Classifications

• • 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
  • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

• the present invention relates to biologicall active macromolecular conjugates, in particular, t conjugates of biologically active polypeptides an glycopolypeptides with water-soluble polymers.
• polypeptides wit water-soluble polymers such as polyethylene glycol (PEG)
• PEG polyethylene glycol
• the coupling of peptides an polypeptides to PEG and similar water-soluble polymers is disclosed by U.S. Patent No. 4,179,337 to Davis et al.
• Davis et al. discloses that physiologically active polypeptides modified with PEG exhibit dramatically reduced immunogenicity and antigenicity.
• the PEG-protein conjugates when injected into a living organism, have been shown to remain in the bloodstream considerably longer than the corresponding native proteins. Accordingly, a number of PEG-conjugated therapeutic proteins were developed exhibiting reduced immunogenicity and antigenicity and longer clearance times, while retaining a substantial portion of the protein's physiological activity.
• PEG-conjugated therapeutic proteins include tissue plasminogen activator, insulin, interleukin 2 and hemoglobin.
• Dreborg et al., Crit. Rev. Therap. Drug Carrier Syst.. 6 , 315-65 (1990) disclose that covalent modification of potent allergen proteins with PEG often can be effective in reducing their allergenicity.
• Sehon, et al., Pharmacol. Toxicol. Proteins. 65, 205-19 (1987) disclose that such PEG-conjugated allergen proteins having reduced allergenicity can then be utilized as tolerance inducers.
• covalent attachment of the polymer is effected by reacting PEG-succinimide derivatives with amino groups on the exterior of protein molecules.
• the amino groups of many proteins are moieties responsible for polypeptide activity that can be readily inactivated as a result of such modification.
• the conjugation of such proteins is not desirable, because it results in the reduction of physiological activity.
• Other proteins may have only a small number of available amino groups, and consequently very few polymer anchoring sites. As a result, many proteins of interest cannot be conjugated with PEG in this manner.
• U.S. Patent No. 4,179,337 discloses the reaction of an amino-PEG derivative with l-ethyl-3-(3-dimethylamino-propyl) carbodiimide(EDC)- activated carboxylic acid groups of trypsin and other proteins.
• the selectivity of this reaction is rather poor because the reactivity of amino-PEG is similar to that of the lysyl residues of proteins, with both the amino-PEG and protein amino groups competing to react with the activated carboxylic acid groups. This results in intermolecular as well as intramolecular crosslinking and a loss of protein activity.
• the results from TNBS assays are meaningless when determining the degree of conjugation of proteins when the polymer is covalently attached to alternative functional groups. In such instances, the number of free amino groups will not vary between conjugated and non-conjugated protein species.
• the conjugated protein can also be digested in small fragments with an enzyme and separated by column chromatography followed by preparation of a peptide map for comparison to a map of the unmodified protein, with the fragments having altered elution times indicative of the location of polymer attachments.
• this procedure consumes large quantities of product and is not suitable for use with polypeptides of limited availability.
• Radioactive labeling represents another alternative, but this alternative is not suitable for materials being prepared for therapeutic end uses for which the determination of degree of conjugation is most critical. Yamasaki et al., Agric. Biol. Chem. , 52(8) f
• Mater., 17, 208-9 (1990) also disclose the use of a norleucine spacer in PEG-succinimide derivatives covalently bonded to protein amino groups, noting that the use of such an unnatural amino acid helps in the characterization of the adduct because a single amino acid analysis would give both protein concentration and number of polymer chains bound to the amino groups.
• each single norleucine residue acid represents a polymer chain bound to an exterior amino grou .
• water-soluble polymers can be conjugated with biologically active polypeptides and glycopolypeptides utilizing acyl hydrazine derivatives of the water-soluble polymers.
• the acyl hydrazine derivatives of the water-soluble polymers covalently link to either the oxidized carbohydrate residues of the glycopolypeptides or the reactive carbonyl or activated carboxylic acid groups o peptide moieties of polypeptides or glycopolypeptides
• This invention extends the realm of water-solubl polymer-peptide conjugation to those polypeptide an glycopolypeptide materials that could not have bee modified heretofore by conventional methods
• pK a about 3 acyl hydrazine containing polymers of this inventio possess higher reactivity than the amino groups o polypeptides (pK a about 10.5), therefore minimizing an in most cases eliminating the competing reactions o these
• biologically active macromolecular conjugate is provide of a biologically active polypeptide or glycopolypeptid and one or more water-soluble polymer molecule covalently bonded thereto at a reactive carbonyl o carboxylic acid group of a peptide moiety on th polypeptide or glycopolypeptide by a linkage containin a hydrazide or hydrazone functional group.
• the linkag is formed by reacting an acyl hydrazine derivative o the water-soluble polymer with a polypeptide o glycopolypeptide having an activated carboxylic aci group or a reactive carbonyl group generated thereon.
• the present invention also provides biologically active macromolecular conjugate of biologically active glycopolypeptide and one or mor water-soluble polymer molecules covalently bonde thereto at an oxidized carbohydrate moiety of th glycopolypeptide by a linkage containing a hydrazide o hydrazone functional group bound to the polymer via short peptide sequence.
• the oxidation of th carbohydrate moiety produces reactive aldehydes.
• Th hydrazone linkage is formed by reacting an acy hydrazine derivative of the water-soluble polyme containing the peptide sequence with these aldehyde groups.
• the hydrazone can be further stabilized by reduction to a very stable alkyl hydrazine derivative.
• the peptide sequence influences the lability of the linkage to proteolytic enzymes and also allows convenient characterization of the polymer conjugates by amino acid analysis of their hydrolysates. By using state-of-the-art techniques of amino acid analysis, the quantity of peptide sequences, and consequently the degree of conjugation, can be determined for picomolar concentrations of the conjugate.
• the peptide sequences also be utilized with the polypeptide conjugates of the present invention to bind the linkages containing a hydrazide or hydrazone functional group to the water-soluble polymer.
• FIG. 1 is a GF-HPLC chromatogram comparison of mPEG-beta-alanine-bovine serum albumin conjugate to native bovine serum albumin.
• FIG. 2 is a GF-HPLC chro atogram comparison of mPEG-beta-alanine-ovalbumin conjugate to native ovalbumin.
• FIG. 3 is a GF-HPLC chromatogram comparison of PEG-beta-alanine-IgG, conjugated via oxidized carbohydrate moieties, to native IgG.
• FIG. 4 is a GF-HPLC chromatogram comparison of PEG-beta-alanine-rhG-CSF, conjugated via carboxylic acid groups of rhG-CSF, to native rhG-CSF. Best Mode of Carrying Out the Invention
• the macromolecules of the present invention are biologically active polypeptides or glycopolypeptides having one or more water-soluble polymer molecules covalently bonded thereto.
• biologically active is used consistently with the meaning commonly understood to those of ordinary skill in the polypeptide and glycopolypeptide art, which meaning is not limited to physiologically or pharmacologically activities of the polypeptides or glycopolypeptides in the therapeutic sense.
• physiologically active polypeptides such as enzymes, the water-soluble polymer conjugates of which have therapeutic applications, are also able to catalyze reactions in organic solvents.
• therapeutic uses exist for water-soluble polymer conjugates of proteins such as concanavalin A, immunoglobulins, and the like, the polymer conjugates of these proteins are also useful as laboratory diagnostic tools.
• Enzymes of interest for both biological applications in general and therapeutic applications in particular include the oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases disclosed by U.S. Patent No. 4,179,337, the disclosure of which is hereby incorporated herein by reference thereto.
• examples of specific enzymes of interest include asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase and bilirubin oxidase.
• Carbohydrate-specific enzymes are also of interest—for example, glucose oxidase, glucosidase, galactosidase, glucocerebrosidase, glucuronidase, etc.
• proteins of general biological or therapeutic interest include, but are not limited to, Factor VIII and polypeptide hormones such as insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothamic releasing factors, antidiuretic hormones and prolactin.
• glycopolypeptides of interest include, but are not limited to, immunoglobulins, chorionic gonadotrophin, follicle-stimulating hormone, thyroid-stimulating hormone, ovalbumin, bovine serum albumin (BSA) , lectins, tissue plasminogen activator, numerous enzymes and glycosilated interleukins, interferons and colony stimulating factors.
• Immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD and fragments thereof.
• glycopolypeptides such as the interleukins, interferons and colony stimulating factors also exist in non-glycosilated form, usually the result of preparation by recombinant protein techniques.
• the structure of such versions may not contain carbohydrate moieties.
• the non-glycosilated versions are still capable of conjugation at reactive carbonyl or carboxylic acid groups of the peptide moieties.
• allergen proteins and glycoproteins having reduced allerginicity when conjugated with water-soluble polymers and consequently suitable for use as tolerance inducers include those allergens disclosed by Dreborg et al., Crit. Rev. Therap. Drug Carrier Syst.. discussed above, the teachings of which are hereby incorporated herein by reference thereto.
• allergens disclosed by this article are Ragweed Antigen E, honey bee venom, mite allergen, and the like.
• the water-soluble polymers suitable for attachment to the polypeptides and glycopolypeptide include polyalkylene oxides, polyoxyethylenated polyols, polyacrylamides, polyvinyl pyrrolidone, polyvinyl alcohol, dextran, and other carbohydrate-based polymers.
• the polymer must be soluble in water at room temperature.
• Polyalkylene oxide homopolymers meeting this requirement are polyethylene glycol (PEG) and copolymers thereof.
• Block copolymers of PEG with polypropylene glycol or polypropylene oxide are also suitable for use with the present invention, provided that the degree of block copolymerization is not so great as to render the polymer insoluble in water at room temperature.
• polyoxyethylenated polyols examples include polyoxyethylenated glycerols, polyoxyethylenate sorbitols, polyoxyethylenated glucoses, and the like.
• the molecular weight of the polymer is no critical, and will depend mainly upon the end use of particular polymer conjugate. Those of ordinary skil in the art are capable of determining molecular weigh ranges suitable for their end use applications. I general, the useful range of molecular weight is number average molecular weight between about 600 an about 100,000 daltons, and preferably betwee about 2,000 and about 20,000 daltons.
• One or more polymer units can be attache covalently to the polypeptide or glycopolypeptide b reacting an acyl hydrazine derivative of the polyme with a polypeptide or glycopolypeptide having a reactiv carbonyl group or an activated peptide carboxylic aci group.
• th reactive carbonyl group is defined as being either ketone or aldehyde group, excluding othe carboxyl-containing groups such as amides.
• Aldehyd groups are preferred, because they are more reactiv than ketones.
• the carbonyl group can be generated either o a peptide or a saccharide unit.
• Dixon, J. Protein Chem.. 3., 99 (1984) has reviewed some of the methods to generate reactive carbonyl groups on the N-terminus of a polypeptide molecule.
• Carbonyl groups can be generated on peptides, for example, by reacting a polypeptide or glycopolypeptide with a suitable heterobifunctional reagent such as a reactive ester of formyl benzoic acid, disclosed by King et al.. Biochemistry, 25. 5774 (1986) , the teachings of which are hereby incorporated herein by reference thereto.
• Carbonyl groups can be generated on saccharide units of glycopolypeptides, for example, by oxidizing vicinal diols of carbohydrate moieties of glycopolypeptides with excess periodate or enzymatically e.g. by use of galactose oxidase.
• the polymer acyl hydrazine reacts with the reactive carbonyl group on the polypeptide or glycopolypeptide to form a hydrazone linkage between the polymer and the polypeptide or glycopolypeptide.
• the hydrazone can be reduced to a more stable alkyl hydrazide by using for example NaBH 4 or NaCNBH 3 .
• the activated peptide carboxylic acid group can be derived either from a C-terminus carboxylic acid group or a carboxylic acid group of aspartic or glutamic acid residues.
• the polymer acyl hydrazine reacts with the activated peptide carboxylic acid group to form a diacylhydrazine linkage between the polymer and the polypeptide or glycopolypeptide.
• Activated carboxylic acid groups are carboxylic acid groups substituted with a suitable leaving group capable of being displaced by the polymer acyl hydrazine.
• suitable leaving groups are disclosed by Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, New York, 1984) , the disclosure of which is hereby incorporated herein by reference thereto.
• Such leaving groups include, but are not limited to, imidazolyl, triazolyl, N-hydroxysuccin- imidyl, N-hydroxynorbornenedicarboximidyl and phenolic leaving groups, and are substituted onto the peptide carboxylic acid group by reacting the polypeptide or glycopolypeptide in the presence of an activating reagent with the corresponding imidazole, triazole , N-hydroxysuccinimide, N-hydroxynorbornene dicarboximide and phenolic compounds.
• Suitable activating reagents are also well-known and disclosed by the above-cited Bodanszky, Principles of Peptide Synthesis, the disclosure of which is hereby incorporated herein by reference thereto.
• Examples of such activating reagents include, but are not limited to, water-soluble carbodiimides such as ethyl dimethyla ino-propyl carbodiimide (EDC) and 3-[2-morpholinyl-(4)-ethyl] carbodiimide, p-toluene sulfonate, 5-substituted isoxazolium salts, such a Woodward's Reagent K, and the like.
• acyl hydrazine polymer derivatives of th present invention will have the general structure (I) :
• R is one of the above-disclosed water-solubl polymers
• Z is 0, NH, S or a lower alkyl grou containing up to ten carbon atoms
• X is a termina group on the polymer.
• X can be a hydroxyl group, i which case the polymer has two labile groups per polyme moiety capable of reacting to form a derivative that can be covalently linked with a polypeptide or glycopolypeptide.
• X can therefore also be a group into which the terminal hydroxyl group may be converted, including the reactive derivatives of the prior art disclosed in U.S. Patent Nos.
• heterobi ⁇ functional polymers can be prepared by methods known to those skilled in the art, including the methods disclosed by the present specification with reference to the preparation of acyl hydrazine derivatives, as well as the methods disclosed by Zalipsky and Barany, Polym. Prepr.. 27(1.. 1 (1986) and Zalipsky and Barany, J. Bioact. Compat. Polym.. 5 , 227 (1990), the disclosures of which are hereby incorporated herein by reference thereto.
• X is a functional group useful for covalently linking the polymer with a second polypeptide or glycopolypeptide
• X can be a solid support or a small molecule such as a drug, or an acyl hydrazide derivative of the formula (II) :
• Such double polymer substitution can result in either intra- or intermolecular crosslinking of the polypeptide and glycopolypeptide moieties, which, in some cases, can be useful.
• Such crosslinking can be controlled by the amount of polymer used and the concentration of reacting species, which methods are well-known to those of ordinary skill in the art.
• Crosslinking of the polypeptide or glycopolypeptide moieties can also be prevented by using a pre-blocked polymer having only one labile hydroxyl group per polymer moiety.
• X would represent a blocking group such as an alkoxy group of one to four carbon atoms.
• the preferred blocking group is a methoxy group.
• the selectivity of the acyl hydrazines for the reactive carbonyl or activated carboxylic acid groups over the peptide amino group prevents intermolecular crosslinking between peptide amino groups and the reactive carbonyl groups and activated carboxylic acid groups, limiting occurrences of such crosslinking to instances when bifunctional polymer derivatives are employed.
• X can also represent an antibody or solid support covalently coupled to the polymer by methods known to those skilled in the art.
• solid supports covalently coupled to water-soluble polymers and methods of coupling water-soluble polymers to solid supports are disclosed in Published European Patent Application No. 295,073, the disclosure of which is hereby incorporated herein by reference thereto.
• the acyl hydrazine derivative is prepared by reacting, for example, the terminal -OH group of methoxylated PEG (mPEG-OH) with phosgene to form mPEG-chloroformate as described in U.S. Patent Appln.
• a more preferred form of the present inventio uses polymer hydrazides of the general formula (III) :
• AA represents an amino acid or a peptide sequence.
• AA can be a peptide sequence of any of the common amino acids, or at least one amino acid residue. In the case of AA being one amino acid residue, it is preferable that it is a residue that does not appear naturally in proteins. Examples of such unusual residues include, but are not limited to, alpha- or gamma- amino butyric acid, norleucine, homoserine, beta-alanine, epsilon-caproic acid, and the like.
• the linkage is a urethane linkage, which is very stable at ambient temperature in a variety of buffers, even at extreme pH's, but is readily split under conditions normally used for protein hydrolysis, thus allowing determination of amino acid components of AA by amino acid analysis.
• the peptide sequence can serve two roles. First, it can provide for convenient characterization of the modified protein by quatitation of the sequence by amino acid analysis. In this instance, the peptide sequence preferably is as short as possible and preferably contains unusual amino acid residues. For characterization of the modified protein, the peptide sequence most preferably contains but one amino acid.
• AA can also contain a labeled amino acid residue (chromophore, fluorophore, or radioisotope containing) , or an amino acid that could be easily labeled (e.g. tyrosine can be iodinated) .
• a labeled amino acid residue chromophore, fluorophore, or radioisotope containing
• an amino acid that could be easily labeled e.g. tyrosine can be iodinated
• the peptide sequence can optimize the lability of the covalent linkage between the water-soluble polymer and the polypeptide to proteolytic enzymes.
• the peptide sequence is preferably as long as possible and preferably contains natural amino acid residues.
• the polymer conjugates can be used to deliver physiologically active polypeptides or glycopolypeptides to specific sites, such as cancer cells having elevated concentrations of certain proteolytic enzymes to which the peptide sequence is labile.
• the length and sequence of the peptide in this second instance can be fine-tuned depending on the system of use and specificity of the target enzyme. Usually, three to seven amino acid residues would be required. Using modern techniques of peptide chemistry such short peptide sequences can be readily assembled.
• X can also contain a second peptide sequence residue.
• X is an acyl hydrazine derivative, X would have the general formula
• the acyl hydrazine polymer derivativ containing a peptide sequence can be synthesized b first preparing the polymeric chloroformate as describe above.
• the polymeric chloroformate is then reacted wit the peptide or an amino acid derivative in a solvent i which the polymeric chloroformate is soluble, such a ethylene chloride.
• the peptide or amino acid i preferably in the form of the ester of the C-terminu acid group, more preferably methyl or ethyl esters.
• This reaction is also operative under mil conditions and typically runs to completion at roo temperature and * the resulting product can be readil converted to a hydrazide by hydrazinolysis.
• the acy hydrazine polymer derivative containing a peptid sequence is then recovered and purified by conventiona methods, such as repeated precipitation of the polymer product.
• the acyl hydrazine polyme derivative containing a peptide sequence or an amin acid can be prepared by reacting the peptide sequenc with a succinimidyl carbonate active ester of th polymer, as disclosed by the above-mentioned Zalipsky, U.S. Patent Appln. No. 340,928 or by directly reactin isocyanate derivatives of an amino acid with th terminal hydroxyl group of the polymer as disclosed b Zalipsky et al.. Int. J Peptide Protein Res.. 30. 740 (1987) , the disclosures of both of which are hereby incorporated herein by reference thereto.
• Either of the above polymer-polypeptide derivatives can be readily converted to a hydrazide by the hydrazinolysis method disclosed above to yield an acyl hydrazine.
• the preparation of peptide sequences is essentially conventional and disclosed by the above-cited Bodanszky, Principles of Peptide Synthesis, the disclosure of which is hereby incorporated herein by reference thereto.
• the hydrazone can be reduced to the more stable alkyl hydrazide by reacting the hydrazone with, for example, NaBH 4 or NaCNBH 3 .
• R3-C-OH e.g., EDC R 3 -C-R 4
• R again represents the above-described water-soluble polymers, and Z is the same as described above for Formulae I-IV.
• R 3 represents a polypeptide containing aspartic acid, glutamic acid or a C-terminus carboxylic acid residues.
• R 4 represents one of the above-described leaving groups substituted on the peptide carboxylic acid when the carboxylic acid group is activated as described above.
• R 4 and Z are the same as described above with respect to
• the conjugation of a polypeptide or glycopolypeptide with a water-soluble polymer first involves either oxidizing carbohydrate moieties of the glycopolypeptide or activating carboxylic acid groups of peptide moieties of the polypeptides or glycopolypeptides.
• the carbohydrate moieties can be oxidized by reacting the glycopolypeptide in aqueous solution with sodium periodate or enzymatically usin galactose oxidase or combination of neuraminidase an galactose oxidase as disclosed by Solomon et al., J. Chromatographv. 510. 321-9 (1990) .
• the reaction runs rapidly to completion at room temperature.
• the reaction medium is preferably buffered, depending upon the requirements of the polypeptide or glycopolypeptide.
• the oxidized glycopolypeptide is then recovered and separated from the excess periodate by column chro atography.
• Carboxylic acid groups of peptide moieties can be activated by reacting the polypeptide or glycopolypeptide with an activating reagent such as a water-soluble carbodimide such as EDC.
• the reactants are contacted in an aqueous reaction medium at a pH between about 3.0 and 8.0, and preferably about 5.0, which medium may be buffered to maintain the pH. This reaction is taking place under mild conditions (typically 4 to 37 C) that are tolerated well by most proteins.
• Polypeptides or glycopolypeptides having peptide units on which reactive carbonyl groups have been generated may be directly reacted with the acyl hydrazine polymer derivatives in an aqueous reaction medium.
• This reaction medium may also be buffered, depending upon the pH requirements of the polypeptide or glycopolypeptide and the optimum pH for the reaction, which pH is generally between about 5.0 and about 7.0 and preferably about 6.0.
• the optimum reaction media pH for the stability of particular polypeptides or glycopolypeptides and for reaction efficiency, and the buffer in which this can be achieved is readily determined within the above ranges by those of ordinary skill in the art without undue experimentation.
• the operativeness of the within reactions under mild conditions is defined as meaning that the preferred temperature range is between about 4 and about 37 X C.
• the reactions will run somewhat faster to completion at higher temperatures, with the proviso that the temperature of the reaction medium cannot exceed the temperature at which the polypeptides or glycopolypeptides begin to denature.
• polypeptides and glycopolypeptides will require reaction with the polymer acyl hydrazine derivatives at reduced temperatures to minimize loss of activity and/or prevent denaturing.
• the reduced temperature required by particular polypeptides and glycopolypeptides is preferably no lower than 4 ⁇ C and in no event should this temperature be lower than 0 C. The reaction will still take place, although longer reaction times may be necessary.
• the polypeptide or glycopolypeptide is reacted in aqueous solution with a quantity of the acyl hydrazine polymer derivative in excess of the desired degree of conjugation. This reaction also proceeds under mild conditions, typically at 4 to 37 X C.
• the reaction medium may be optionally buffered, depending upon the requirements of the polypeptide or the glycopolypeptide, and the optimum pH at which the reaction takes place.
• the conjugated product is recovered and purified by diafiltration, column chromatography or the like.
• the degree of polymer conjugation of the polypeptide or glycopolypeptide can then be determined by amino acid analysis.
• acyl hydrazine polymer derivatives of the present invention possess the optimum balance of reactivity and selectivity so that polymer conjugates can be formed with non-amino functional groups of polypeptides and glycopolypeptides with virtually no competition between the acyl hydrazines and the peptid amino groups for the non-amino functional groups.
• crosslinking is prevented and the activity of th polypeptide or glycopolypeptide is preserved.
• Methoxy-PEG (mPEG) is available fro Union Carbide.
• the solvents used, as well as beta-alanine ethyl ester HCL, hydrazine, P2°5' EDC , N-hydroxy-5-norbornene-2,3-dicarboximide (HONb) , NaCNBH 3 and NaI0 4 are available from Aldrich Chemicals of Milwaukee, Wisconsin. Chymotrypsin was obtained from Worthington Chemical. BSA, ovalbumin and human immunoglobulin G (IgG) are available from Sigma Chemical of St. Louis, Missouri. G-CSF was obtained from Amgen of Thousand Oaks, California.
• EXAMPLE 1 SYNTHESIS OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-ALANINE: mPEG (MW n 5,000, 100 g, 20 mmol) was dissolved in toluene (250 mL) and azeotropically dried for two hours under reflux. The solution was brought to 25 ⁇ C, diluted with methylene chloride (50 mL) and then treated with phosgene (30 mL of 20 percent toluene solution, 56 mmol) overnight. The solvents and the excess of phosgene were removed by rotary evaporation under vacuum.
• the mPEG-beta-alanine ethyl ester (62 g, 12 mmol) was dissolved in pyridine (120 L) and treated with hydrazine (12 mL, 0.375 mole) under reflux for six hours. The solution was rotary evaporated to dryness and the residue crystallized twice from isopropanol and dried in vacuo over P 2 0 5 . The yield was 60 g (97%) .
• TNBS gave 0.2 mmol/g (103% of theoretical) .
• the beta-alanine content of the polymer was 0.205 mmol/g (105% of theoretical) as determined by amino acid analysis of a completely hydrolysed (6N HC1, 110 C, 24 h) aliquot of the product.
• Example 2 The same conjugation protocol as Example 2 was employed, in the presence of HONb (28.7 mg, 0.16 mmol).
• the PEG-chymotrypsin obtained had an average 2.7 molecules of mPEG per molecule of protein, based on quantitation of beta-alanine by amino acid analysis. This demonstrates that the conjugation process is only slightly enhanced by the presence of HONb.
• EXAMPLE 4 COUPLING OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-ALANINE TO EDC-ACTIVATED CARBOXYL GROUPS OF BSA: A solution of BSA (20 mg) and a mPEG-beta-alanine hydrazide derivative of Example 1 (800 mg, 0.16 mmol) in 50 mM NaCl (10 mL) was treated with EDC (15 mg, 0.078 mmol) overnight at pH 5.0, 25 C as in Example 2. Excess reagents were removed by extensive diafiltration of the reaction solution at 4 ⁇ C against phosphate buffer (50 mM, pH 7.7).
• phosphate buffer 50 mM, pH 7.7
• the content of beta-alanine in the conjugate corresponded to 8.1 residues of mPEG per molecule of BSA.
• a GF-HPLC comparison of the PEG-conjugate to native BSA was performed with a BIOSEP SEC 4000 column, the results of which are depicted in FIG. 1.
• the elution conditions were 10% (vol/vol) methanol/40 mM phosphate buffer.
• FIG. 1 depicts good homogeneity of the PEG-conjugate 1, with a substantially increased molecular weight as compared to the native BSA 2.
• Ovalbumin (20 mg, 4.4 x 10 ⁇ 7 mole) dissolved in Phosphate Buffered Saline (PBS) buffer, pH 6.0 (1.8 mL) was treated with NaI0 4 (0.2 mL of 200 mM aqueous solution) . The reaction was allowed to proceed in the dark at 4 ⁇ C. After one hour, the oxidized glycoprotein was separated from the excess of periodate by passing the reaction solution through a 12 mL Sephadex G-25 column equilibrated with acetate buffer to pH 5.0. Additional samples were prepared and the procedure was repeated equilibrating the column with PBS buffer at pH 6.0 and phosphate buffer at pH 7.0. This resulted in three separate reaction mixtures having different buffering systems.
• PBS Phosphate Buffered Saline
• Example 1 To each mixture was added the mPEG-beta-alanine-hydrazide derivative of Example 1 (150 mg, 2.9 x 10 ""5 mole). Each of the three reaction mixtures was divided into two equal portions and NaCNBH 3 (0.3 mL of 6.6 mg/mL solution, 3.15 x 10 ⁇ 5 mole) was added to one portion of each. The reactions were allowed to proceed overnight at 4 C. Each solution was diafiltered using phosphate buffer pH 7.7 until all the unreacted reagents were removed. The conjugates in the solutions to which the NaCNBH 3 was added formed
• FIG. 2 Depicted in FIG. 2 is the GF-HPLC analysis using a TSK G 4000SW column and a 10% (vol/vol) methanol/40 mM phosphate buffer pH 7.5 mobile phase, which showed good homogeneity of the mPEG-ovalbumin conjugate 3, and a substantially increased molecular weight as compared to the native ovalbumin 4.
• FIG. 3 depicts good homogeneity of the PEG-conjugate 5, with a substantially increased molecular weight as compared to the native IgG 6.
• the amount of beta-alanine was determined by amino acid analysis of a hydrolyzed (6 N HCl, 110 C, 24 h) aliquot of the PEG-IgG conjugate to correspond to six residues of mPEG per protein molecule.
• EXAMPLE 7 ATTACHMENT OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-ALANINE TO THE CARBOHYDRATE MOIETY OF IMMUNOGLOBULIN G WITHOUT REMOVAL OF EXCESS PERIODATE:
• EXAMPLE 8 ATTACHMENT OF mPEG-HYDRAZIDE DERIVATIVE TO CARBODIIMIDE- ACTIVATED CARBOXYL GROUPS OF G-CSF: The mPEG-beta-alanine-hydrazide of Example 1
• the average number of mPEG residues in the PEG-G-CSF was 5.8, as determined by measuring the amount of beta-alanine in an hydrolyzed (6 N HCl, 110 C, 24 h) aliquot of the conjugate.
• TNBS assay confirmed that both native and PEG-modified G-CSF-1 had the same number of amino groups, indicating that the EDC activated carboxylic acid groups of the protein did not react with amino groups of the protein.
• the preparation of mPEG-G-CSF gave four separate bands on SDS-PAGE (PhastGel-, Homogenous 12.5, Pharmacia) in the range from 29,000 to 67,000 daltons.
• the present invention is applicable to the production of polymers conjugated with various biologically active and pharmaceutically active compounds representing a novel form of drug delivery.

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  • 1992-03-12 CA CA002101918A patent/CA2101918A1/en not_active Abandoned
  • 1992-03-12 JP JP4508914A patent/JPH06506217A/ja active Pending
  • 1992-03-12 WO PCT/US1992/002047 patent/WO1992016555A1/en not_active Application Discontinuation
  • 1992-03-12 AU AU16769/92A patent/AU1676992A/en not_active Abandoned
  • 1992-03-12 EP EP19920909326 patent/EP0576589A4/en not_active Withdrawn

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