selected from the group consisting of morphine, morphine-6-glucuronide, morphine-3-glucuronide, and hydromorphone.
10. The polypeptide-opioid conjugate of any one of claims 1 -9, wherein said linker is selected from the group consisting of succinyl, glutaryl, 3,3-dimethylglutaryl, adipyl, pimelyl, suberyl, azelayl, trans- -hydromuconyl, 3,6,9-trioxaundecanedioyl, a poly (ethylene glycol) (PEG) linker, carboxybutylether, carboxyethylether, a disulfide-containing linker, ethyl disulfide, ethyl-disulfide-ethyl, a cis-buten-mal linker, a hex-mal linker, a PEG3-mal linker, a triazole-containing linker, and methyl- triazolyl-propyl.
11. The polypeptide-opioid conjugate of any one of claims 1-10, wherein one or more of said opioid moieties is linked to a lysine residue in said polypeptide.
12. The polypeptide-opioid conjugate of any one of claims 1-10, wherein one or more of said opioid moieties is linked to a cysteine residue in said polypeptide.
13. The polypeptide-opioid conjugate of any one of claims 1-10, wherein one of said opioid moieties is linked to the N-terminus of said polypeptide.
14. The polypeptide-opioid conjugate of any one of claims 1-10, wherein said polypeptide is linked to exactly one opioid moiety.
15. The polypeptide-opioid conjugate of any one of claims 1-10, wherein said polypeptide is linked to exactly three opioid moieties.
16. The polypeptide-opioid conjugate of any one of claims 1-10, wherein said polypeptide contains at least one D-amino acid.
17. The polypeptide-opioid conjugate of claim 16, wherein said polypeptide contains exactly three D-amino acids.
18. The polypeptide-opioid conjugate of claim 1 , wherein said polypeptide-opioid conjugated from the group consisting of:
Figure imgf000051_0001
Figure imgf000052_0001
51
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
54
Figure imgf000056_0001
55
Figure imgf000057_0001
or a pharmaceutically acceptable salt thereof.
19. A nanoparticle, wherein said nanoparticle is conjugated to a plurality of polypeptides, wherein at least one of said polypeptides comprises an amino acid sequence having at least 70% sequence identity to any of the sequences set forth in SEQ ID NO: 1-105, SEQ ID NO: 107-1 17, SEQ ID NO: 120- 138, or SEQ ID NO: 166-178; and wherein said nanoparticle is bound to or contains an opioid moiety.
20. The nanoparticle of claim 19, wherein said amino acid sequence identity is at least 90%.
21. The nanoparticle of claim 19, wherein said polypeptide contains at least 70% sequence identity to SEQ ID NO:97.
22. The nanoparticle of claim 21 , wherein said polypeptide contains at least 90% sequence identity to SEQ ID NO:97.
23. The nanoparticle of any one of claims 19-22, wherein said opioid moiety is morphine or
M6G.
24. A method of treating or prophylactically treating pain in a subject, said method comprising the step of administering a composition containing the polypeptide-opioid conjugate of any one of claims 1-18 or the nanoparticle of any one of claims 19-23 to said subject in an amount sufficient to treat said pain.
25. The method of claim 24, wherein said pain is selected from the group consisting of postoperative pain, cancer pain, chronic pain, acute pain, somatic pain, neuropathic pain, visceral pain, inflammatory pain, migraine-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritic pain, skeletal pain, joint pain, gastrointestinal pain, muscle pain, angina pain, facial pain, pelvic pain, claudication, post traumatic pain, obstetric pain, labor pain, gynecological pain, and chemotherapy-induced pain.
26. The method of claim 24 or 25, wherein said opioid conjugate is administered in an amount that reduces at least one opioid-induced side effect as compared to the same effective dosage of the corresponding unconjugated opioid.
27. The method of claim 26, wherein said opioid-induced side effect is selected from the group consisting of constipation, respiratory depression, apnea, circulatory depression, respiratory arrest, shock, cardiac arrest, lightheadedness, dizziness, sedation, nausea, vomiting, sweating, dysphoria, euphoria, weakness, headache, agitation, tremor, uncoordinated muscle movements, seizure, alterations of mood, dreams, muscle rigidity, transient hallucinations and disorientation, visual disturbances, insomnia, increased cranial pressure, dry mouth, biliary tract spasm, laryngospasm, anorexia, diarrhea, cramps, taste alterations, flushing of the face, tachycardia, bradycardia, palpitation, faintness, syncope, hypotension, hypertension, urine retention or hesitance, reduced libido and/or potency, pruritus, skin rashes, edema, diaphoresis, antidiuretic effects, paresthesia, muscle tremor, and blurred vision.
28. The method of claim 26, wherein said opioid-induced side effect is constipation.
29. The method of claim 24 or 25, wherein said conjugate is administered at a lower equivalent dosage than would be required to treat said pain with a corresponding unconjugated opioid.
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