AU2003231664B2 - Hemoglobin-haptoglobin complexes - Google Patents

Description

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P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: "HEMOGLOBIN-HAPTOGLOBIN COMPLEXES" The following statement is a full description of this invention, including the best method of performing it known to me/us: TITLE: Hemoglobin-Haptoglobin Complexes FIELD OF THE INVENTION This invention relates to protein complexes and use thereof in medical applications. More specifically, it relates to complexes of hemoglobin compounds with therapeutic substances such as drugs, genes etc. which have a therapeutic action on specific parts and/or organs of the body, and means for targeting such complexes to specific body parts and body organs.

Also within the scope of the invention are complexes of hemoglobin with diagnostic substances, such as imaging agents.

BACKGROUND OF THE INVENTION The use of hemoglobin and modified hemoglobin as a drug delivery means has been proposed previously. Hemoglobin, as a natural component of red blood cells, present and circulating throughout the body in relatively large quantities, has well-established bioacceptability and the potential-to deliver drugs throughout the body.

Thus, Kluger et al., U.S. Pat. No. 5,399,671 describe a hemoglobin compound which has been cross-linked to effect intramolecular stabilization of the tetrameric structure thereof, but which contains a residual functional group on the cross-linker residue to which drugs for delivery can be covalently attached.

Anderson et al., U.S. Pat. No. 5,679,777, describe complexes of hemoglobin compounds and polypeptide drugs, in which the polypeptide drug is bound to a globin chain through a disulfide linkage to a cysteine unit inherent in or genetically engineered into the globin chain.

Haptoglobins (Hp) constitute part of the oX2 -globin family of serum glycoproteins. Haptoglobins are present in mammalian plasma, and constitute about one-quarter of the c2 -globulin fraction of human plasma. Each individual has one of three phenotypic forms of haptoglobin, of close structural and chemical identity. Haptoglobins are composed of multiple o3p dimers and the phenotypes are conventionally denoted Hp 1-1, Hp 2-1 and Hp 2-2. The P3 chains are identical in all haptoglobin phenotypes, but the a chains vary (ax 1 and a 2 The amino acid sequences of all chains are known. Hp 1-1 is -2composed of two ol3 dimers and has a molecular weight of about 98 kDa.

The structure of Hp 2-1 and Hp 2-2 can be written as follows: (a1P) 2

(C

2 13), where n=0,1,2 and (c 2 P)m where m=3,4,5, respectively.

Delivery of drugs to a patient suffering from a disease or disorder affecting primarily one body part or one body organ is best accomplished by choosing a delivery method which targets the part or organ in need of treatment with a high degree of specificity. Such a delivery system makes most effective use of the active drug, so as to reduce the necessary dosage level, and reduces side effects of the drug.

One function of haptoglobin is to bind extracellular hemoglobin, arising from red blood cell lysis, to form essentially irreversible haptoglobinhemoglobin complexes that are recognized by specific receptors on hepatocytes in the liver. In this way, hemoglobin is targeted to the liver for metabolism.

Control and manipulation of genes and gene products are potentially powerful means of treating various diseases and genetic disorders. When specifically introduced into the cells, genes can use the host cell biosynthetic machinery for the expression of the therapeutic biomolecules they encode.

For successful gene therapy, one must devise a successful method of in vivo gene delivery. One such technique developed in recent years is receptormediated delivery. This has the advantage of high specificity of delivery to the cells which express the targeted receptor.

The specific targeting of low molecular weight therapeutic and diagnostic agents to tissues is enhanced greatly through the use of receptormediated delivery. Diagnostic agents such as fluorescent or radiolabeled substances indicate the location and quantity of cells bearing the targeted receptors when such agents are administered as complexes with ligands for those receptors. These complexes are also useful in characterizing the binding and transport properties of receptors on cells in culture. Such information is useful in detection of and/or design of therapy for tissues containing the cells being recognized, either in vitro or in vivo.

SUMMARY OF THE INVENTION In one aspect the present invention provides a means and composition for specifically targeting hepatocytes or other cells having receptors for hemoglobin-haptoglobin complexes with therapeutically active substances or diagnostic agents.

In another aspect the present invention provides a novel complex of a substance selected to exert a beneficial effect on a mammalian patient's liver, in vivo, and a substance that specifically targets hepatic cells.

The present invention describes haptoglobin-hemoglobin constructcomplexes to which hepatocyte-modifying agents are attached. Such haptoglobin-hemoglobin construct-complexes serve as effective hepatocytetargeting vehicles for the attached agents, for delivery of specific hepatocytemodifying agents (drugs, diagnostics, imaging compounds, etc) to the liver, and to other cells having the appropriate hemoglobin-haptoglobin receptors.

The expression "construct-complex" is used herein to refer to the combination of haptoglobin with hemoglobin to which a bioactive, therapeutic or diagnostic agent is attached. The present invention provides constructcomplexes composed of a hemoglobin compound, a haptoglobin and a hepatocyte-modifying substance of interest such as a drug, a diagnostic agent or a gene. In one aspect of the present invention, the construct-complex is prepared extracorporeally and then administered to the patient. In another aspect, a complex of hemoglobin-hepatocyte modifying substance is prepared extracorporeally, administered to the patient, and forms the construct-complex of haptoglobin-hemoglobin-hepatocyte modifying substance with haptoglobin that is naturally present in the patient's serum. In a further aspect, the patient's haptoglobin level may be supplemented by haptoglobin administration, a known procedure, either before, during or after administration of the hemoglobin-hepatocyte modifying substance-constructcomplex. In any case, the construct-complex specifically targets and binds as a ligand to the hepatocyte receptors, owing to the presence of the haptoglobin and hemoglobin portions of the construct-complex.

The construct-complexes of the present invention, formed ex vivo or in vivo, target any cells having receptors for Hb-Hp complexes, and this includes metastases arising from primary hepatoma. It is normally difficult to identify and treat metastases because of the systemic distribution and small size of such cancers. Secondary hepatic metastases, i.e. hepatoma cells outside the liver which have such receptors are targeted by the construct-complexes of the present invention, as well as cells of the liver, and should be regarded as "hepatocytes" as the term is used herein.

Further, the construct-complexes of the present invention may exert beneficial effects on neighboring cells, if the hepatocyte modifying substance is, for example, a drug which is active towards neighboring cells even if they are not hepatocytes. They may also modulate or initiate the activity of other therapeutic or diagnostic agents delivered by other methods for hepatocyte modification, such as prodrugs, enzymes or genes coding for enzymes and requiring activation to cause an effect. Agents effecting such action resulting in hepatocyte modification or effect on other agents or cells are hepatocyte modifying agents according to this invention.

The construct-complex according to the present invention can be generally represented by the formula: (Hp)a (Hb)b (Lc -Ad)e where a=1 to about to about c=0 to about d=1 to about e=1 to about Hp is haptoglobin as described herein; Hb is a hemoglobin as described herein; in one embodiment, it is a nonintramolecularly cross-linked Hb; L is a linker as described herein; and A is a hepatocyte modifying agent as described herein, in which the stoichiometry of Hp to Hb in the complex is dictated by the available number of binding sites on the two proteins, but is generally of the order of 1:05 to 1:2.

In one form the invention provides a hemoglobin construct which binds to haptoglobin comprising a non-intramolecularly cross-linked hemoglobin and an anti-viral substance bound to the hemoglobin.

In another form the invention provides a hemoglobin construct which binds to haptoglobin comprising a non-intramolecularly cross-linked hemoglobin and an anti-neoplastic substance bound to the hemoglobin.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings in which: FIG. 1 is a reaction scheme illustrating diagrammatically a process for producing one embodiment of a construct-complex of the present invention; FiG. 2. Panels A, B, C, and D are size exclusion chromatography results, in the form of plots of absorbance at 280 nm and 414 nm against elution time, indicating the molecular weight distribution of the four products of Example 2. Complexes were formed using poly(L-lysine) of molecular weight 4 kDa, 7.5 kDa, 26 kDa and 37 kDa.

FIG. 3 is a similar plot, for the product complex utilizing 26 kDa poly(Llysine) after 24 hours incubation with haptoglobin, produced in Example 2; FIG. 4 represents are depictions of gel mobility shift assays of DNA in the presence of THb and THb-poly(L-lysine) produced according to Example 4; FIG. 5 is a dye fluorescence assay of the products of Example 4; -6- FIG. 6 is a depiction of the gel mobility shift assay of the products of Example 4; FIG. 7 is a fluorescence assay of another product of Example 4; FIG. 8 is a size exclusion chromatogram of the product of Example 6; FIG. 9. Panels A, C and D show size exclusion chromatograms of the products of Example 9, utilizing haptoglobin 1-1, haptoglobin 2-1, (D) haptoglobin-2-2; Panel B shows the UV-visible spectra of the products of Example 9; FIG. 10 is a size exclusion chromatogram of the product of Example 11; FIG. 11 is a size exclusion chromatogram of the product of Example 13; FIG. 12 shows anion exchange chromatograms (overlaid) of products and starting materials of Example 14; FIG. 13 shows overlaid size exclusion chromatograms of the products of Example FIG. 14 shows size exclusion chromatography elution profiles with detection at 280 (solid lines) and 414 nm (broken lines) for products of Example 16; haptoglobin 1-1, 64 kDa ORHb, haptoglobin-[64-kDa ORHb], >64 kDa ORHb, haptoglobin-[>64 kDa ORHb].

FIG. 15 shows size exclusion chromatograms of products of Example 17; FIG. 16. Panels A-D are graphical presentations of analyses of results obtained in Example 18; FIG. 17 is a graphical presentation of further analyses of results obtained according to Example 18.

FIG. 18 Anion-exchange HPLC of Hb-ribavirin conjugate using a pH gradient (pH 8.5 6.5) under non-denaturing conditions.

FIG. 19 is a MALDI-TOF mass spectrometry of Hb-ribavirin conjugate.

The increase in MW of each of the a and p globin chains by multiples of the ribavirin phosphate mass (308 Da) confirms addition of multiple drugs per globin chain.

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FIG. 20 illustrates biological activity of ribavirin from Hb-ribavirin conjugate. Human HepG2 and mouse AM12 hepatic cells were treated with ribavirin and ribavirin cleaved and purified from Hb-ribavirin conjugate The cells were assayed for uptake of bromodeoxyuridine (BrdU) as a measure of cell proliferation. Inhibition of cell proliferation is represented as inhibition of control untreated cells.

FIG. 21 illustrates in vitro uptake of Hb-ribavirin conjugate by hepatic and non-hepatic cells. Human hepatoma HepG2, mouse AM12 hepatocyte, and 5637 bladder carcinoma cells were assayed for internalization of fluorescein-labeled Hb and Hb-ribavirin conjugate at 37°C for 2 hr. Results are expressed as fold increase in relative fluorescence units (RFU) over equivalent samples not treated at 37°C.

FIG. 22 HPLC illustrating the conversion of Ara-C to the imidazolide derivative.

FIG. 23 Anion exchange chromatography illustrating the formation of Hb-Ara-A and Hb-Ara-C conjugates.

FIG. 24 Size exclusion chromatography illustrating Hp binding of Hb- Ara-A and Hb-Ara-C -32 kDa Hb species (Hb-drug conjugates) elute at approximately 36 minutes. Polymers of these elute as an earlier shoulder to this peak. Hp complexes elute in the 20-25 minute range.

FIG. 25 C4 RP HPLC of Hb-Dox conjugate. The Dox-modified beta globin chain observed at 220 nm (top) corresponds to the major fluorescent species (bottom), indicating conjugation of Dox (fluorescent) to Hb.

Fluorescent species eluting before 20 minutes are low MW Dox species, removed later by dialysis.

FIG. 26 is a mean fluorescence Intensity versus time graph illustrating the in vitro uptake of Hb-Dox conjugate. WT cells do not express the Hb-Hp receptor, while nonWT cells do. Uptake is significant only in cells bearing the Hb-Hp receptor, and only for complexes with Hp.

FIG. 27 is a bar graph illustrating Hp-HbDox toxicity on SUDHL cells.

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-8- DETAILED DESCRIPTION OF THE INVENTION A wide range of hepatocyte modifying substances may be used in complexes of the present invention. These can be therapeutic agents, diagnostic agents, markers or the like capable of interacting with hepatocytes and consequently capable of acting in vivo at the liver. They can be designed for treatment of normal liver cells or such cells undergoing metastases. Thus, the hepatocyte-modifying substances can be antineoplastic substances (doxorubicin, daunorubicin, ricin, diphtheria toxin, diphtheria toxin A, for example), antiviral substances (ara-AMP, trifluorothymidine, interferon, antisense oligonucleotides, ribavirin, cytarabin (ara-C), ara-CMP, acyclovir, didonosine, vidarabine (ara-A), adefovir, zalcitabine, lamivudine, fialvridine, and other nucleoside analogs, for example), anti-inflammatory substances, anti-parasitic substances, antimicrobial substances, antioxidant substances, hepatoprotective agents, imaging and diagnostic agents, nucleic acids and their compounds for effecting gene therapy, agents effecting lipid metabolism, anti-toxicants, proteins, enzymes, enzyme and prodrug combinations, and the like).

Examples of diagnostic agents useful in construct-complexes in this invention include radiolabeled lysine and putrescine, and the fluorescent compounds monodansyl cadaverine and fluorescein. Low molecular weight therapeutic agents can also be selectively targeted to the cells to minimize side effects at non-targeted tissues and vascular clearance. Examples of therapeutic agents in this application include putrescine, a modulator of cell growth and activity, and primaquine, an anti-malarial substance.

More specifically, hepatocyte modifying substances which can be used in construct-complexes according to the present invention include agents for treating or preventing hepatic fibrosis, a dynamic process from chronic liver damage to cirrhosis, and for treating or preventing other chronic liver disorders including viral hepatitis and alcoholic and cryptogenic liver diseases.

These hepatocyte modifying substances include cytoprotective drugs such as S-adenosyl-L-methioine, prostaglandin E1,E2,12 and their analogues, colchicine and silymarin, all of which have been demonstrated to be effective -9in protecting the liver from damage and having anti-fibrotic properties. Other liver protectant substances which are hepatocyte modifying substances within the scope of this invention include free radical scavengers/anti-peroxidants such as glutathione, SA 3443 (a cyclic disulphide), S-adenosylmethionine, superoxide dismutase, catalase, a-tocopherol, vitamin C, deferoxamine, (+)cianidanol-3, mannitol, tryptophan, pantetheine, pantotheinic acid, cystamine, cysteine, acetylcysteine, folinic acid, uridine monophosphate, zinc sulphate, schizandrin B and kopsinine; lipoxygenase inhibitors such as the aforementioned prostaglandins and their analogs dimethyl PGE, misoprostol and enisoprost, and prostacyclin PGI2 and its analog iloprost; calcium channel blockers such as trifluoroperazine, verapamil, nifedipine and related dihydropyridine compounds, and dilitiazem; proteinase inhibitors; atrial natriuretic peptide; a2 -macrofetoprotein;synthetic linear terpenoid; putrescine; cholestyramine; .epsilon.-aminocaproic acid,; phenylmethylsulfonyl fluoride; pepstatin; glycyrrhizin; fructose 1,6-biphosphate; and ursodeoxycholic acid.

The hemoglobin compound useful as a component of the complexes of the present invention can be substantially any hemoglobin compound providing the necessary degree of biocompatibility for administration to a patient or animal, the necessary sites for attachment of the hepatocyte modifying substance of interest, and having sufficient binding affinity for haptoglobin. Within these limitations, it can be a naturally occurring hemoglobin from human or animal sources. It can be a modified natural hemoglobin, e.g. an intramolecularly cross-linked form of hemoglobin to minimize its dissociation into dimers, an oligomerized form or a polymerized form. It can be a hemoglobin derived from recombinant sources and techniques, with its naturally occurring globin chains or such chains mutated in minor ways. It can be comprised of subunits or fragments of Hb, or derivatives thereof, which have affinity for haptoglobin. It can be a hemoglobin in which individual amino acids of the globin chains have been removed or replaced by site specific mutagenesis or other means. Certain modifications which are known to decrease the affinity of hemoglobin for binding to haptoglobin are preferably avoided in hemoglobin compounds used in the present invention.

One type of preferred hemoglobin compounds are those which comprise hemoglobin tetramers intramolecularly cross-linked to prevent their dissociation into dimers, and which leave functional groups available for chemical reaction with the hepatocyte modifying substance, either directly or through a chemical linker molecule. Such hemoglobin compounds have the advantage that they provide a known, controlled number of reactive sites specific for the therapeutic substance of interest, so that an accurately controlled quantity of the therapeutic substance can be attached to a given amount of hemoglobin compound. They also have the added advantage that they avoid utilizing sites on the globin chains for linkage to the therapeutically active substance, so as to minimize conformation disruption of the globin chains and minimize interference with the hemoglobin-haptoglobin binding and with binding of the construct-complex to the receptor protein on a hepatocyte cell.

Human hemoglobin, e.g. that obtained from outdated red blood cells, and purified by the displacement chromatography process described in U.S.

Pat. No. 5,439,591 Pliura et al. is one preferred raw material for preparation of the hemoglobin product for use in the complex of the present invention. This material may be cross-linked with a trifunctional cross-linking agent as described in aforementioned U.S. Pat. No. 5,399,671, Kluger et al., namely a reagent which utilizes two of its functional groups for intramolecular crosslinking between subunits of the hemoglobin tetramer, and leaves its third functional group available for subsequent reaction with a nucleophile. A specific example of such a cross-linking reagent is trimesoyl dibromosalicylate), TTDS, the chemical formula of which is given in the attached FIG. 1, and the preparation of which is described in the aforementioned Kluger et al. U.S. Pat. No. 5,399,671.

When cross-linked hemoglobin, i.e. stabilized tetrameric hemoglobin is used as a component of the complex, the hepatocyte modifying substance is bound to the hemoglobin, either directly or through a chemical linker or -11 spacer, and then this complex may be administered to the patient so that the haptoglobin-hemoglobin binding takes place in vivo. The entire constructcomplex, (haptoglobin-hemoglobin-hepatocyte modifying substance) can, if desired, be formed extracorporeally and then administered to the patient, and this can under some circumstances lead to better control of the amounts of active substance finally being delivered to the hepatocytes. However, such a procedure is not normally necessary, save for those exceptional patients having zero or low levels of haptoglobin, e.g. in conditions of acute hemolysis.

Such patients can be administered haptoglobin before, during and/or after administration of the construct-complex of the invention. Usually, however, there is sufficient haptoglobin in the patient's plasma to form the constructcomplex in situ and effect its delivery to the hepatocytes. Preparation of the two-part complex and administration of that to the patient, to form the threepart complex in situ is generally cheaper and less complicated.

Use of intramolecularly crosslinked hemoglobins will give rise to high molecular weight polymers containing more than one hemoglobin and/or haptoglobin owing to the presence of two binding sites on each of these proteins. There may be advantages to using non-crosslinked hemoglobin as a component of the construct-complexes of the present invention. Such a hemoglobin, with a hepatocyte-modifying substance bound to it, will dissociate into dimeric hemoglobin of approximate molecular weight 32 kDa, and two such dissociated dimeric hemoglobin products bind to a single molecule of haptoglobin to give a complex according to the present invention. The formation of high molecular weight haptoglobin-hemoglobin complexes is thus avoided. Haptoglobin binding to ap-dimers is generally a much faster reaction than haptoglobin binding to crosslinked hemoglobin. The lower molecular weight complexes resulting from the use of non-crosslinked hemoglobin may show improved hepatocyte receptor binding and uptake.

Where hemoglobin of a form which will dissociate into dimers is used as a component of the present invention, or where hemoglobin dimers themselves are used, for example, where the dimers have been modified such that they cannot reform 64 kDa hemoglobin, it is preferred to form the -12 construct-complex according to the invention extracorporeally, and then to administer the finished construct-complex to the patient, so as to avoid the risks attendant on administering to the body a molecular species of too small a molecular weight, namely, clearing the drug too rapidly through excretion.

Administration of Hb dimers bearing therapeutic or diagnostic agents may be possible without prior binding to haptoglobin in cases where complex formation in vivo is adequate prior to clearance of the modified dimer.

A further example of a hemoglobin compound useful in constructcomplexes of the present invention is dimeric hemoglobin bearing a modifying group containing thiol, preferably a terminal side chain thiol, of the type described in U.S. Provisional Patent Application of Kluger and Li, entitled "Hemoglobin With Chemically Introduced Disulfide Crosslinks and Preparation Thereof", filed Nov. 3, 1997. Hepatocyte modifying substances can be ligated to such dimeric hemoglobin, either by direct reaction with the exposed thiol, or by direct reaction with an activated form of the thiol, or by mixed disulfide formation, or through a linker molecule. Construct-complexes of this type are made extracorporeally and administered to a patient in this form. The hemoglobin-hepatocyte modifying substance conjugate can also be administered for in vivo Hp binding. The use of dissociable hemoglobin (32 kDa molecular weight) has the advantage over the use of cross-linked hemoglobin tetramers in that they provide an exposed dimer-dimer interface which facilitates haptoglobin binding.

The construct-complexes of the present invention may also utilize hemoglobin which has been modified in a manner which results in impaired nitric oxide binding. Such modified hemoglobins are known in the art.

Reduced NO binding may reduce the tendency of the hemoglobin to effect modifications to a patient's blood pressure upon administration, an effect which has been noted with some hemoglobins, even in small amounts.

In forming the construct-complex, it may be necessary to interpose between the reactive site on the hemoglobin chosen and the hepatocyte modifying substance, a chemical linker or a spacer group. This depends upon the nature of the available chemical group on hemoglobin for linking, and on -13 the chemical groups available on the hepatocyte modifying compound, for this purpose. For example, a polycationic segment such as polylysine is appropriately attached to the electrophilic site of the TTDS modified hemoglobin to provide a binding site for DNA through electrostatic interactions. Linear polymers of lysine provide appropriate cationic segments for this purpose.

A construct-complex according to a preferred embodiment of the present invention comprises a haptoglobin molecule, which may be haptoglobin 1-1 or any other phenotype, bonded to one or more molecules of a hemoglobin compound by means of strong non-covalent interaction. The hemoglobin may be cross-linked, oligomerized or unmodified, as described above.

FIG. 1 diagrammatically illustrates the chemical steps involved in preparing a cross-linked hemoglobin, for reaction with a linker and/or agent, and with haptoglobin to form a construct-complex according to various embodiments of the invention. TTDS is reacted with hemoglobin, whereupon two of the three 3,5-dibromosalicylate groups leave. Primary amine groups at Lys-82 and P-Lys-82 on the hemoglobin are bonded by an amide linkage to the cross-linker, forming an intramolecularly cross-linked and stabilized tetrameric hemoglobin with the third dibromosalicylate group intact and available for further reaction. In the second step, the cross-linked hemoglobin is reacted with the agent or a linker (in the case of Example 1, polylysine) necessary for later attachment of the agent. In other cases, the hepatocyte modifying substance, or active agent, takes the place of the polylysine in the scheme of FIG. 1, to form the construct. The complex is then ready for administration to the patient to form a construct-complex in situ, or alternatively haptoglobin can be reacted with the complex so formed extracorporeally, so that the haptoglobin binds to the hemoglobin portion of the complex to form the three part complex ready for administration to the patient. Alternatively, the TTDS-modified hemoglobin with a linker attached can be reacted with haptoglobin and agent attached as a final step. After administration, the construct-complex will bind to the hepatocytes, where the

L

-14haptoglobin-hemoglobin mediates binding to the selective receptors thereof and allows the hepatocyte-modifying substance to be delivered to and enter into the hepatocyte utilizing the hepatocyte receptors selective for haptoglobin-hemoglobin complex.

The following non-limiting examples are illustrative of the present invention:

EXAMPLES

Example 1 Conjugation of TTDS Cross-linked Hemoglobin (THb) to Poly(Llvsine) Poly(L-lysine) conjugates of TTDS cross-linked hemoglobin (THb-Kn) were synthesized by adding poly(L-lysine) to THb-DBS (TTDS cross-linked hemoglobin with one unhydrolyzed 3,5-dibromosalicylate functionality) at 1:1 molar ratio to promote formation of conjugates in which only one molecule of hemoglobin is attached to a single poly(L-lysine) chain. The poly(L-lysine) used in this experiment is a linear polymer with an amide linkage between the carboxyl group and the a.-amino group of lysine. Polymers with an average molecular weight of 4 kDa (K4kDa), 7.5 kDa (K75kDa), 26 kDa (K26koa) and 37 kDa (K37kDa) were conjugated to THb.

TTDS (13.9 mg) in ethanol (100 ILL) was added to deoxyhemoglobin mL, 8.5 g/dL) in 50 mM borate pH 9.0. The reaction mixture was stirred at OC. under nitrogen for 45 min. The hemoglobin was then charged with CO (the solution was kept on ice) and the excess of the cross-linking reagent was removed by passing the hemoglobin solution through a Sephadex column (200 mm L.times.25 mm D) equilibrated with 50 mM borate pH The resulting hemoglobin solution (3.6 g/dL) was again charged with CO.

Poly(L-lysine) solutions were prepared in 50 mM borate pH 8.0 and added to hemoglobin (3.6 g/dL, 1.9 mL) as indicated in Table 1 below. The molar ratio of poly(L-lysine) to hemoglobin was 1:1 for all four polymers. The THb-poly(Llysine) conjugates (THb-Kn) were sealed in serum bottles, recharged with CO and left at room temperature for two days. Hemoglobin concentrations in these samples were determined using Drabkin's reagent.

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TABLE 1 Poly(L-lysine) Amount of poly(L-lysine) added to THb (mg) K4 kDa 4.2 K75 kDa

K

2 6 kDa 27.5

K

37 kDa 39.3 Anion Exchange Chromatography: Crude THb-K n complexes were analyzed using anion exchange chromatography on a SynChropak AX-300 column (250 mm L.times.4.6 mm D, SynChrom, Inc.). A sodium chloride gradient was used to elute various modified hemoglobins. The effluent was monitored at 280 nm.

By the time of analysis all unreacted THb-DBS had hydrolyzed to give THb. The reaction resulted in a mixture of products all of which, as expected, migrated before the THb on the anion exchange chromatography media. The yields were calculated by adding the peak areas of the early eluting peaks and comparing them to the total peak area. Yields of poly(L-lysine) modified hemoglobin calculated in this way were: 37, 37, 81 and 84% for K.4kDa, K.7.5kDa, K.26kDa and K.37kDa, respectively.

Purification of THb-K.n conjugates: THb-K., conjugates were separated from unconjugated THb by anion exchange chromatography on a POROS column (52 mm L, 14 mm D) equilibrated with 25 mM Tris-HCI buffer pH 8.4. Modified Hbs were eluted with a sodium chloride gradient. The effluent was monitored at 280 nm and pooled fractions containing THb-K.n conjugates were concentrated using an AmiconTM diafiltration device and a kDa cutoff membrane.

Size Exclusion Chromatography: The molecular weight distribution of purified THb-K..n conjugates and their haptoglobin complexes was determined using size exclusion chromatography (SEC) on a SuperdexTM-200 column (300 mm L.times.10 mm D, Pharmacia) equilibrated and eluted with 0.5 M magnesium chloride containing 25 mM Tris-HCI pH 7.2 at a flow rate of 0.4

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-16mL/min. The effluent was monitored at 280 nm and 414 nm. Hemoglobin to poly(L-lysine) stoichiometry ranged from 1:1, using 4 kDa poly(L-lysine), to heterogeneous constructs with stoichiometries up to 4:1 using the higher molecular weight poly(L-lysine) linkers, according to corresponding elution times with molecular weight standards. No unmodified THb was present.

These constructs were stable under the high salt conditions of chromatography.

Example 2 Complex Formation Between THb-Kn and Haptoglobin 1-1 The following stock solutions were used for the preparation of the complexes: 1.74 mg/mL haptoglobin 1-1 (Hp) in water and 1.0 mg/mL solutions of the THb-Kn (all THb-Kn concentrations represent hemoglobin concentrations) in 50 mM sodium borate pH 9.0. Haptoglobin (14 uL) was added to THb-Kn in potassium phosphate pH 7.0 to give the following final concentrations: 0.12 mg/mL (1.22 uM) haptoglobin and 0.19 mg/mL (2.9 pM) THb-Kn in 25 mM potassium phosphate pH 7.0 (200 p.L final volume). After incubation for 180 min. at room temperature, the samples were analyzed using SEC.

THb-K.n complexes with haptoglobin 1-1: The formation of THb-Kn complexes with haptoglobin can be followed using size exclusion chromatography (SEC). FIG. 2A shows the composition of the THb-K 4 kDa mixture with Hp after incubation at room temperature for 180 min. A new, high molecular weight peak appears at 25.5 min. Plots of the ratio of absorbance at 280 and 414 nm (A 280

/A.

414 over the elution period indicate the relative proportions of haptoglobin and hemoglobin in the construct-complexes and other peaks. The absorbance ratio (A 280

/A.

41 4 throughout the new peak is 0.9 indicating that both haptoglobin and hemoglobin components are present in this complex. Haptoglobin 1-1 migrates at 29.7 min. and is easily identified by high A 280

/A

414 ratio. FIG. 2B shows SEC of the THb-K7.5kDa mixture with Hp after incubation at room temperature for 180 min. Again, a new peak appears at 25.1 min. with a A 28 0

/A

41 4 ratio of 0.73, followed by haptoglobin at 29.7 min.

and THb-K 7 5 kDa at 35.8 min. with A 280

/A

414 ratio of 0.3. The analysis of the -17- SEC of THb-K 2 6kD and THb-K 37 kDa complexes with haptoglobin is more complicated due to their broad molecular weight distribution. The results are presented in FIGS. 2D and 2D respectively. It is evident from FIGS. 2C and 2D that both THb-K26kDa and THb-K 37 kDa form complexes with haptoglobin. The

A

280 /A414 ratio is 0.64 for THb-K26kDa -Hp and 0.69 for THb-K 37 kDa -Hp.

Degree of THb-K 2 6 kDa -Hp complex formation: To determine whether all structurally different components of the THb-Kn bind to haptoglobin, THb- K26kDa was incubated with a 15% excess of haptoglobin for various lengths of time and then analyzed using SEC. The following stock solutions were used for the preparation of the complex: 1.74 mg/mL haptoglobin 1-1 in water and 7.4 mg/mL solutions of THb-K26kDa in potassium phosphate pH 7.0 to give the following final concentrations: 0.74 mg/mL (7.5 mM) haptoglobin and 0.41 mg/mL (6.4 mM) THb-K 26 kDa (1.2:1 molar ratio of Hp to Hb) in 25 mM potassium phosphate pH 7.0. After incubation at room temperature for various lengths of time, the mixtures were analyzed using SEC. The progress of the reaction was followed by monitoring the disappearance of haptoglobin peak on a SEC profile. 85% of the THb-K26kDa was bound by haptoglobin after 24 hours. The resulting THb-K26kDa -Hp complex has a broad molecular weight distribution ranging from 370 kDa to app. 1000 kDa (FIG. 3).

Example 3 DNA Binding to THb-K., and THb-K.n -Hp. Gel Mobility Shift Assay Gel mobility shift assays were conducted to evaluate the stoichiometry of binding of plasmid DNA (pCMVbeta) to the THb-K.n conjugates. This gel electrophoretic method is based on the observation that the migratory properties of the DNA are altered upon binding protein. Neither proteins nor DNA-protein complexes in which protein constitutes a significant part of their mass enter 1% agarose gels. If mixtures with an increasing THb-Kn to DNA ratio are analyzed, it is observed that the DNA band disappears at and above the ratio that corresponds to the stoichiometry of the complex. For each of the four conjugates and for the THb-K26kDa -Hp complex, solutions containing from 0.4 to 6400 ng of the conjugate (this weight based on the hemoglobin -18component) in 32 pL of 20 mM HEPES pH 7.3 containing 150 mM NaCI were prepared. The plasmid DNA (560 ng in 28 pL of 20 mM HEPES pH 7.3 containing 150 mM NaCI) was added drop wise to each sample and the mixtures were incubated for 1 hour at room temperature. The samples (15 pL) were analyzed on a 1% agarose gel containing ethidium bromide (0.2 pg/mL).

The amount of conjugate which prevented DNA entry into the gel was determined. Results are described in the following Example.

Example 4 DNA Binding to THb-K26kDa and THb-K 26 kDa -Hp Complex: Thiazole Orange Fluorescence Quenching Method This dye fluorescence assay is based on the observation that a DNA intercalating dye (thiazole orange)is fluorescent only if bound to DNA.

Complex formation between THb-Kn and DNA causes the displacement of the intercalating dye from DNA and the decrease of total fluorescence.

The following stock solutions were used in this experiment: 0.05 mg/mL DNA (pCMVbeta), 0.010 mg/mL, THb-K 26 kDa or THb-K26kDa -Hp complex, 1.75.times.10 6 M thiazole orange (0.1 mg/mL solution in 1% methanol was diluted 190 times with water), 20 mM HEPES pH 7.3 containing 0.15 M NaCl.

Plasmid DNA (10 pL), THb-K 26 kDa (volumes varying from 2.5 to 60 IL) and buffer (to the final volume of 200 piL) were mixed in a generic 96 well plate and incubated for 2.5 hours at room temperature. Sample containing thiazole orange in HEPES buffer was also prepared and used as a background control. Fluorescence was measured on a Packard FluoreCount. plate reader using excitation at 485 nm and emission at 530 nm. The THb-K 26 kDa Hp complex was prepared as described above and used without purification.

It was diluted with 20 mM HEPES pH 7.3 containing 0.15 M NaCI to give a final concentration of 0.010 mg Hb/mL.

The gel mobility shift assay and the fluorescence quench assay both demonstrated that THb-Kn binds to DNA. FIG. 4A (left) and 4B (right) are depictions of gel mobility shift assays of haptoglobin-hemoglobin-DNA conjugates produced according to Example 4. One hundred and forty ng of DNA were added to increasing amounts of THb or THb-K26kDa. Lane 1 -19of both gets contain DNA molecular weight markers. Hb content in other lanes: (A2) 50 ng, (A3) 100 ng, (A4) 200 ng, (A5) 400 ng, (A6) 800 ng, (A7) 1600 ng, (A8) empty, (B2) 25 ng, (B3) 50 ng, (B4) 100 ng, (B5) 200 ng, (B6) 400 ng, (B7) 800 ng, (B8) DNA only. As regards the gel mobility shift assay, increasing the proportion of THb-Kn in the DNA samples affected DNA migration as seen in FIG. 4. FIG. 4A shows the migratory properties of DNA after incubation with increasing amount of THb ranging from 50 to 1600 ng of protein. In this concentration range THb does not bind DNA, since no change in DNA migration can be detected. THb-K26kDa is most effective at binding DNA. One hundred ng of THb-K 26 kDa (THb-K 26 kDa to DNA ratio=0.7, w/w) completely prevents the DNA from entering the agarose gel (FIG. 4B).

Approximately 400 ng of the other THb-Kn preparations were required to bind all DNA. The results for THb-K 2 6kDa are in good agreement with the fluorescence quench assay which indicated 86% of fluorescence decrease at the same THb-K26kDa to DNA ratio. FIG. 5 shows the effect of THb-K 26 kDa on DNA-thiazole orange fluorescence. On FIG. 5, the amount of THb- K.sub.26kDa is based on the hemoglobin component only.

The THb-K26kDa -Hp complex also binds DNA. It was found that 200 ng of THb-K 26 kDa -Hp completely prevented 140 ng of DNA from entering the agarose gel (THb-K26kDa -Hp to DNA ratio=1.4, FIG. 6 shows THb- K26kDa -Hp binding to DNA by gel mobility shift assay. One hundred and forty ng of DNA were added to increasing amounts of THb-K 2 6kDa -Hp: 25 ng (lane 50 ng 100 ng 200 ng 400 ng 800 ng weights based on the hemoglobin component. Molecular weight standards were loaded in lane 1 and 140 ng of DNA in lane 8. At the same THb-K 2 6 kDa -Hp to DNA ratio (1.4:1 w/w) the fluorescence assay indicates only 42% of fluorescence decrease and 81% fluorescence decrease at 2.8 ratio. The fluorescence assay is shown in FIG. 7 (the weight of the conjugate is based on the hemoglobin component thereof only). Comparison of the gel mobility shift assays for THb-K26kDa -Hp indicates that approximately twice as much protein-bound poly(L-lysine) is required to prevent DNA from migrating into the gel when the haptoglobin complex is used. Since the amount of hemoglobin conjugated poly(L-lysine) was identical in both experiments, the decreased DNA binding ability of THb- K26kDa -Hp is probably due to steric crowding in the THb-K 26 kDa -Hp-DNA complex.

In these examples, there has been synthesized and characterized a construct having all the necessary components for in vivo targeted gene delivery to human hepatocytes through haptoglobin receptors. Poly(L-lysine) was conjugated to the TTDS cross-linked hemoglobin to provide a site for binding DNA through electrostatic interactions of its positively charged .epsilon.-amine groups with the negative charges of phosphate groups on DNA. It has been previously demonstrated that when more than 90% of DNA's negative charges are neutralized, the linear DNA strand is compacted into a toroid structure, a form which is more stable and more amenable to internalization by cells. Optimal gene expression has been reported for the DNA to poly(L-lysine) ratios which result in electroneutral complexes.

The gel mobility shift and the fluorescence assays have demonstrated that THb-K 26 kDa -Hp complex binds the plasmid DNA thus completing the assembly of a construct potentially capable of delivering oligonucleotides by haptoglobin receptor-mediated endocytosis.

Example 5 Synthesis of Crosslinked Hemoglobin Bearing Tritiated or Nontritiated Lysine A solution of L-[ 3 H]-lysine was evaporated under a stream of nitrogen to obtain 59.5 nmole (5 mCi) of solid material. 59.5 nmole of non-radiolabeled L-lysine was prepared in a similar manner. TTDS (39.8 mg) was dissolved in ethanol (270 imL) and 200iL of this solution was added to deoxyhemoglobin mL, 9.2 g/dL) in 50 mM borate pH 9.0. The reaction mixture was stirred at room temperature under nitrogen for one hour, then oxygenated. Excess cross-linker was removed from half of the mixture by gel filtration and then the solution was CO charged and frozen, giving cross-linked Hb with an activated ester on the crosslinker (THb-DBS, 62 mg/mL) as described by Kluger (U.S.

Pat. No. 5,399,671). Unreacted crosslinker was removed from the other half of the crude reaction mixture by gel filtration using 0.1 M L-lysine/L-lysine -21 hydrochloride elution buffer (pH The eluate was CO charged and left at room temperature overnight. Using this process, lysine became conjugated to the linker via the activated ester, giving THb-Lys. Freshly thawed THb-DBS (29.5 nmole, 30.5 L) was added to the radiolabeled and the non-radiolabeled lysines each day for three days. THb-Lys (700 iL) was then added to both mixtures and the products desalted. Completion of the reaction was confirmed by anion exchange chromatography.

Example 6 Haptoglobin-THb-Lvs Complex Haptoglobin (1.61 mg/mL haptoglobin 1-1 in water, 11 p.L) was added to THb-Lys (38 mg/mL in 50 mM sodium borate pH 9.0) to give the following final concentrations: 0.68 mg/mL (6.9 pM) haptoglobin and 0.41 mg/mL (6.4 pM) THb-Lys were made up to a final 200 p.L volume at 25 mM potassium phosphate pH 7.0. Within 18 hours, the haptoglobin-THb-Lys complex was observed by SEC as a high molecular weight species, with absorption at 280 and 414 nm, eluting separately from native haptoglobin and the original THb- Lys product (FIG. The construct-complex was purified by SEC. The column was equilibrated and eluted with phosphate-buffered saline (PBS).

Example 7 Haptoglobin-THb-[ 3 HI-Lys Complex THb-[ 3 H]-Lys (75 p.L, 41 mg/mL, 0.657 Ci/mmole) was added to a solution of partially purified haptoglobin 1-1 (0.273 mL, 3.7 mg/mL) in PBS pH 7.4. The mixture was incubated at room temperature overnight. The THb- 3 H]-Lys-Hp complex was purified using SEC equilibrated and eluted with PBS pH 7.4. Radioactivity was associated primarily with a high molecular weight species identified by SEC, having absorption at 280 and 414 nm and eluting separately from native haptoglobin and the original THb-Lys product, and with a retention time corresponding to the non-radiolabeled product of Example 6.

Example 8 Synthesis of Fluorescein-hemoglobin Conjugate (FL-Hb) fluorescein (5-IAF, 11 mg, 21 pmol) solution in N,Ndimethylformamide (DMF, 50 pL) was slowly added to oxyhemoglobin j -22mg/mL, 5 mL) in 50 mM potassium phosphate pH 7.0 with stirring at 4 0

C.

After three hours of reaction at the excess of 5-IAF was removed by extensive dialysis against 50 mM potassium phosphate pH 7.2 until no could be detected in the dialysate. The UV-visible absortion spectrum of the product showed a characteristic fluorescein absorption band at 496 nm.

Example 9 Complexes of FL-Hb with Haptoglobin 1-1, 2-1 and Mixed Phenotype Haptoqlobin FL-Hb (6 mg/mL in 50 mM potassium phosphate pH 7.2, 40 pL) was added to haptoglobin 1-1, 2-1 or mixed phenotype (Hpmix) (2.8 mg/mL in water, 39 pL) to give the following final concentrations: 0.6 mg/mL (6.2 pM) Hp and 1.3 mg/mL (21 p.M) FL-Hb in 180 .p.L final volume of 25 mM potassium phosphate pH 7.0. The mixture was analyzed by SEC after incubation at room temperature for 10 min. FL-Hb complex with haptoglobin 1-1 migrates at 33 min.(FIG. 9A--overlaid SEC chromatograms of Hp 1-1 and Hp 1-1 complex with FI-Hb) and can be clearly distinguished from haptoglobin by its absorbance at 414 nm. FL-Hb migrates at 42.9 min. (FIG. 9A). FL-Hb complexes with Hp 1-1 and Hpmix were isolated and analyzed by UV-Vis spectroscopy (FIG. 9B--UV-Vis spectrum of haptoglobin 1-1 and Hpmix complexes with FL-Hb, the arrow indicates the band characteristic of fluorescein) and fluorimetry. This material shows fluorescence with excitation at 480 nm and emission at 520 nm, and a characteristic absorption band for fluorescein with Xmax at 496 nm. FL-Hb complexes with Hp 2-1 and Hpmix are shown in FIGS. 9C and 9D, respectively. The construct-complexes were purified by SEC eluted with PBS buffer.

Example 10 Synthesis of Cross-linked Hemoglobin Bearing Tritiated Putrescine 200 mL of purified Hb was diafiltered into 50 mM borate buffer pH then deoxygenated and the concentration adjusted to 7.1 g/dL. Hb was crosslinked at a 2:1 ratio of TTDS to Hb for 45 min at 30.degree. C. and then desalted using 50 mM borate pH 9.0 buffer yielding a final concentration of -23- 3.1 g/dL. 1.43 mL of the desalted Hb was added to each of two 1 mL aliquots of radiolabeled putrescine (1 mCi/mL, 6.94.times.10 5 mmol/mL) and reacted at room temperature for 1.5 hours (10:1 Hb:putrescine ratio). 0.9 mg of cold putrescine (40 fold excess over radiolabeled putrescine) was reacted with 17 mL of the THb-DBS at a ratio of 1.5:1 THb-DBS:putrescine. 5 mL of this solution was added to each of the two reactions and mixed overnight at room temperature. Both mixtures were then added to freshly cross-linked and desalted THb-DBS (5.3.times.10 5 moles) and reacted at room temperature for hours. A 20 fold excess of cold putrescine (172 mg) was then added and reacted overnight. The THb-[ 3 H]Pu was then diafiltered into Ringers Lactate.

The specific activity was 1.5 Ci/mole, 90 mg/mL.

Example 11 Haptoglobin 1-1 Complex with THb-[ 3 HIPu Haptoglobin (3.0 mg/mL in water, 51 pL) was added to THb-[ 3 H]Pu mg/mL in PBS pH 7.2, 20 iL) to give the following final concentrations: 1.4 mg/mL (14 jM) haptoglobin and 1.8 mg/mL (28 jiM) THb-[ 3 H]Pu in a final 110 .mu.L volume of 25 mM potassium phosphate pH 7.0. The mixture was analyzed by SEC after incubation at room temperature for 2 hours. Fractions (0.4 mL) of the effluent were collected and analyzed by scintillation counting.

THb-[ 3 H]Pu-Hp complex migrates as a high molecular weight species with elution time from 20 to 28 min. (FIG. 10) and is well separated from haptoglobin band at 30 min. and THb-[ 3 H]Pu at 37 min. THb-[ 3 H]Pu -Hp absorbs both at 280 nm and 414 nm (A 28 0nm /A 4 1 4 nm =0.74) and has specific radioactivity (cpm/mg Hb) similar to that of THb-[ 3 H]Pu. The constructcomplex was purified by SEC.

Example 12 Synthesis of Cross-linked Hemoglobin Bearing Monodansyl Cadaverine Purified Hb (8.0 g/dL, 100 mL, 1.25 x10 4 moles) was diafiltered into mM borate buffer, pH 9.0, then oxygenated and deoxygenated. A deoxygenated solution of TTDS (2 fold molar excess over Hb, 0.26 g, 2.5 x -4 moles) was added and the mixture was stirred for 1 hour at 35 0 C.,then

L

-24charged with CO. Ion exchange chromatography at this time indicated only a small amount of unreacted Hb A 15-fold molar excess of monodansylcadaverine (MDC) in .about.20 mL of 0.1 M HCI adjusted to mL with 50 mM borate, pH 9.0 was added to the crosslinked Hb (0.63 g, 1.88 x 10- 3 moles). After 60 hours at room temperature, the MDC-Hb was diafiltered against 10 mM borate, pH 9.0. The product was purified by gel filtration and diafiltered into Ringers Lactate.

Example 13 Haptoqlobin 1-1 Complex with THb-MDC THb-MDC (20 mg/mL in Lactated Ringer's solution pH 7.2, 3.5 jpL) was added to haptoglobin 1-1 (1.1 mg/mL in water, 200 pL) to give the following final concentrations: 1.1 mg/mL (11 pM) Hp and 0.34 mg/mL (5.4 pM) THb- MDC. The mixture was analyzed by SEC after incubation at room temperature for 24 hours. THb-MDC complex with haptoglobin migrates as a high molecular weight species with elution time from 21 to 29 min (FIG. 11). This material migrates separately from haptoglobin (30.9 min.) and absorbs at both 280 nm and 414 nm (A 2 80nm /A 4 14nm THb-MDC elutes at 37.9 min. with

A

2 8 0nm /A41 4 nm =0.29. The construct-complex can be purified by SEC.

Example 14 Synthesis of Cross-linked Hemoqlobin Bearing Primaquine (THb-PO) TTDS (14.0 mg) in ethanol (100 g.L) was added to deoxyhemoglobin mL, 58 mg/mL) in 50 mM borate pH 9.0. The reaction mixture was stirred at room temperature under nitrogen for one hour. The excess of the crosslinker was then removed by gel filtration eluted with 50 mM borate pH 9.0 and the product (THb-DBS, 43 mg/mL) was charged with CO. Primaquine diphosphate (0.5 g, 1.1 mmol) was dissolved in 50 mM borate pH 9.0 (10 mL) and the pH of the resulting solution was adjusted to 8.5 with 10 M NaOH (primaquine partially precipitated). THb-DBS (10 mL) was added to primaquine and the reaction mixture was stirred in the dark at room temperature overnight. The product was then filtered and the filtrate dialyzed extensively against 50 mM borate pH 9.0. Anion exchange chromatography of the product (FIG. 12) indicates that THb-PQ constitutes 68% of all hemoglobin components in the mixture. THb-DBS conjugated with primaquine constitutes 64% of all p-chains when the product is analyzed using reversed phase chromatography.

Example 15 Haptoglobin 1-1 Complex with THb-PQ THb-PQ (15 mg/mL in 50 mM borate pH 9.0, 67 was added to haptoglobin 1-1 (4.0 mg/mL in water, 500 ML) to give the following final concentrations: 2.0 mg/mL (20 gM) Hp and 1.0 mg/mL (15.7 pM) THb-PQ.

The mixture was analyzed by SEC and anion exchange chromatography after incubation at room temperature for 21 hours. THb-PQ complex withhaptoglobin migrates as a high molecular weight species with elution time from 21 to 29 min. (FIG. 13). This material migrates separately from haptoglobin complexed with uncross-linked hemoglobin (29.9 min.) and haptoglobin (30.6 min.) and absorbs at both 280 nm and 414 nm (A 2 8onm

/A

4 1 4 nm Anion exchange chromatography indicated that all unmodified hemoglobin and 74% of both THb-PQ and THb have reacted with Hp. This result is in good agreement with the SEC analysis which indicates that 78% of hemoglobin has reacted with Hp.

Example 16 Haptoglobin-[polv-O-raffinose-Hbl and Haptoglobin-[64 kDa-O-raffinose-Hbl ComplexesHbA0 was cross-linked and polymerized using oxidized raffinose (OR) according to the procedure of Pliura Pat.

No. 5,532,352). Molecular weight species greater than 64 kDa, representing polymerized Hb (>64 kDa OR-Hb), where separated from 64 kDa species (64 kDa OR-Hb) by size exclusion chromatography. Hb preparation were combined separately with human haptoglobin 1-1 in water to a final concentration of 0.2 mg Hb/mL and 0.125 mg haptoglobin/mL (final Hb:Hp approximately The mixtures were incubated for one hour at 22.degree.

then analyzed by size exclusion chromatography under dissociating, nondenaturing elution conditions (0.5 M MgCl 2 25 mM Tris pH FIG. 14, which shows size exclusion chromatography elution profiles with detection at -26- 280 (solid lines) and 414 nm (broken lines), indicates binding of the modified hemoglobins with haptoglobin. Incubation of the modified hemoglobins with haptoglobin results in high molecular weight species which do not correspond to either the modified hemoglobin or haptoglobin, and which have absorption at 414 nm indicating hemoglobin content.

Example 17 Binding of Modified Human Hb 3 H]-NEM-Hb) to Rat Haptoglobin in Plasma 1 mCi of 3 H-N-ethylmaleimide 3 H]-NEM) in pentane was evaporated in 0.5 mL phosphate buffer, and 25 mg of Hb in 1 mL buffer was added giving a final NEM:Hb ratio of 0.06:1, or 37 .mu.Ci/mg Hb. RP HPLC analysis after 24 hours at 4.degree. C. indicated incorporation of the majority of the radiolabel into a modified beta peak. After 47 hr, a 15-fold excess (over pCys93 thiol) of non-radiolabeled NEM was added. Salts and unbound NEM were removed by gel filtration, and the final concentration adjusted to 10.2 mg Hb/mL. A small portion of this material 3 H-NEM-Hb) was then combined with rat serum containing haptoglobin to determine if all radiolabeled components bound to Hp. The Hb-binding capacity of the rat serum was adjusted to 670 pg Hb/mL serum. 0.5 and 2.0 equivalents of 3 H-NEM-Hb, based on Hbbinding capacity, were combined with serum and analyzed -by size exclusion chromatography eluted under dissociating, non-denaturing conditions using M MgCl 2 25 mM Tris pH 7.4 (FIG. 15). In the 3 H-NEM-Hb preparation, all radioactivity was associated with a 32 kDa peak. At 0.5 eq. 3 H-NEM-Hb, all radioactivity appeared in the Hb-haptoglobin peak (31 minutes). At 2.0 eq., haptoglobin is saturated and excess 3 H-NEM-Hb remains unbound (41 minutes). 7.3% of the radioactivity combining with plasma components appears in a high MW peak at 22 minutes. These findings demonstrate that all components of the modified human Hb, 3 H-NEM-Hb, are capable of binding rat haptoglobin in plasma.

-27 Example 18 Biodistribution of Modified Hb and Haptoqlobin Complexes in Rat The ability of Hp to target modified Hb to the liver was measured in a radioisotope biodistribution study. Two test articles were prepared from purified human HbAo modified with tritium-labeled N-ethylmaleimide 3

H]-

NEM-Hb): 3 H]-NEM-Hb alone in Ringer's lactate, and 3 H]-NEM-Hb complexed to a slight excess of rat haptoglobin in rat plasma. Three treatment groups were analyzed; normal rats received the modified Hb-haptoglobin complex in plasma, normal rats received the modified Hb only (approximately twice the Hb-binding capacity of the rat), and haptoglobindepleted rats received the modified Hb only. Approximately 3 mg of Hb were administered to conscious Sprague-Dawley rats in each case. Liver and plasma samples were collected at 30, 60 and 120 minutes post-administration and radioactivity counted after solubilization and quenching. Values were converted to percentages of total dose and concentration/dose, and various analyses are shown in FIG. 16. This shows radioactivity contents, indicative of dose percentages. FIG. 16A shows the percentage of dose in plasma. FIG.

16B shows the percentage of dose in liver. FIG. 16C shows the percentage of dose in liver +plasma. FIG. 16D shows the liver/plasma concentrations. Star designations and show differences (p<0.05) within treatment groups at different times. Crosses (t and t) show differences (p<0.05) within time points for different treatment groups.

Plasma retention was highest in group A, and both groups A and B were higher than group C. The greatest difference in plasma content was at 120 minutes at which time group A plasma contained 3 times the radioactivity of group C and 3.5 times that of group B. Liver content in groups A and B was higher than in group C at all time points. At 30 minutes, groups A and B had approximately 20% of the total dose in the liver compared to 11% in group C.

Liver content was the same at 30 and 60 minutes in groups A and B, and declined by the 120 minute time point. By 120 minutes, group A and B liver contents were 5- and 2-fold higher than group C, respectively. Groups A and B contained 60% of the dose in the plasma and liver compartments at -28 minutes, compared with roughly half that amount in the Group C animals.

Liver to plasma concentration/dose ratios increased with time in all groups, with liver concentration approximately 4 times that of plasma in groups A and B by 120 minutes, roughly twice the ratio of group C at the same time. The improvement in plasma retention and liver targeting is further demonstrated by comparison of mean combined liver and plasma contents between groups, presented in FIG. 17, namely the ratios of mean combined liver and plasma percentages of total dose. Shaded bars are derived from Group A/C, solid bars from Group B/C, and open bars from group A/B. Group A and B combined liver and plasma contents were consistently greater than in group C, with group A having a combined content 4 times greater than in group C at 120 minutes. Areas under the distribution curves were calculated without extrapolation to time zero (Table 2) and indicated that liver uptake in groups A and B was approximately twice that of group C. The data overall demonstrate a greater ability to concentrate product in the liver when Hp is present, either in a pre-formed complex with the modified Hb, or in the form of endogenous Hp where it is capable of forming a complex with administered Hb. There is also a clear indication that plasma retention of Hb conjugates is increased through combination with haptoglobin, such that a drug conjugate would be available for tissue uptake for a greater length of time.

TABLE 2 Areas under distribution curves for plasma and liver in rat AUC* AUC* (ug Hb .multidot. (ug Hb .multidot.

min/mL/dose) min/g/dose) Group Plasma Liver A 370.0 455.3 B 284.2 510.4 C 163.0 234.0 *dose ug Hb/g body weight

L

-29- Thus it has been demonstrated that agents can be conjugated to both 32 kDa hemoglobin dimer and to 64 kDa intramolecularly cross-linked Hb, using either attachment to side chain functionalities, to an intramolecular cross-linker or to a secondary linker attached to the intramolecular crosslinker. All of these constructs bound to haptoglobin. There has further been demonstrated the selective targeting of such a construct-complex, formed in vivo or ex vivo, to the liver and the extension of circulating half-life.

Examples 19- 22 In Examples 19 22, a non-intramolecularly cross-linked hemoglobin preparation was used hemoglobin isolated from red blood cells). The preparation was made using techniques known in the art.

Example 19 Preparation of Hb-Ribavirin Conjuqate Synthesis of Ribavirin Phosphate Imidazolide.

Ribavirin phosphate was synthesized by derivatisation of ribavirin at its primary hydroxyl group using phosphooxychloride and dimethylphosphate (Allen, et al., J Med Chem. 1978 Aug;21(8):742-6.), and monitored for ribavirin modification by C18 reverse-phase HPLC. Following completion of the reaction, the ribavirin phosphate (1 mmol) was mixed with 10 g of fine charcoal (100-400 mesh). The charcoal-reaction mixture was centrifuged at 2000 g for 15 min and the supernatant recovered. The wash steps were repeated until no inorganic phosphate could be detected in the supernatant as assayed using the Ames method (Ames BN (1966). Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8: 115118).

The charcoal was extracted with EtOH/water/NH 4 0H (10:10:1) and the pooled extract evaporated to dryness. The resulting ribavirin phosphate ammonium salt was converted to its free acid (Streeter et al, Proc. Natl. Acad. Sci. USA 1973 Apr; 70(4):1174-8). Purity of the ribavirin phosphate was evaluated using 2 assays. Acid phosphatase was used for complete enzymatic cleavage of ribavirin from ribavirin phosphate, followed by quantitation of the released ribavirin by C18 reverse-phase HPLC. C18 reverse-phase HPLC was performed on a C18 Phenomenex Luna column (4.6x250) using isocratic elution with water/TFA 0.1% pH 2.9, flow 1 ml/min. Total inorganic phosphate content of ribavirin phosphate was measured using the Ames method (described below). Purified ribavirin phosphate was converted to its imidazolide (Fiume et al., Anal Biochem. 1993 Aug 1:212(2):407-11), with slight modifications. The reaction was performed under dry N 2 using dry solvents. Typically, 324 mg (1 mmol) of ribavirin phosphate was dissolved in 10 ml anhydrous DMF. 5 mmol carbonyldiimidazole in 5 ml DMF was added, followed by 5 mmol imidazole in 5 ml DMF. The solution was stirred at RT for min and the DMF evaporated. The remaining oil/solid was dissolved in 2 ml anhydrous EtOH, followed by precipitation with the addition of 20 ml anhydrous ether. The precipitate was washed twice with ether, and residual ether was removed with a gentle stream of dry N 2 The ribavirin phosphate imidazolide was used immediately for conjugation to Hb.

Conijuation of Ribavirin Phosphate Imidazolide with Hb.

0.06 tmol ml of Hb (CO form, 100 mg/ml) was mixed with 6.6 pmol ribavirin phosphate imidazolide (0.8 pM) in 0.1 M NaHCO3/Na2CO 3 pH The pH of the reaction mixture was monitored over the first hr, and maintained at pH 9.5 9.6 by addition of 0.2 M Na2CO 3 After the pH of the reaction had stabilized, the reaction mixture was charged with CO for 15 min and the reaction allowed to continue under CO pressure at 370C for 96 hr. Hb was monitored for drug modification using anion-exchange chromatography.

Anion exchange chromatography was performed on a Poros H/HQ (4.6/100) column, using a pH gradient 8.3-6.3 over 10 min (mobile phase 25 mM Tris pH 8.3, 25 mM bisTris pH 6.3) with a flow rate of 4 mi/min. All Hb was modified as evidenced by later elution on anion exchange media relative to unmodified Hb control, due to the added net negative charge resulting from modification of lysine side chain amino groups with the phosphate containing conjugant (Figure 18). The conjugate was dialysed (MWCO 10 KD) against -31 Ringer's Lactate (3X 0.5 sterile filtered (0.2 tim) and charged with CO prior to storage at Determination of Molar Drug Ratio.

The molar drug ratio of Hb-ribavirin conjugate was determined by quantitation of ribavirin released by enzymatic cleavage using the acid phosphatase assay and determination of total inorganic phosphate using the inorganic phosphate assay. The molar concentration of Hb protein was determined using the Drabkins assay kit for Hb (Sigma). MALDI-TOF mass spectrometric analysis of the Hb-ribavirin conjugate indicated up to at least ribavirin phosphate groups attached to both alpha and beta chains of the Hb (Figure 19). For the acid phosphatase assay, 5 nmol (0.3 mg) of the Hbribavirin conjugate was diluted into 0.3 ml of 1 mM NaOAc/HAc buffer, pH 4.8.

3 units of a freshly prepared acid phosphatase (Type IV-S, potato) was added, and the enzymatic reaction allowed to proceed at 37°C for 2 hr. Hb precipitate was removed by centrifugation and the supernatant analysed for ribavirin by C18 reverse-phase HPLC. For evaluation of biological activity of released ribavirin, the supernatant was dialysed against PBS and concentrated prior to analysis. For the inorganic phosphate assay, total inorganic phosphate was determined using the method of Ames, 1966. The volume of 10% Mg(N0 3 2 in EtOH was optimized to 80 tl for assay of 5 nmol Hb-ribavirin conjugate.

Hp Binding Assay. Hb-RV section To determine retention of Hp binding by Hb-ribavirin conjugate, a complex was allowed to form with a 10% molar excess of human Hp, at RT for a minimum of 30 min. SEC analysis was used to detect for Hb 414 nm absorption that was shifted due to formation of a higher MW complex with Hp.

SEC was performed using a Pharmacia Superdex 200 column using MgCI2/25 mM Tris, pH 7.2 at a flow rate of 0.4 ml/min. Formation of the complex of conjugate with Hp was confirmed by elution of Hb-containing species which appeared as peaks eluting earlier than the non-complexed Hb-drug conjugates -32- Preparation of the Fluorescein-Hb-Ribavirin double conjugate.

A 1 mM solution of fluorescein maleimide (Pierce 46130) was prepared in PBS. Hemoglobin or hemoglobin-ribavirin conjugate was added to a concentration of 100 pM, and incubated 4 hr in the dark at RT with shaking.

The sample was then dialyzed extensively against PBS to remove any unbound fluor. Based on the concentration of fluor and protein in the purified conjugate, determined by fluorimetry and Coomassie analysis, respectively, the ratio of fluor label to Hb-RV was approximately 1. RP HPLC analysis coupled with fluorescence detection showed all fluorescence to be associated with the p-chain, indicating the expected attachment of the fluorescein maleimide to the surface reactive pCys93 thiol group. Binding of the fluorescein labelled Hb-Rv to Hp was also verified by size exlusion HPLC analysis. Binding to Hp was verified the a shifting of the Hb derivative peaks to an earlier retention time corresponding to an increase in molecular weight upon formation of the Hp-Hb(FI)-RV complex.

In vitro Bioactivity Assay of Ribavirin from Hb-Ribavirin Coniugate.

Ribavirin recovered from acid phosphatase cleavage of Hb-ribavirin conjugate was evaluated for bioactivity in an in vitro cell proliferation assay using the Cell Proliferation ELISA Bromodeoxyuridine (BrdU) kit (Roche, 1 647 229). Human hepatoma HepG2 cells and mouse hepatocyte AML12 cells were plated at a density of 4 x 104 cells/well, and 1 x 104 cells/well, respectively, in flat bottom 96-well plates. The cells were allowed to grow for 24 hours, at which time they were treated in quadruplicate with ribavirin or ribavirin from Hb-ribavirin conjugate for 6 hours. The treatments were removed, and fresh media containing BrdU was added to the wells and the incubation continued for 18 hr. The standard BrdU EL1SA assay was then followed according to the kit protocol. Cleaved ribavirin activity was equivalent to unmodified ribavirin control (Figure 20), demonstrating the ribavirin is not detrimentally altered by the conjugation and cleavage processes, and suggesting that activity of ribavirin cleaved from the conjugate in vivo will have activity similar to free ribavirin.

-33- Internalization Assay.

To evaluate uptake of Hb-ribavirin conjugate by hepatic cells, internalization assays were performed using fluorescein-tagged Hb and Hbribavirin conjugate (Zuwala-Jagiello and Osada, 1998). HepG2 cells and 5637 bladder carcinoma cells were plated in 12-well plates at 2.5 x 10 6 cells/well, AML12 cells at 2 x 10 5 cells/ well, and the cells allowed to grow for 48 hours.

Media was removed and the cells were washed with HBSS containing 2 mg/ml BSA (HBSS/BSA). Hb or Hb-ribavirin conjugate labelled with fluorescein was complexed with Hp (1:1 molar ratio) in HBSS/BSA, and added to cells to a final concentration of 500 pg/ml. The labelled complexes were allowed to bind for 2 hr at 4°C. ATP was added to 1 mM and receptormediated internalization was initiated by incubation at 370C for various times.

The cells were washed with HBSS/BSA and surface-bound ligands were stripped from cells by incubation in 0.2 M acetic acid/0.5 M NaCI for minutes. The cells were washed with PBS and lysed with 2 M NaOH. The solubilized cell extract was transferred to a flat bottom 96 well plate, and fluorescence measured (485 nm excitation/530 nm emissision) using a fluorometric plate reader (Packard Fluorocount). Both the Hb and Hbribavirin, complexed to Hp, were taken up by the liver derived cell lines and neither was effectively internalized by the non-liver cell line, demonstrating the selective targeting of the Hb-ribavirin complex to cells bearing receptors the Hb-Hp complex (Figure 21). The labelled albumin control was not significantly internalized by any of the cell lines, thereby confirming that the level of Hb uptake in the liver cell lines was not due to passive transport of macromolecules.

Figure 21 illustrates In vitro uptake of Hb-ribavirin conjugate by hepatic and non-hepatic cells. Human hepatoma HepG2, mouse AM12 hepatocyte, and 5637 bladder carcinoma cells were assayed for internalization of fluorescein-labeled Hb and Hb-ribavirin conjugate at 37 0 C for 2 hr. Results are expressed as fold increase in relative fluorescence units (RFU) over equivalent samples not treated at 37"C.

1 -34- Example 20 Conjugation of Ara-AMP and Ara-CMP to Hb Preparation of Ara-AMP-Imidazolide and Ara-CMP-lmidazolide Reactions were conducted under dry N2 using anhydrous reagents.

Solutions of 6.4 mg, t 20 umol, Ara-AMP in 1 mL DMF or 24.1 mg, 90 umol, Ara-CMP in 3 mL DMF were added to 1 ml dry DMF were prepared under N 2 Carbonyldiimidazole, CDI, 156 mg, z 950 umol was in 4 ml dry DMF. 28.5 mg, 420 mmol, Imidazole was dissolved in 2 ml dry DMF. 0.3 ml of the CDI and 0.5 ml of the Imidazole solution were added to the Ara-AMP suspension.

1 ml of the CDI and 1.5 ml of the Imidazole solution were added to the Ara- CMP suspension. Reactions were stirred for 3 hours. The formation of the Imidazolide was followed on HPLC (C18 RP Aqua column, mobile phase 66 mMol phosphate buffer, pH 7.35, flow 1 ml min, UV abs. at 254 and 280 nm).

The peak corresponding to starting nucleotide was converted to a later eluting species (Figure 22). DMF was evaporated and the crude reaction products and the resulting oils were dissolved in EtOH. Any undissolved material was removed by centrifugation. The EtOH solutions were precipitated with dry ether at -20 Precipitates were isolated by centrifugation, washed with ether and dried under N 2 and shown to be pure by HPLC.

Hb-Ara-A and Hb-Ara-C conjuqates Ara-AMP-Im, 20 umol, was dissolved in 300 ul carbonate buffer, pH 9.3. 125 ul CO-Hb solution was added (10 g/dL, 200 nmol). Ara-CMP-Im, 90 umol, was dissolved in 600 ul carbonate buffer, pH 9.3. 200 ul Hb solution was added (10 g/dL, 320 nmol). The pH of the reaction mixtures was adjusted between 7.7 and 9.05, and reaction proceeded at 37 Anion exchange chromatography showed the formation of Hb species containing greater negative charge over time, indicating attachment of nucleotide to the Hb (Figure 23). Reactions were dialyzed against lactated Ringer's solution at 4°C. Pre- and post-dialysis anion exchange profiles were similar, indicating stability of the conjugate during dialysis. Peaks corresponding to nonconjugated nucleotide species were eliminated by dialysis.

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Hb concentrations of the conjugates were determined with the Drabkin method and the amount of modification was estimated with the Pi assay.

Molar drug ratios (nucleotide:Hb) were 15 and 9 for the Ara-A and Ara-C conjugates, respectively. MALDI-TOF mass spectrometry confirmed the presence of at least 3 nucleotides on each of the alpha and beta chains of the Hb in both conjugates.

Table 3 Conjugate Hb concentration Pi Molar drug ratio (g/dL) (nmol/20 ul) (nucleotide:Hb) Hb-Ara-A 1.5 69.7 15:1 Hb-Ara-C 1.4 35.8 9:1 Example 21 Preparation of haptoglobin complexes of Hb-drug conjucates Hb-drug conjugates, prepared according to the example 20, were combined with at least one equivalent of haptoglobin. Size exclusion chromatography was used to confirm the ability of conjugates to bind haptoglobin. In all cases, conjugates bound to haptoglobin to form higher molecular weight complexes that eluted earlier than the non-complexed Hbdrug conjugates (Figure 24). A portion of any polymeric species in the Hbdrug conjugates did not bind to haptoglobin.

Example 22 Hb-Dox and Hb-iminothiolane-Dox Conjugates Activation of Doxorubicin (Dox) with sulfo-LC-SPDP.

A 2.3 mM solution of Dox was prepared in PBS at pH 7.5 by dissolving with stirring at RT over 2h. Once dissolved, pH was adjusted to 7.5 with NaOH. Sulfo-LC-SPDP was added to 4.55 mM (2x Dox equivalents). The solution was stirred overnight at room temperature (RT) in the dark. Activated Dox precipitated and was washed several times with water, and the resulting

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-36red solid was dissolved in methanol. Dox content was quantified by the aglycone assay.

Thiolation of Hemoglobin (Hb-IT) To increase surface thiol content of Hb, a solution of 2.42 mM iminothiolane (IT) hydrochloride and 0.46 mM CO hemoglobin to in Bis Tris pH 7.3 containing 10 mM EDTA was stirred at 4°C for 18h. Dithiothreitol (DTT) was added to 62.5 uM and allowed to react at RT for 2h. Hb-IT was purified using Amersham Pharmacia PD-10 columns (Piscataway, NJ), eluted with PBS pH 7.5 containing 10 mM EDTA. Red eluent was collected and stored at 4°C under CO headspace until further use.

Coniugation of Dox-LC-SPDP to Hemoglobin/Hemoglobin-IT Reaction solution was prepared as follows. Hb-IT in PBS was combined with the methanolic Dox-LC-SPDP solution to a final 30% (v/v) composition of methanol. The solution was adjusted to pH 7.5, and the molar ratio of Hb-IT to Dox-LC-SPDP was 7:1. A similar reaction was conducted using unmodified Hb (no IT group attached). The reaction was protected from light and incubated for 6h (Hb-IT) or 24 h then centrifuged in IEC Centra® GP8R tubes at 3300 rcf for 15 min. Retentate was transferred to Millipore 10K Centripreps® and concentrated to 5 to 10 ml, then dialyzed under CO vs. 0.5 M MgCI2, pH 7.4, then PBS, through a 10 K membrane to remove residual free Dox-LC-SPDP. Any solids were removed from the retentate by centrifugation, and the dissolved conjugate was then concentrated to a Hb concentration of 3-6 g/dl. Conjugation was confirmed by reverse phase HPLC (Vydac® C4 column (4.6/250) using a gradient of 20% 60% acetonitrile/water with 0.1% TFA over 90 min, and a flow rate of 0.9 ml/min, Figure 25). In the case of Hb-Dox, the native beta globin peak was decreased in size and a later-eluting, fluorescent peak formed, corresponding to the conjugation of the fluorescent Dox molecule to the beta chain.

Aglycone analysis of the Hb-IT-Dox and Hb-Dox conjugates indicated a drug loading of 3:1 and 1.5 -2:1 molar ratio of Dox:Hb, respectively.

1 -37- Aglycone Assay: Quantification of Hemoglobin Modification with Dox The aglycone assay was used to quantify the amount of Dox in activated Dox and conjugated Dox solutions. 50 ul conjugate was combined with 50 ul EtOH and 14 ul of 6N HCI in an a plastic tube. Dox standards were similarly prepared. After 2h at 55°C, solutions were analyzed by reverse phase HPLC (C18 Beckman Ultrasphere® column (4.6x150) using isocratic elution of mobile phase with 43% acetonitrile in water containing 0.64% SDS and 0.11% H 3 P0 4 pH 2.4) with fluorescence detector with excitation set at 460 nm and emission read at 540 nm. Aglycone eluted at 5.7 min. Any free unmodified Dox eluted at -25 min.

Hp Binding Assay.

To determine retention of Hp binding by Hb-Dox conjugate, a complex was allowed to form with a 10% molar excess of human Hp at RT for a minimum of 30 min. SEC analysis demonstrated Hp binding by the conjugate as shown by a shift of Hb-containing peaks to earlier retention times, consistent with formation of higher MW species as expected upon Hp binding.

In vitro Uptake of Hb-Dox Conjugates.

Wild type (WT) cells, with no receptor for Hb-Hp, and modified cells (nonWT) bearing receptors for the Hb-Hp complex, were laid out in 24 well plates at le5 cells/well in RPMI 10% FBS with no Geneticin®. The cultures were then allowed to recover for 24 hours prior to the assay. Hb or conjugate were combined with Alexa fluor labelled Hp at a 1:1 molar ratio and allowed to bind for 1 hour prior to dilution to 25 pg/ml with respect to Hb concentration in AIM-V medium. The media was removed from the cells and the cells washed with Dulbeccos PBS. 200 pl of the Hb-Hp samples were then added to the wells and the cells were incubated for 1 to 4 hours at 37 OC. The media was removed. The cells were washed with PBS and released from the plate by trypsinization. The cell suspensions were transferred to a fresh tube and the wells rinsed with 500 pl of PBS that was then added to the tube. The cells were then washed with 1 ml of PBS, pelleted and resuspended in 500 pi of PBS. Hb-Hp uptake was then determined by flow analysis (Figure 26). Only those cells with Hb-Hp receptor took up conjugate to a significant degree. Hb

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-38and Hb-Dox were only taken up when complexed to Hp. These observations confirm the selective uptake of the Hp-Hb-Dox conjugate complex in cells bearing receptors for Hb-Hp.

In vitro Cytotoxicity of Hb-Dox Coniuqates.

SUDHL cells normally express the Hb-Hp receptor. U937 cells normally do not, but can be induced to express receptors for Hb-Hp with PMA and dexamethasone (DEX). SUDHL, U937 and U937 (PMA-DEX treated) were assayed for Hb-Dox cytotoxicity as described below. 5 x 105 cells were incubated for 30 minutes at 37 0 C with 200 pg/ml of conjugate or native Hb either free or complexed with Hp. The cells were washed and harvested by centrifugation. The final cell pellet was resuspended in 500 pi of PBS. Cell viability was determined by incubating samples for 20 minutes at room temperature with 7-AAD (a naturally fluorescing molecule that is excluded from live cells). Conjugate was not cytotoxic to U937 cells which did not express the Hb-Hp receptor (Figure 27). SUDHL and U937 treated with PMAIDEX experienced significant cell mortality, demonstrating specific cytotoxicity for cells bearing the Hb-Hp receptor.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (35)

1. A hemoglobin construct which binds to haptoglobin comprising a non- intramolecularly cross-linked hemoglobin and an anti-viral substance bound to the hemoglobin.

2. The hemoglobin construct of claim 1 wherein the anti-viral substance is selected from the group consisting of: ara-AMP, trifluorothymidine, interferon, antisense oligonucleotides, ribavirin, cytarabin, ara-CMP, acyclovir, didonosine, vidarabine, adefovir, zalcitabine, lamivudine, fialvridine.

3. The hemoglobin construct of claim 2 wherein the anti-viral substance is selected from the group consisting of: ribavirin, ara-AMP, and cytarabin.

4. The hemoglobin construct of claim 3 wherein the anti-viral substance is ribavirin. The hemoglobin construct of claim 3 wherein the anti-viral substance is ara-AMP.

6. The hemoglobin construct of claim 3 wherein the anti-viral substance is cytarabin.

7. The hemoglobin construct of any one of claims 1 to 6 wherein the hemoglobin is a natural or modified hemoglobin, or a derivative thereof that has an affinity for haptoglobin.

8. The hemoglobin construct of claim 7 wherein the hemoglobin is derived from recombinant sources.

9. The hemoglobin construct of any one of claims 1 to 6 wherein the anti- viral substance binds to the hemoglobin at a site independent from the haptoglobin binding site. The hemoglobin construct of any one of claims 1 to 6 or claim 9 wherein the anti-viral substance is bound to the hemoglobin through the intermediary of a chemical linker.

11. The hemoglobin construct of claim 10 wherein the linker is a polycationic linker enabling binding of the agent through electrostatic interactions.

12. The hemoglobin construct of claim 11 wherein the linker is polylysine.

13. The hemoglobin construct of anyone of claims 1 to 6,or 9 or 10 further comprising a diagnostic agent.

14. The hemoglobin construct of claim 13 wherein the diagnostic agent is an imaging agent. The hemoglobin construct of claim 14 wherein the diagnostic agent is a radiolabelled compound or a fluorescent compound.

16. The hemoglobin construct-complex comprising the hemoglobin construct of any one of claims 1 to 15 and a haptoglobin bound to the hemoglobin portion thereof.

17. The hemoglobin construct-complex of claim 16 formed in vivo by administering to a mammalian non-human patient the said hemoglobin construct wherein the construct binds to the haptoglobin in vivo.

18. The hemoglobin construct-complex of claim 16 formed ex vivo by reaction of the said hemoglobin construct with haptoglobin ex vivo.

19. The hemoglobin construct-complex of any one of claims 16 to 18 which is capable of interacting with a hemoglobin-haptoglobin receptor or a cell comprising said receptor. Use, in the preparation of a medicament for treating a viral infection in a mammalian patient, of a hemoglobin construct of any one of claims 1 to 6.

21. Use in the preparation of a medicament for diagnosis of a viral infection in a mammalian patient, of a hemoglobin construct as defined in any one of claims 13 to -41

22. Use of a hemoglobin construct of claim 20 or claim 21 wherein the construct binds to haptoglobin in vivo to form a hemoglobin construct- complex and subsequently interacts with a hemoglobin-haptoglobin receptor of a cell and acts in vivoat the cell having said receptor.

23. Use of a hemoglobin construct-complex of any one of claims 16 to 19 in the preparation of a medicament for treating a viral infection in a mammalian patient.

24. Use of a hemoglobin construct-complex comprising the hemoglobin construct of anyone of claims 13 to 15 and a haptoglobin bound to the hemoglobin for diagnosing a viral infection. A hemoglobin construct which binds to haptoglobin comprising a non- intramolecularly cross-linked hemoglobin and an anti-neoplastic substance bound to the hemoglobin.

26. The hemoglobin construct of claim 25 wherein the anti-neoplastic substance is selected from the group consisting of: doxorubicin, daunorubicin, ricin, diphtheria toxin and diphtheria toxin A.

27. The hemoglobin construct of claim 26 wherein the anti-neoplastic substance is doxorubicin.

28. The hemoglobin construct of claim 27 wherein the ratio of doxorubicin: hemoglobin is 1.5- 3:1.

29. The hemoglobin construct of claim 28 wherein the ratio is 1.5-2:1. The hemoglobin construct of any one of claims 25 to 29 wherein the hemoglobin is a natural or modified hemoglobin, or a derivative thereof that has an affinity for haptoglobin.

31. The hemoglobin construct of claim 30 wherein the hemoglobin is derived from recombinant sources.

32. The hemoglobin construct of anyone of claims 25 to 29 wherein the anti- -42- neoplastic substance binds to the hemoglobin at a site independent from the haptoglobin binding site.

33. The hemoglobin construct of any one of claims 25 to 32 wherein the anti-neoplastic substance is bound to the hemoglobin through the intermediary of a chemical linker.

34. The hemoglobin construct of claim 33 wherein the linker is a polycationic linker enabling binding of the agent through electrostatic interactions. The hemoglobin construct of claim 34 wherein the linker is polylysine.

36. The hemoglobin construct-complex comprising the hemoglobin construct of any one of claims 25 to 35 and a haptoglobin bound to the hemoglobin portion thereof.

37. The hemoglobin construct-complex of claim 36 formed in vivo by administering to a mammalian non-human patient the said hemoglobin construct wherein the construct binds to the haptoglobin in vivo.

38. The hemoglobin construct-complex of claim 36 formed ex vivo by reaction of the said hemoglobin construct with haptoglobin ex vivo.

39. The hemoglobin construct-complex of anyone of claims 36 to 38 which is capable of interacting with a hemoglobin-haptoglobin receptor or a cell comprising said receptor.

40. Use, in the preparation of a medicament for treating a neoplastic condition in a mammalian patient, of a hemoglobin construct of anyone of claims 25 to 33.

41. Use of a hemoglobin construct of claim 40 wherein the construct binds to haptoglobin in vivo to form a hemoglobin construct-complex and subsequently interacts with a hemoglobin-haptoglobin receptor of a cell and acts in vivo_at the cell having said receptor.

42. Use of a hemoglobin construct-complex of any one of claims 36 to 38 in L-- -43- the preparation of a medicament for treating a neoplastic condition in a mammalian patient. DATED this eighth day of August 2003 HEMOSOL INC By their Patent Attorney s FISHER ADAMS KELLY