GB2098219A - Daunorubicin-protein conjugates - Google Patents
Daunorubicin-protein conjugates Download PDFInfo
- Publication number
- GB2098219A GB2098219A GB8203523A GB8203523A GB2098219A GB 2098219 A GB2098219 A GB 2098219A GB 8203523 A GB8203523 A GB 8203523A GB 8203523 A GB8203523 A GB 8203523A GB 2098219 A GB2098219 A GB 2098219A
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- Prior art keywords
- daunorubicin
- protein
- drug
- conjugate
- protein conjugate
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229960000975 daunorubicin Drugs 0.000 claims abstract description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 15
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical group O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 claims abstract description 15
- KQRWYZDETCDVGB-TZSSRYMLSA-N (7s,9s)-7-[(2r,4s,5s,6s)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-9-(2-bromoacetyl)-6,9,11-trihydroxy-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CBr)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 KQRWYZDETCDVGB-TZSSRYMLSA-N 0.000 claims abstract description 4
- 235000018102 proteins Nutrition 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 239000005018 casein Substances 0.000 claims description 3
- 108060002885 fetuin Proteins 0.000 claims description 3
- 102000013361 fetuin Human genes 0.000 claims description 3
- 238000005342 ion exchange Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 108010076119 Caseins Proteins 0.000 claims description 2
- 108010033040 Histones Proteins 0.000 claims description 2
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 claims description 2
- 102000005891 Pancreatic ribonuclease Human genes 0.000 claims description 2
- 108010044715 asialofetuin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- -1 lysoxyme Proteins 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 description 19
- 239000003814 drug Substances 0.000 description 19
- 230000000694 effects Effects 0.000 description 9
- 230000000259 anti-tumor effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 230000001472 cytotoxic effect Effects 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 230000001332 colony forming effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 230000027455 binding Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- IQHSSYROJYPFDV-UHFFFAOYSA-N 2-bromo-1,3-dichloro-5-(trifluoromethyl)benzene Chemical group FC(F)(F)C1=CC(Cl)=C(Br)C(Cl)=C1 IQHSSYROJYPFDV-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010062580 Concanavalin A Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000003817 anthracycline antibiotic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
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- 230000004700 cellular uptake Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
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- 150000001875 compounds Chemical class 0.000 description 1
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- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
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- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/252—Naphthacene radicals, e.g. daunomycins, adriamycins
-
- 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
-
- 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
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- 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
- A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
- A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Daunorubicin-protein conjugates in which the daunorubicin is bound to the protein through 9-acetyl side-chain of the daunorubicin, rather than through the 3'- primary amino group are provided. They may be prepared by reaction of 14-bromo-daunorubicin with an excess of a protein in aqueous- methanolic solution at ambient temperature for 2 to 4 hours. Neutrality is maintained by addition of dilute sodium hydroxide as necessary.
Description
SPECIFICATION
Daunorubicin-protein conjugates
DESCRIPTION
The invention relates to the preparation of daunorubicin-protein conjugates useful as antitumoural agents.
It is common knowledge that the major problem in cancer chemotherapy still remains the lack of selectivity of the available drugs. For increasing the selective toxicity of antitumour drugs for the tumour cells, and consequently their therapeutic effectiveness, linkage or association of those drugs to a carrier macromolecule has been reported (A. Trouet, Europ. J. Cancer 14: 105-111, 1978; M.Szerkerke and J.S. Driscoll, ibid, 13: 529-537, 1977). Anthracycline antibiotics have been used in the immunochemical approach in the attempt to increase the selectivity of those cytoxic agents, but the use of antibodies as specific carriers has been limited by difficulties to obtaining antitumour antibodies.Moreover all reported procedures for linking daunorubicin covalently to antitumour antibodies or also to less specific proteins, involve the oxidation or modification of the primary amino group of the sugar moiety of the antibiotic. Since the presence and stereochemistry of the unsubstituted amino function in the antibiotic molecule is a critical determinant of both DNA binding and biological activity, the reported methods produce generally less potent drugs as a consequence of loss of basic character and electrostatic binding component in the drug.
To avoid this disadvantage, we have found and developed an alternative procedure for the preparation of daunorubicin-protein conjugates in which 14-bromo-daunorubicin (Belgian
Patent No. 731,398) is used to bind the drug, via ita acetyl side-chain, to various proteins.
The invention provides a process for the preparation of a daunorubicin-protein conjugate, the process comprising reacting 1 4-bromo-daunorubicin with an excess of a protein in aqueousmethanolic solution at ambient temperature for a period of from 2 to 4 hours while keeping the solution at neutrality by addition of dilute sodium hydroxide. A pure solution of daunorubicinprotein conjugate may be obtained from the reaction mixture by removing any preciptate, for example by centrifugation, and purifying the supernatant liquid. The purification may be effected by passage over an ion-exchange or adsorption resin, the former when negatively charged proteins are used and the latter when positively charged proteins are used.
The resultant daunorubicin-protein conjugates, in which the utilization of the primary amino group of the antibiotic, essential for its cytoxic activity, is still retained, show a cytotoxic selectivity either in "vitro" by inhibition of the colony forming ability of Hela cells or in "vivo" against Ehrlich ascites tumour.
Since its assumed that the 1 4-bromo derivative of daunorubicin reacts with the free amino groups or with the caaboxylic groups of the involved proteins, a molar excess of protein (with respect to the amino acids residues) has been used for the preparation of the new daunorubicinprotein conjugates as reported in the following Example which illustrates the invention.
EXAMPLE:
20 mg of protein were dissolved in 2 ml of water and the pH of the solution was adjusted to 8.5 by adding a dilute aqeuous solution of sodium hydroxide. 2 mg of 14-bromo-daunorubicin (freshly dissolved in 0.1 ml of methanol) were then added and the mixture was left under stirring, at room temperature, for 120-240 minutes while contemporaneously adjusting the pH of the solution to neutrality by a cautious addition of a dilute aqueous solution of sodium hydroxide to neutralize the hyrobromic acid during the reaction. When the reaction was over (pH
= 7), the mixture was centrifuged at 5000 rpm for 1 5 minutes and the clear supernatant, in the case of negatively charged proteins, was passed through an ion-exchange column filled with
Dowex 5 OW-X2 resin (Trade Mark) to remove any free antibiotic.When positively charged proteins were used, for avoiding the binding of the protein to the ion-exchange resin, the eventual free drug was removed by adsorption chromatography on Amberlite XAD-2 resin (Trade
Mark). The resultant pure solutions of daunorubicin-protein conjugates (no free drug detected by
HPLC) were used as such for the evaluation of their biological activity.
BIOLOGICAL ACTIVITY
The inhibition of the colony forming ability of HeLa cells was used to quantitate activity of the drug linked to various proteins. In general, the activity of the conjugates was lower than that of the free daunorubicin, since typically four times (or more) higher concentrations of conjugates (expressed as daunorubicin content) than that of free antibiotic are required to produce 50% inhibition. This reduction is expected since the limited cellular uptake of protein macromolecules reduce intracellular drug accumulation.
However the effect of the various carrier proteins on the cytotoxic activity of daunomycin is different. Although the actual mechanism of antitumour activity of the macromolecular derivative of cytotoxic drug remains to be established, it is clear that the use of some proteins offered a consistent advantage in most cases. In the case of daunorubicin-proteins conjugates, if the cytotoxicity is correlated to the drug content, a considerable variation in the inhibitory potency of the conjugates (Table I) suggests some protein specific effects.
Daunorubicin linked to casein, ribonuclease A, asialo fetuin, immunoglobulin and concanavalin A is appreciably more active than drug linked to bovine serum albumin, fetuin, lysozyme, histones. The cytoxic effect cannot be attributed to the protein component of the conjugates since unmodified proteins when tested in the absence of daunorubicin, did not show any cytoxic effect at similar or higher concentration (up to 201lg/ml.
Daunorubicin bound to immunogloblin retained a relevant activity.
Table 1
BIOLOGICAL ACTIVITY OF DAUNORUBICIN-PROTEIN CONOUGATES
Daunorubicin-Protein Drug/protein Drug/proein Concentration required for 50% inhibition of
Conjugate weight ratio molar ratio colony-forming abiliy of HeLa cells.
Concentrations are expressed as daunorubicin
content in the conujugates.
Daunosrubicin (Da) 6.5(4-12.5)
Da-CASEIN 1:38 1.21 35
Da-RIBONUCLEASE A 1:26 0.93 37.5(25-50)
Da-ASIALO FETUIN 1:17 5.2 42,5(35-50)
Da-IMMUNOGLOBULIN 1:28 9.5 50(40-60)
Da-CONCANAVALIN A 1:27 1.77 71(40-115)
Da-FETUIN 1:26 > 117
Da-BSA 1:28 4.18 100 (70-130)
Da-LYSOZYME 1:34 0.75 105(100-110)
Da-HISTONE 1:30 0.95 175(150-200)
HeLa cells were exposed to drug for 8 h. The drugs were removed and cells were diluted and plated into 50 mm tissue culture dishes. Colonies were countes after 8 days of incubation. Data were deduced from dose-response curves in experiments performed at 4 different concentration leveis (5 replicate dishes per concentration). The dats are presented as mean of value deduced from at least 3 replicate experiments (in brackets, range of individual experiments).
In vivo antitumour studies
CDL mice (22-28 g) were inoculated intraperitoneally with Ehrlich ascites tumour cells, 5x106 cells per animal) 24 hours prior to treatment with a single dose. Under these conditions, the tumour is lethal to 100% of the untreated mice in 16-18 days. Each experimental group consisted of at least ten mice. Preliminary results obtained with the daunorubicin-immunoglobulin (bovine, non specific) conjugate indicated that the protein-bound drug retained, at least in part , its activity at higher doses. However, the reduction of potency is associated with an appreciable reduction of toxicity. (Table ill). Daunorubicin linked to the synthetic polypeptide, poly-l-lysine, showed a reduction of antitumour potency, as in the case of the daunorubicinimmunoglobulin conjugate.However, the daunorubicin-poly-l-lysine conjugate was as active (or more active) as the free drug at the maximal dose tested (16 mg/kg). Again the protein-bound drug was less toxic than free drug. The antitumour activity could not be due to the protein component of the conjugate, since the ummodified protein, at the same dose was completely inactive.
ACTIVITY of danorubicin-protein conjugates on Ehrlich ascites tumour
Table II
Compound Dosea MSTb
Daunorubicin (DA) 4 1 88 5 197
6 138
10.5 75 Da-immunoglobulinc 10.5 143
14 153
Immunoglobulind - 100 Da-poly-i-lysineC 1 2 1 66 16 222
Poly-l-lysined 100 aTreatment i.p. on day 1 (mg/kg of body weight) bMedian survival time expressed as percent of untreated control.
CDose is expressed as daunorubicin content in the conjugate dProteins were tested at the same dose as the daunorubicin-protein conjugate.
Claims (5)
1. A daunorubicin-protein conjugate in which the daunorubicin is bound through its 9-acetyl side-chain to the protein.
2. A daunorubicin-protein conjugate according to claim 1 in which the protein is casein, ribonuclease A, asialo fetuin, immunoglubulin, concamavlain A, fetuin, bovine serum albumin, lysoxyme, histone or poly-Iysine.
3. A process for the preparation of a daunorubicin-protein conjugate, the process comprising reacting 14-bromo-daunorubicin with an excess of a protein in aqueous-methanolic solution at ambient temperature for a period of from 2 to 4 hours while keeping the solution at neutrality by addition of dilute sodium hydroxide.
4. A process according to claim 3 further comprising removing any precipitate and purifying the supernatant liquid by passing it over an ion-exchange or adsorption resin, the former when negatively charged proteins are used and the latter when positively charged proteins are used.
5. A process for the preparation of a daunorubicin-protein conjugate, the process being substantially as described herein with reference to the Example.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8203523A GB2098219A (en) | 1981-05-08 | 1982-02-08 | Daunorubicin-protein conjugates |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8114167 | 1981-05-08 | ||
| GB8203523A GB2098219A (en) | 1981-05-08 | 1982-02-08 | Daunorubicin-protein conjugates |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| GB2098219A true GB2098219A (en) | 1982-11-17 |
Family
ID=26279386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8203523A Withdrawn GB2098219A (en) | 1981-05-08 | 1982-02-08 | Daunorubicin-protein conjugates |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2098219A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2148299A (en) * | 1983-09-01 | 1985-05-30 | Hybritech Inc | Antibody compositions of therapeutic agents having an extended serum half-life |
| EP0167761A1 (en) * | 1984-05-22 | 1986-01-15 | Ajinomoto Co., Inc. | Anthracycline compounds and anticancer agents |
| EP0392487A3 (en) * | 1989-04-13 | 1991-04-03 | Takeda Chemical Industries, Ltd. | Stabilized composition of anthracyclines |
| WO1992002255A1 (en) * | 1990-08-03 | 1992-02-20 | Farmitalia Carlo Erba S.R.L. | New linker for bioactive agents |
| USD515873S1 (en) * | 2004-08-27 | 2006-02-28 | Wilton Industries, Inc. | Dish |
| USD517372S1 (en) * | 2004-08-27 | 2006-03-21 | Wilton Industries, Inc. | Dish |
| USD524604S1 (en) * | 2004-08-27 | 2006-07-11 | Wilton Industries, Inc. | Dish |
-
1982
- 1982-02-08 GB GB8203523A patent/GB2098219A/en not_active Withdrawn
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2148299A (en) * | 1983-09-01 | 1985-05-30 | Hybritech Inc | Antibody compositions of therapeutic agents having an extended serum half-life |
| EP0167761A1 (en) * | 1984-05-22 | 1986-01-15 | Ajinomoto Co., Inc. | Anthracycline compounds and anticancer agents |
| EP0392487A3 (en) * | 1989-04-13 | 1991-04-03 | Takeda Chemical Industries, Ltd. | Stabilized composition of anthracyclines |
| WO1992002255A1 (en) * | 1990-08-03 | 1992-02-20 | Farmitalia Carlo Erba S.R.L. | New linker for bioactive agents |
| US5387578A (en) * | 1990-08-03 | 1995-02-07 | Farmitalia Carlo Erba S.R.L. | New linker for bioactive agents |
| US5547667A (en) * | 1990-08-03 | 1996-08-20 | Farmitalia Carlo Erba S.R.L. | Linker for bioactive agents |
| USD515873S1 (en) * | 2004-08-27 | 2006-02-28 | Wilton Industries, Inc. | Dish |
| USD517372S1 (en) * | 2004-08-27 | 2006-03-21 | Wilton Industries, Inc. | Dish |
| USD524604S1 (en) * | 2004-08-27 | 2006-07-11 | Wilton Industries, Inc. | Dish |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |