EP0702553A1 - Agents de reticulation a base de polyamides non immunogene hydrosoluble - Google Patents

Agents de reticulation a base de polyamides non immunogene hydrosoluble

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Publication number
EP0702553A1
EP0702553A1 EP95906090A EP95906090A EP0702553A1 EP 0702553 A1 EP0702553 A1 EP 0702553A1 EP 95906090 A EP95906090 A EP 95906090A EP 95906090 A EP95906090 A EP 95906090A EP 0702553 A1 EP0702553 A1 EP 0702553A1
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Prior art keywords
polyamide
group
water
soluble
product
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP95906090A
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German (de)
English (en)
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EP0702553A4 (fr
Inventor
Tom That Hai
Deanna J. Nelson
David Eugene Pereira
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Baxter International Inc
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Baxter International Inc
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Publication of EP0702553A4 publication Critical patent/EP0702553A4/fr
Publication of EP0702553A1 publication Critical patent/EP0702553A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups

Definitions

  • the present invention relates to water-soluble, substantially nonimmunogenic polyamide cross-linking agents.
  • the present invention also relates to covalent binding of water-soluble polyamides to proteins, polynucleotides and other biological substrates to form substantially nonimmunogenic water- soluble products.
  • the present invention also relates to proteins, polynucleotides and other biological substrates which are cross-linked, conjugated, polymerized or decorated with water-soluble polyamides to form substantially nonimmunogenic products.
  • Cross-linking reagents are used for a variety of purposes, including the investigation of the spatial arrangement and functions of various macromolecular entities, the identification of binding sites (receptors) for ligands, the preparation of affinity matrices, and the modification and stabilization of diverse macromolecular structures (Methods in Enzymology, Volume 91, pages 580 to 609 (1983)) .
  • Cross-linkers have been designed to preserve electrostatic charge; to alter electrostatic charge; to decrease immunogenicity; to increase and decrease susceptibility to proteolysis; to introduce fluorescent labels, spin labels, radiolabels, and electron-dense substituents; to attach several different types of carbohydrate moieties; to modify enzyme specificity; and to introduce intramolecular and/or intermolecular cross-links, both to couple already associated species and to join various proteins in order to combine the properties of both into a single molecule (G. E. Means and R. E. Feeney, Bioconjugate Chemistry, Volume 1, page 2 to 12 (1990)) .
  • a large number of cross-linking reagents have been developed to serve these and a variety of other purposes. Many of these reagents are commercially available.
  • Cross-linking of proteins and their immobilization has been employed to increase the stability of proteins or of certain conformational relationships in proteins; to couple two or more different proteins; to identify or characterize the nature and extent of certain protein- protein interactions or to determine distances between reactive groups in or between protein subunits.
  • Proteins may be immobilized to facilitate their use and their separation from other products.
  • Cross- linking therapeutic proteins or polypeptides has been shown to decrease immunogenicity and to increase the lifetime of the cross-linked product in the blood stream.
  • cross-linking agents consist of an organic bridge between activated termini. The termini bind to biological macromolecules to form a link.
  • organic bridges are recognized in the art, including peptides, carbohydrates (e.g., dextran, starch, and hydroxyethylstarch) , fatty acids, polyglycolides, polypeptides (e.g., gelatin or collagen), polyalkylene units, and polymers such as poly(vinylalcohol) , polyvinylpyrrolidone, and polyethylene glycol (also known as polyoxyethylene) .
  • homobifunctional and heterbifunctional cross-linking agents range in size from about 6 to 16 A. Their solubility in water decreases with chain length. Yet the efficiency of cross-linking is increased with chain length as steric hindrance is reduced.
  • Peptides composed of three to nine amino acid residues are commonly used as cross-linking agents.
  • these suffer from the following disadvantages: the chemistries used in peptide synthesis are complex, involving selective blocking and deblocking of functional groups and specific coupling conditions. Care must be taken not to racemize the amino acid components. Peptides must be chosen carefully so that they have no biological activity. Finally, they are subject to enzymatic hydrolysis, which limits their period of utility, particularly during circulation in vivo.
  • a synthetic polymer cross- linker desirably has the following characteristics: (1) The polymer must be water-soluble and exhibit a narrow, definite molecular weight distribution. (2) It should provide attachment/release sites or the possibility of the incorporation of such sites. (3) The polymer should be compatible with the biological environmental, i.e., non-toxic, non-antigenic, and not provocative in any other respect. (4) It should be biodegradable or eliminated from the organism after having fulfilled its function (Duncan and Kopecek, Advances in Polymer Science, Volume 97, pages 53 to 101 (1984)) .
  • U.S. Patent No. 5,122,614 to Zalipsky describes the use of polyethylene gycol as a cross-linking agent.
  • U.S. Patent No. 5,053,520 to Bieniarz describes polyamino acid based coupling agents which are not water-soluble.
  • U.S. Patent No. 4,182,695 to Horn describes protein bound to polyamides .
  • Russian Patent Application No. SU 1659433 discloses water- soluble polyamides with luminescent groups in the chain.
  • U.S. Patent No. 5,110,909 to Dellacherie discloses water-soluble macromolecular conjugates of hemoglobin.
  • PCT Application WO 92/08790 to Cargill discloses the use of polyamide polymers bonded to a linker group which is bonded to a protein.
  • Many potentially therapeutic proteins have undesirable characteristics such as short half life in vivo, poor solubility, vulnerability to enzymatic degradation in vivo, or immunogenicity.
  • the polyamides of the present invention when coupled to such proteins overcome these disadvantages.
  • the present invention is water-soluble, substantially nonimmunogenic polyamides having number average molecular weights of about 300 to about 20,000 grams per mole; where the amide repeat units are comprised of: (i) a water-soluble organic acid subunit having at least one carboxylate group and fifteen or fewer atoms separating the amide functionalities in the polyamide; covalently linked as an amide to (ii) a water-soluble organic amine subunit having at least one primary amino group and fifteen or fewer atoms separating the amide functionalities in the polyamide.
  • the polyamide of the present invention is a water-soluble, substantially nonimmunogenic polyamide selected from the formulas I, II, and III:
  • n is the number of amide repeat units in the polyamide; and (v) where the acid subunits of the amide repeat units are (a) organic acids having fifteen or fewer atoms in the chain and having one or more heteroatoms selected from the group consisting of O other than carboxyl or carbonyl O, S, P or tertiary N present as substituents on or atoms in the chain, or (b) two or more of such organic acids bridged by water-soluble organic diamines; and (vi) where the amine subunits of the amide repeat units are organic, water-soluble amines having at least one primary
  • the present invention includes one or more such polyamides used to cross-link, conjugate, decorate or polymerize proteins, antibodies, haptens, polypeptides, polynucleotides or other biological substrates.
  • the cross-linked, conjugated, polymerized or decorated product is water-soluble, substantially nonimmunogenic and retains all or a useful portion of the physiological activity of the substrate.
  • Figure 1 shows the polycondensation of ethylene glycol bis (methoxycarbonylmethyl ether) and 1,4- diaminobutane.
  • Figure 2 shows the reaction conditions, product characteristics, and yield of the reactions shown in Figure 1.
  • Figure 3 shows the experimental data, including oxygen binding function of diaspirin cross-linked hemoglobin polymerized and decorated with PAS-2400.
  • Figure 4 shows the size exclusion chromatographic profiles of diaspirin cross-linked hemoglobin polymerized and decorated with PAS-2400.
  • Figure 5 shows the reverse phase HPLC profiles of diaspirin cross-linked hemoglobin polymerized and decorated with PAS-2400.
  • Figure 6 depicts the components of polyamide synthesis.
  • Figure 7 depicts the synthesis of BMDAB (a polyamide component) .
  • Figure 8 depicts the polycondensation of BMDAB with diamine, to form a polyamide.
  • Figure 9 depicts the synthesis of polyamide activated esters PAS-3037 and PAS-4200.
  • Figure 10 depicts the synthesis of maleimide- capped polyamide, designated PAM-4080.
  • Figure 11 depicts the size exclusion profiles following polymerization of diaspirin cross-linked hemoglobin with PAS-3037.
  • Figure 12 depicts size exclusion chromatography following polymerization of diaspirin cross-linked hemoglobin with PAS-4200.
  • Figure 13 depicts the reverse phase HPLC profiles following polymerization of diaspirin cross- linked hemoglobin with PAS-4200.
  • Figure 14 depicts the size exclusion chromatography profiles following polymerization of diaspirin cross-linked hemoglobin with PAM-4080.
  • Figure 15 depicts reverse phase HPLC following polymerization of diaspirin cross-linked hemoglobin with PAM-4080.
  • Figure 16 shows the experimental data following polymerization of diaspirin cross-linked hemoglobin with PAS-3070.
  • Figure 17 shows the experimental data following polymerization of diaspirin cross-linked hemoglobin with PAS-4080.
  • Figure 18 shows the experimental data following polymerization of diaspirin cross-linked hemoglobin with PAM-4080.
  • the polyamides of the present invention are substantially non-immunogenic, water-soluble polyamides having number average molecular weights of about 300 to about 20,000 grams per mole.
  • the amide repeat units of these polyamides are composed of a water-soluble organic acid subunit having at least one carboxylate group and fifteen or fewer atoms separating the amide functionalities in the polymer, covalently linked as an amide to a water-soluble organic amine subunit having at least one primary amino group and fifteen or fewer atoms separating the amide functionalities in the polymer.
  • polyamides may be employed directly or after activation, for the purposes of cross-linking, conjugating, polymerizing and/or decorating biological substrates such as proteins, polypeptides, antibodies, haptens, carbohydrates or polynucleotides to give products which are water-soluble, substantially nonimmunogenic, and which retain all or a useful portion of the substrate's physiological activity. They may also be used to attach substrates to detection agents or solid matrices.
  • substantially non-immunogenic indicates that the polyamide does not elicit a humoral or cell-mediated immune response, either in vivo or in vi tro .
  • water-soluble indicates that the polyamide has a solubility in water that exceeds 500 mg per 100 mL.
  • the term also indicates that the polyamide does not act as a detergent and does not form aggregates such as micelles in water.
  • activation means converting a group located on a polyamide terminus to a more reactive coupling group.
  • the polyamide may be linear or branched.
  • substrate means the molecule to which the polyamide of the present invention is bound.
  • Substrates include but are not limited to proteins such as enzymes, growth factors, antibodies or blood proteins; polynucleotides such as complementary DNA fragments; steroids and hormones; immunoconjugates; carbohydrates; and conjugates of any of these substrates.
  • the substrate may also be a solid support or bead.
  • Substrates include molecules having therapeutically useful biological activity.
  • a substrate is said to be "decorated” when multiple polyamides are bound to the substrate by one terminus of each polyamide and all other termini of the polyamide are not bound to a different substrate molecule.
  • the water-soluble polyamides of this invention may be prepared by methods known in the art. Known methods for the preparation of polyamides are incorporated here by reference as useful methods for the preparation of the polyamides of the present invention.
  • N. Ogata et al. Polymer Journal, Volume 5, pages 186ff (1973) and N. Ogata and Y. Hosoda, Journal Polymer Science, Polymer Lett. Ed., Volume 12, pages 355ff (1974) describe the polycondensation of diesters activated by ether or hydroxyl groups with diamines.
  • N. Ogata et al. Journal Polymer Science, Polymer Chemistry Ed., Volume 14, pages 783ff (1976), N. Ogata et al.
  • the acid subunits of the amide repeat units are selected from the group of organic acids having fifteen or fewer atoms in the chain and having heteroatoms (O, S, P, N) present either as substituents on or atoms in the chain.
  • the acid subunits of the amide repeat units may consist of two or more such organic acids joined to bridging water-soluble, organic diamines.
  • the amine subunits of the amide repeat units are selected from among the group of organic amines having fifteen or fewer atoms in the chain and having heteroatoms (0, S, P, N) present as substituents on or atoms in the chain.
  • Polyamides of similar and/or dissimilar • structure may be linked by a central polyacid, polyamine or polyamino acid to form branched, water- soluble polyamides.
  • X is a polyamide selected from (B-A) n , (A-B) n , and branched polyamides formed by linking (B-A) n or (A-B) n to a central polyacid, polyamine or poly(amino acid) .
  • the acid subunits of the polyamide repeat units are two or more organic acids each of said organic acids having fifteen or fewer atoms in the chain and having one or more heteroatoms selected from the group consisting of 0 other than carboxyl or carbonyl 0, S, P or tertiary N present as substituents on or atoms in the chain bridged by a water-soluble diamine having the formula -NH-R ⁇ _-NH- where R ] _ is a substituted or unsubstituted aliphatic chain having from about 4 to about 5 carbon atoms .
  • the water-soluble diamine having the formula -NH-R]_-NH- is 1,4- diaminobutane.
  • the acid subunits of the amide repeat units are organic acids having the formula -OC-CH-CH 2 -S-(CH 2 ) m -X 1 -CH -CH 2 -X 2 -(CH2) m -S-CH 2 CH-CO-
  • R2 R 2 where m is from about 2 to about 4, X]_ and X 2 are independently a heteroatom selected from the group consisting of 0 other than carboxyl or carbonyl O, S, P or tertiary N, and R 2 is H or acetamide.
  • the sulfur group is located in a position ⁇ to each of the terminal carboxyl groups.
  • X]_ ancj X 2 are both O.
  • X]_ anc j X 2 are both O and m is 2.
  • Compounds in accordance with this aspect of the present invention have several advantageous features when used with substrates having therapeutic biological activity. These compounds are less reactive which permits greater control following activation to minimize hydrolysis. This, in turn, optimizes coupling with protein substrates. These polyamides are also effective oxygen radical quenchers.
  • the acid subunits of the amide repeat units are organic acids having the formula Y OC- -2-CH-S- (CH ) m -Xl-CH2-CH 2 -X2- (CH 2 ) m -S-CHCH 2 -COYi
  • X ⁇ i and X2 are independently a heteroatom selected from the group consisting of 0 other than carboxyl or carbonyl O, S, P or tertiary N, and R3 is a lower alkyl having from about 1 to about 2 carbon atoms.
  • X]_ and X2 are both O.
  • X ⁇ i and X2 are both O, m is 2, and R3 is methyl.
  • A' is an ⁇ , ⁇ -di-acid having the formula Y]_-A-Y]_, where Y ⁇ has the formula -OC (CH2) p -NH- where A is an ⁇ , ⁇ -di- acid as described herein above.
  • Any of the known coupling chemistries may be used to activate polyamides of this invention to decorate, link, polymerize and/or conjugate substrates. Many examples of such coupling chemistries are given in "Chemistry of Protein Conjugation and Cross-linking," S. Wong, CRC Press,
  • Such chemistries include reacting the polyamides with bi- or poly- functional protein reagents such as dialdehydes, N-hydroxysuccinimide esters, functionalized acetals, bis-maleimides, bifunctional imino esters, diepoxides, and dicarboxylic acid chlorides.
  • bi- or poly- functional protein reagents such as dialdehydes, N-hydroxysuccinimide esters, functionalized acetals, bis-maleimides, bifunctional imino esters, diepoxides, and dicarboxylic acid chlorides.
  • the choice of coupling chemistries will depend upon the substrate molecule being cross-linked, conjugated, polymerized and/or decorated. The coupling chemistry should be selected so that it does not alter the biological or chemical activity of the substrate molecule.
  • substrates such as amino acids, peptides, proteins, nucleotides, polynucleotides, pharmaceutic agents, and diagnostic agents have functional groups which may be covalently bound to the pendant functional groups of the polyamide backbone and functionalized derivatives thereof.
  • the order of reaction is not important.
  • the pendant functional group(s) of the polyamide may be activated appropriately, if so required, and then attached to the substrate.
  • the substrate may be activated appropriately, if necessary, and then attached to the polyamide.
  • amino, hydroxy, carbonyl, carboxyl, or thiol substituents are commonly found as part of the structure of amino acid, peptide, protein, nucleotide, polynucleotide, and diagnostic agent compounds.
  • the polyamide may be synthesized to incorporate reactive termini such as these substituents.
  • the substrate may be joined to the polyamide by chemistries such as those cited below, or by other chemistries such as those disclosed in Bodanszky and Bodanszky, “The Practice of Peptide Synthesis,” Springer-Verlag, New York, (1984); Lundblad, “Chemical Reagents for Protein Modification,” CRC Press, Boca Raton, Florida, (1991); Mosbach “Methods in Enzymology, Volume XLIV, Immobilized Enzymes,” Academic Press, New York, (1976); or Uhlmann and Peyman, “Antisense Oligonucleotides: A New Therapeutic Principle," Chemical Reviews, Volume 90, No. 4, pages 543 to 585 (June 1990) .
  • biotin is recognized as a diagnostic probe that is selectively retained by complexation with avidin.
  • Biotin contains a carboxyl group that may be activated as a succinimidyl ester and attached to a polyamide having a amino terminus. Either prior to or following covalent bonding to biotin, the other terminus of the polyamide may be covalently bonded to a peptide, protein or other biochemical agent. Under these conditions, the polyamide serves as a spacer group that concurrently maintains or increases the aqueous solubility of the product. The biochemical agent is thereby labeled with a diagnostic probe that is positioned at the end of the polyamide spacer to facilitate interaction with avidin.
  • deferoxamine is a pharmaceutic agent that is used therapeutically as an antidote to iron poisoning.
  • the duration of therapeutic action of deferoxamine is short, because it is rapidly excreted via the kidney. It has been recognized that if deferoxamine is conjugated to a larger molecular weight entity such as a dextran or albumin, it will be retained in the vascular circulation for longer periods of time.
  • a polyamide may be used as a spacer group that concurrently maintains or increases the aqueous solubility of the product.
  • One terminus of the polyamide may be converted to a carbonyl functional group and attached to the amino sub ⁇ tituent of deferoxamine by reductive amination, and the other terminus of the polyamide may be converted to an activated ester (e.g. a succinimidyl ester) and attached to albumin.
  • an activated ester e.g. a succinimidyl ester
  • All the components of the polyamides of the present invention are selected so as to preserve water-solubility. They are water-soluble, hydrophilic over the entire chain length.
  • the length of the polyamide is chosen to facilitate interaction between the substrate and the polyamide.
  • the cross-linking of a large substrate will require a longer polyamide since it will minimize steric interactions between two large substrate molecules.
  • An immunogenic substrate should generally be highly decorated and should have relatively long chain polyamides.
  • polyamides of the present invention In reacting the polyamides of the present invention to biologically active substrates, such as enzymes, care is taken to avoid destroying the activity of the substrate.
  • biologically active substrates such as enzymes
  • care is taken to avoid destroying the activity of the substrate One skilled in the art will understand that varying the degree of decoration and/or polymerization will allow one to prepare a product having a useful biological activity.
  • the polyamides of the present invention are not polymers of ⁇ -amino acids, so they are not subject to enzymatic hydrolysis.
  • polyamides of the present invention may be used to render substrates soluble in organic solvents such as methanol, ethanol or acetonitryl.
  • the polyamides of the present invention may be used as polymerization agents.
  • the termini of a polyamide have been modified as maleimide groups, suitable for reaction with thiol substituents of proteins.
  • Bis (maleimide) polyamide was employed to polymerize human hemoglobin via the cysteine-E93 thiol residues of that protein.
  • the termini of a polyamide were converted to bis (succinimidyl) esters or bisaldehydes, suitable for reaction with amino substituents of proteins. Both the bis (succinimidyl) polyamide and the bisaldehyde polyamide have been employed to polymerize human hemoglobin via the ⁇ -amino groups of lysine residues of the protein.
  • polyamides of the present invention may be used to link probes (e.g., fluorescent, radioactive, etc.) to a substrate to be detected.
  • probes e.g., fluorescent, radioactive, etc.
  • the polyamide is bis (maleimidoacyl) polyamide.
  • the polyamide is bis (maleimidoglycyl) polyamide.
  • a polyamide identified as PAM-3800 is a polyamide bis (maleimide) having a molecular weight of about 3800 Daltons.
  • each component is assigned on the basis of relative migration (Rf) and reactivity toward ninhydrin spray reagent.
  • Rf relative migration
  • polyamides with diester end-groups have the largest Rf, followed by components with mono- ester/mono-amine end-groups, and di-amine end-groups, respectively. Only components having an amine end- group are reactive toward ninhydrin.
  • the structure of the mono- and di-esters is confirmed by base-catalyzed hydrolysis and TLC of the resulting products; under these conditions esters are hydrolyzed to acids and the Rf of the material decreases. Finally, the molecular weight is estimated by amino end-group analysis using fluorescamine.
  • Precisely and accurately weighed polyamide samples are dissolved in methanol/phosphate buffer, derivatized by adding fluorescamine dissolved in acetone, and then analyzed by flow injection with a HPLC system equipped with a fluorescence detector.
  • Equivalent weights are determined by comparison of responses for standard solutions of dia inohexane/PEG/ethyl acetate in methanol/phosphate buffer. Equivalent weights are converted to number average molecular weights based on the average number of amines per molecule.
  • the NMR spectrum can be used to estimate the number average molecular weights of the polyamides, as follows: the first step is to divide the structure of the polyamide into end groups and repeating units.
  • each part is calculated.
  • one identifies unique components in each part and correlates the corresponding NMR resonance with that component.
  • Polyamides have a number of well-resolved resonances that can be correlated with specific functional groups. For example, the two pairs of two hydrogens on the succinate group in PAS-4200 give rise to (triplet) resonances at about 2.53 and 2.92 ppm having integrals of 2.197 and 2.605 units, respectively. Similarly, the internal methylene groups of the butanediamine residue give rise to a broad resonance at 1.5 ppm having an integrated area of 16.034 units.
  • the molecular weight of the polyamide is the sum of the molecular weights of each of the end groups (416.44 and 198.14, respectively) and the multiple seven times the molecular weight of the repeat unit (7 x 504.57 or 3532) .
  • the sum is 4146.57 or about 4200 Da. This value was also obtained independently by end-group analysis of the polyamide bisamine precursor of PAS-4200 using fluorescamine.
  • Ethylene glycol bis (methoxycarbonylmethyl) ether (EDE) , which has an ether group as a substituent ⁇ to each ester group, was condensed with 1, 4-diaminobutane (DAB) to produce polyamides. See Figure 1. Two poly- condensation methods were used: the solution method and the melt method.
  • the polycondensations were completed as follows.
  • EDE and DAB in the desired molar ratio were dissolved in methanol, and the solution was heated at 30°C for seventy two hours or at 65°C for twenty four hours.
  • the solvent was evaporated and the residue was treated with acetone and repeatedly evaporated to remove residual methanol. Trituration of the residue with acetone afforded a solid.
  • a mixture of EDE and DAB was heated at 120°C under vacuum with magnetic stirring to remove methanol . After one to two hours the mixture was dissolved in methanol.
  • the solution was evaporated to dryness and the residue was triturated with acetone to give polyamide product.
  • Diamino-polyamide I having a MW in the range of 1,300 to 1,500 Dalton could be prepared either by the solution or the melt method employing a DAB/EDE molar ratio of 1.3 to 1.5.
  • ⁇ , ⁇ -Diester-polyamides III were obtained in good yield by the melt method with equimolar EDE and DAB. Because DAB is a volatile compound, DAB is gradually removed from the reaction mixture when the melt method is utilized, leaving EDE in large excess. Consequently III is obtained as the major product.
  • Example 1 (b) Conversion of polvamide III to an activated cross-linking agent.
  • Example 2 Polymerization of Hemoglobin with PAS-2400.
  • a typical polymerization of diaspirin cross- linked hemoglobin (designated DCLHb) with PAS-2400 was completed as follows.
  • DCLHb was prepared according to the method described in U.S. Patent No. 5,128,452.
  • a solution of DCLHb in 0.1 M HEPES of about pH 7 to 8 was deoxygenated by successive vacuum / nitrogen cycles for one and a half hours at room temperature.
  • PAS-2400 was dissolved in deoxygenated water, and the solution was added immediately to the DCLHb solution.
  • reaction mixture was stirred at room temperature under nitrogen, and the reaction was monitored by size exclusion chromatography using TSK-G4000SW brand and TSK-G3000SW brand columns connected in series with mobile phase consisting of 2-propranol/50mM phosphate buffer, pH 6.5 (1:9, v/v), delivered at a flow rate of 1 mL/minute detection at 280 nm.
  • the latter method demonstrated that the polymerization was completed in less than thirty minutes and that polymerization was accompanied by decoration.
  • the solution was cooled to 5°C and a solution of 1 M NAC (N-acetyl-L-cysteine) (molar ratio of NAC/Hb about 5:1) was added.
  • the data indicate the following.
  • the yield of oligomer is increased with increasing ratios of PAS- 2400 to DCLHb.
  • SEC elution times of DCLHb monomer decrease with increasing molar ratios of PAS-2400, indicating that PAS-2400 decorates DCLHb.
  • Polymerization is fast; it was complete in less than thirty minutes.
  • competitive hydrolysis of the polymerization agent is also fast.
  • As the solution pH is increased higher yields of high molecular weight polymers are obtained.
  • five equivalents of PAS-2400 give 7%, 17%, and gel, respectively, of high molecular weight polymers at values of pH of 7.0, 7.5, and 8.0, respectively.
  • P50 values and n values of DCLHb polymerized with PAS-2400 are in the range of 29 to 33 mm Hg and 1.8 to 2.1, respectively.
  • P50 is the oxygen partial pressure at which hemoglobin is half saturated while the "n" value is a measure of the cooperativity of oxygen binding.
  • the P50 of human hemoglobin in red blood cells is about 28. Th s, the excellent oxygen- binding function of DCLHb is maintained in these polymers.
  • RP-HPLC analysis ( Figure 5) indicates than both ⁇ — and ⁇ -chains are modified. However, ⁇ - chains apparently are more extensively modified than are ⁇ -chains.
  • PAS-2400 can be used to produce decorated, polymerized DCLHb.
  • the short reaction time (thirty minutes) is favorable for large-scale synthesis.
  • Two to four equivalents of PAS-2400 at pH 7.0 are suitable for polymerization.
  • the hemoglobin maintains its biological activity, i.e., oxygen binding and, as described below is nonimmunogenic.
  • Example 3 Methods for the Synthesis of Longer Polyamides.
  • Longer polyamides are obtained if the lengths of the component acid and amine are increased, i.e., polymerization with adipic acid (six carbons) or 1,6- hexanediamine (six carbons) yields longer polymers than does polymerization with succinic acid (four carbons) or 1, 4-butanediamine (four carbons) .
  • increases in chain length using hydrocarbon components would reduce the aqueous solubility of the protein.
  • DAB (1,4-diaminobu ⁇ tane) was allowed to react with two equivalents of glycolic anhydride in N,N-dimethylformamide (DMF) to give an almost quantitative yield of BCDAB [1,4- bis (carboxymethoxyacetamido) butane] .
  • the latter was esterified in methanol in the presence of aqueous HC1 or HCl in dioxane solution.
  • HC1 in solution is the ease of carrying out the reaction, especially in a large scale synthesis, and the observation that an exact amount of HCl can be employed to avoid the formation of by-products. Gaseous HCl was tried, but a by-product was detected in the product mixture.
  • the first attempt to synthesize polyamide bis (N- hydroxysuccinimide) ester was a three step synthesis from BMDAB and EGBE ( Figure 9) .
  • EGBE and BMDAB in a molar ratio of 1.3 to 1.0 were condensed by the solution method using methanol as solvent at 65°C for 24 hours to give a slightly orange solution.
  • the product could be decolorized by adding decolorizing charcoal (NoritTM A) to the solution, filtering, and evaporating to dryness.
  • a white product ( Figure 9, 2a), having a MW of 2700, was isolated by crystallization from methanol-acetone. The product was not stable and turned yellow during storage.
  • the mixture was treated with sodium hydroxide to convert the methyl ester to bis (2- carboxyethylcarbonyl)polyamide, (3a) and then stirred with cation exchange resin (AG50W-X8) to absorb polyamide amine by-product. After removal of the resin by filtration, the filtrate, which contained a single product as indicated by TLC, was concentrated. Pure product bis (2-carboxyethylcarbonyl)polyamide (3a) was obtained by crystallization from methanol/acetone. Third, conversion of the pure product to the activated diester (4a) (designated PAS-3070) was accomplished by treatment with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide (DCC) in DMF.
  • DCC dicyclohexylcarbodiimide
  • the polyamide bis (succinimide ester) was soluble in water but the coupling groups were slowly hydrolyzed. Therefore when dissolved in water the activated polymerization agent was used without delay.
  • the synthesis described above has several drawbacks.
  • the isolated white product, (2a) is not stable; it is oxidized during storage to an unknown yellow product which could not be removed readily by crystallization.
  • recrystallization of bis (2-carboxyethylcarbonyl) polyamide (3a) in methanol/acetone converts some of the di-acid to the corresponding methyl ester ( ⁇ ) .
  • our preferred synthetic strategy is as follows.
  • steps 1 and 2 were carried out as an integrated process in which product 3 in Figure 9, obtained from Norit A treatment, is not isolated but is allowed to react immediately with succinic anhydride to mask the amino groups which tend to be oxidized to colored product.
  • Such a "one-pot" synthesis increased the yield of 3 in Figure 9, because all crude 2 is used for the second step instead of the 50-60% of isolated product 2 that was converted in the method described above.
  • the use of methanol as crystallization solvent for 3 was excluded to avoid the formation of methyl ester of 3.
  • PAS-4200 ( Figure 9, 4b) was prepared using the integrated approach above.
  • crude product 6b obtained by this procedure, was mixed with cation-exchange resin (AG50W-X8) to remove unreacted polyamide amine and the purified polyamide polymerization agent 6b (designated PAM- 4080) was obtained by crystallization of crude product from methanol/acetone.
  • PAM derivatives are stable in water.
  • a typical polymerization of DCLHb with PAS derivatives of the type described in Examples 4 and 5 above or PAM derivatives of the type described in Example 6 above was completed as follows.
  • a solution of DCLHb (10 g/dL for PAS and 20 g/dL for PAM) was deoxygenated by successive vacuum/nitrogen cycles for 1.5 hours at room temperature.
  • Polyamide reagent in deoxygenated water was added immediately to the DCLHb solution.
  • the reaction mixture was stirred at room temperature under nitrogen and the course of the reaction was followed by SEC. Polymerization was completed within 2 to 3 hours for PAS derivatives and overnight for PAM derivatives.
  • the reaction mixture was cooled to 5°C; the solution pH was adjusted to
  • DCLHb polymerization by PAM derivatives may also be summarized.
  • RP-HPLC profiles Figure 15 suggest that reagent reacted specifically. A specific ⁇ '-peak, which could be a modified ⁇ -peak, was detected at all ratios of PAM tested. Specific reaction with the subunits was also supported by the decrease in titrable thiol residues.
  • Reagent PAM is expected to bind specifically to cysteine- ⁇ 93 residues, and about 65% and 90% of thiol groups are modified when 1 and 2 equivalents of PAM are used, respectively.
  • the binding of PAM to the cysteine residue results in a decrease in P50 values of the polymerized products to 18 to 20 mm Hg.
  • ⁇ - Chains are also modified, but much less extensively than the ⁇ -chains.
  • BIOLOGICAL TESTING In examples 8 through 12 we quenched the polyamide PAM-4200 by reaction with N-acetyl-L- cysteine and tested a sterile, non-pyrogenic solution of the polyamide (PAM-4080) in Ringer's lactate solution.
  • the polyamide concentration was 5 g/dL of solution.
  • the pH of the polyamide solution was adjusted to physiologic values.
  • the osmolality of the solution was within the physiologic range.
  • the concentration of the polyamide was selected to exceed projected use levels by at least an order of magnitude.
  • CCL 1 NCTC 929 (clone of strain L cells, mouse connective tissue) were cultured aseptically in sterile media until confluency.
  • the L-929 cell concentration was adjusted to about 1.3 x 10 ⁇ cells/mL, and aliquots were transferred to wells of a tissue culture plate. The plates were covered and incubated for approximately twenty four hours. Then the culture medium was aspirated from each well and aliquots of the test article solution and dilutions having PAM-4080 concentrations of 2.5 and 1 g/dL, respectively, were added to duplicate wells of the prepared plates. After incubation of the plates for approximately forty eight hours, the wells were stained with 2% crystal violet stain.
  • the toxicity was rated on a scale from 0 to 4+, where a rating of 0 corresponded to the presence of discrete intracytoplasmic granules and the absence of cell lysis and a rating of 4+ corresponded to nearly complete destruction of the cell layers. At the highest concentration, a moderate toxicity rating of 2+ applied. At the two lower concentrations, a toxicity rating of 0 applied, i.e., the polyamide caused no adverse biological response.
  • the polyamides of the present invention are expected to be nontoxic when administered as conjugates of therapeutically useful substrates.
  • Acute toxicity testing in rodents Doses of 500 or 1500 mg of quenched PAM-4080/kg body weight were infused at a rate of 1 mL/kg/min. into the tail vein of male, Sprague-Dawley rats. Each test group consisted of six animals; six undosed animals served a ⁇ control ⁇ . All animal ⁇ were monitored for ⁇ eventy two hour ⁇ for ⁇ ign ⁇ of overt toxicity; none were ob ⁇ erved. The animal ⁇ were sacrificed. No evidence of toxicity was seen at the time of necropsy. Tissues from the liver, kidney, lung were subjected to histopathological analysis. No adverse histopathology findings were noted.
  • the aliquot was mixed with 5000 ⁇ L of SEC mobile phase, filtered through a 0.2 ⁇ pore- size filter and injected on a SuperoseTM 12 column for SEC analysis for native hemoglobin.
  • the experimental data indicated that less than 0.1% hemolysis had occurred. This amount of hemolysi ⁇ was considered negligible.
  • Example 11 Compatibility with human peripheral blood mononuclear cells (monocvtes) .
  • the potential of PAM- 4080 for causing white blood cell activation wa ⁇ evaluated.
  • the ⁇ tock polyamide solution was diluted five-fold with lactated Ringer's ⁇ olution. A volume of this preparation was mixed with an equal volume of peripheral blood mononuclear cell preparation and vortexed gently. An aliquot of this test preparation was removed and diluted with trypan blue. Toxicity was determined by microscopic detection of cells that could no longer exclude the dye. Percent viability was measured by a ratio of live/dead cells. PAM-4080 caused no decrease in cell viability. The remaining test preparation was placed in an incubator (37°C) overnight.
  • TNF Tumor Necrosis Factor
  • Interleukin-l ⁇ Interleukin-6
  • PA-DCLHb Compatibility of PA-DCLHb with human peripheral blood mononuclear cells (monocvtes) .
  • Lactated Ringer's solution was used as the control article.
  • the test articles were seven different preparations of PA-DCLHb in lactated Ringer's solution. Test and control solution ⁇ were made by mixing a volume of each te ⁇ t and control article with an equal volume of peripheral blood mononuclear cell preparation.
  • TNF Tumor Necrosis Factor
  • IL-1 Interleukin-1
  • IL-6 Interleukin-6
  • PAS-DCLHb (3:1) is the least decorated and polymerized product mixture, whereas PAS-DCLHb (10:1) is the most extensively decorated and polymerized product mixture.
  • the extent of decoration and polymerization increases with the molar ratio of PAS employed.
  • PAS-Hb products irrespective of the extent of decoration or polymerization, all yield low TNF- and IL-1 responses. None of the sample ⁇ ⁇ how an IL-6 response.
  • the diester was added to the diamine or vice versa.
  • the rate of mixing was controlled by varying the rate of stirring of the ⁇ olution, by varying the rate of addition, or by controlling the solubility of one of the components (the diester) . It was found that product isolation was facilitated and polymer yield was improved by conversion of a polyamide bis (diamine) to a polyamide bi ⁇ ( ⁇ uccinate) .
  • PATS-2800 a water-soluble polyamide having a number average molecular weight of about 2800 Daltons.
  • a slurry of TBB (5.34 g, 12 mmol) in 60 mL chloroform was stirred at a moderate rate while being cooled to 0 ' in an external ice-bath as a solution of DGBE (3.17 g, 1.4 mmol, a 1.2:1 molar ratio relative to TBB) in 12 mL chloroform was added.
  • the re ⁇ ulting mixture was stirred for 15 minutes at 0-5°C and then the ice-bath was removed. Stirring continued at ambient temperatures overnight. Thin-layer chromatographic analysi ⁇ indicated that a polyamide bis (amine) had formed.
  • the precipitate (2.44 g; about 47% of theoretical) was isolated by filtration and was found to have a SEC retention time of about 35.8 minute ⁇ and was characterized by TLC as a polyamide monoamine monocarboxylate having an average molecular weight of about 5700 Daltons.
  • the filtrate was found to contain a polymer (1.14 g; 22% of theoretical) having a SEC retention time of about 38.6 minutes (i.e., the polyamide bis (amine) had a number average molecular weight of about 2800 Daltons) .
  • Synthesis of PATSS-5700 an activated water- soluble polyamide having a number average molecular weight of about 2800 Daltons.

Abstract

La présente invention concerne des agents de réticulation à base de polyamides non immunogène hydrosoluble et leur emploi pour réticuler, polymériser, décorer et conjuguer des protéines, des polynucléotides et autres substrats biologiques afin de constituer des produits hydrosolubles non immunogènes. La présente invention concerne des protéines, des polynucléotides et autres substrats biologiques qu'il est possible de réticuler, conjuguer, polymériser ou décorer au moyen de polyamides hydrosolubles, pour constituer des produits essentiellement non immunogènes.
EP95906090A 1993-12-27 1994-12-20 Agents de reticulation a base de polyamides non immunogene hydrosoluble Withdrawn EP0702553A1 (fr)

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