AU5729594A - Water soluble non-immunogenic polyamide cross-linking agents - Google Patents

Description

WATER SOLUBLE NON-IMMUNOGENIC POLYAMIDE CROSS-LINKING AGENTS

Field of the Invention

The present invention 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.

Background of the Invention

Cross-linking reagents are used for a variety of purposes, including the investigation of the spatial arrangement and functions of various ipacromolecular 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, either by attachment to an insoluble support or by various other means, 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.

In general, cross-linking agents consist of an organic bridge between activated termini. The termini bind to biological macromolecules to form a link. Various 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). Commercially available 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. However, 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.

Synthetic polymers are being developed for use as cross-linking agents. 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)).

The conjugation of biologically active polypeptides with water- soluble polymers such as PEG is well-known. The coupling of biologically active and pharmaceutically active peptides and polypeptides to PEG and similar water-soluble polymers is disclosed by U.S. Patent No. 4,179,377 to

Davis et al. Polypeptides modified with PEG are disclosed as exhibiting dramatically reduced immunogenicity and antigenicity. The PEG conjugates also exhibit a wide range of solubilities and low toxicity, and have been shown to remain in the bloodstream considerably longer than the corresponding native compounds yet are readily excreted. The PEG conjugates have also been shown not to interfere with enzymatic activity in the bloodstream or the conformation of the polypeptides conjugated thereto. Accordingly, a number of PEG-conjugates of therapeutic proteins have been developed exhibiting reduced immunogenicity and antigenicity and longer clearance times, while retaining a substantial portion of the protein's physiological activity.

Attention has also focused upon the conjugation of PEG with therapeutic drugs. Gnanov et al., "Macromolecules," 17, pages 945 to 952 (1984) observed that the attachment of PEG to various drugs led to prolonged pharmacological activity.

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 acromolecular 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.

Summary of the Invention The present invention is water-soluble, nonimmunogenic polyamides having 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.

In other words, the polyamide of the present invention is a water- soluble, substantially nonimmunogenic polyamide selected from the formulas I, π, and HI:

I Y-A-X-Y π z-B-x-z

(i) where terminus Y is H or a carboxyl coupling group; (ii) where terminus Z is H or a coupling group attached to an amine group; and (iii) where X is a polyamide selected from: (B-A)_n, (A-B)_n, (AA)_n and branched polyamides formed by linking (B-A)_n, (A-B)_n or (AA)_n to a central polyacid, polyamine or polyamino acid; and (iv) where A is a o ,ω-di-acid; B is a o ,ω-diamine; AA is a o ,ω-amino acid; 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 O, S, P or 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 amine group and having fifteen or fewer atoms in the chain and having one or more heteroatoms O, S, P or N present as substituents on or atoms in the chain; and (vii) where n is from 2 to about 100. 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, nonimmunogenic and retains all or a useful portion of the physiological activity of the substrate.

Brief Description of the Drawings

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.

Detailed Description of the Invention

The polyamides of the present invention are substantially non- immunogenic, water-soluble polyamides having 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. These 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.

The term "non-immunogenic" indicates that the polyamide does not elicit a humoral or cell-mediated immune response, either in vivo or in vitro.

The term "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. The term "activation" means converting a group of the polyamide terminus to a more reactive coupling group.

The polyamide may be linear or branched.

The term "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.

As used herein, 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., Polym Journal, Volume 5, pages 186ff (1973) and N. Ogata and Y. Hosoda, Journal Polym Science, Polym 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 Polym Science, Polym Chemistry Ed., Volume 14, pages 783ff (1976), N. Ogata et al., Polym Journal, Volume 11, pages 827 to 833 (1979), and H. Sato, et al., Makromol Chemistry, Volume 182, page 755 to 762 (1981) describe the polycondensation of activated diesters containing ether, thioether or hydroxyl groups with diamines. D. Kieley and T-H. Lin have also described polyhydroxypolyamides and a process for making same, U.S. Patent No. 4,833,230. N. Ogata and Y. Hosoda, Journal Polym Science, Polym Chemistry Ed., Volume 18, pages 1159 to 1162 (1978) describe the synthesis of water-soluble polyamides by polycondensation in solutions of ethylene glycol dimethoxycarbonylmethyl ether and hexamethylene diamine. 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. Alternatively, 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 (O, S, P, N) present as substituents on or atoms in the chain. Polyamindes of similar and/or dissimilar structure may be linked by a central polyacid, polyamine or polyamino acid to form branched, water-soluble polyamides. 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, Inc. (1991) which is incorporated by reference herein. 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. 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.

Generally, to decorate a substrate molecule, between about 4 and 50 moles of polyamide should be used per mole of substrate. Larger substrate molecules will require a greater proportion of polyamide. To primarily conjugate, cross-link or polymerize a substrate without decorating requires the knowledge of a chemist skilled in the art as to the chemistries of the coupling agents, the reactive groups on the particular substrate, the size of the substrate, the size of the polyamide, concentration of the substrate, and general reaction parameters.

As will be appreciated readily by those of skill in the art, 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. Those of ordinary skill in the art having the benefit of this disclosure will comprehend the synthetic approaches that may be employed to covalently join the polyamide and the substrate. 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. Likewise, the substrate may be activated appropriately, if necessary, and then attached to the polyamide.

For example, 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. Moreover, 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).

For example, 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.

Similarly, 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. In accordance with the present invention, 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 substituent 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. Through this conjugation, the duration of vascular circulation of conjugated deferoxamine is lengthened and the agent retains its chelating abilities.

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. 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. 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 oc amino acids, so they are not subject to enzymatic hydrolysis.

In addition, the 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. In one example described fully below, the termini of a polyamide have been modified as maleimide groups, suitable for reaction with thiol substituents of proteins. Bis(maleimide) polyamide was empolyed to polymerize human hemoglobin via the cysteine-β93 thiol residues of that protein. Similarly, in another example below, 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 ε -amino groups of lysine residues of the protein. In another embodiment the polyamides of the present invention may be used to link probes (e.g. fluorescent, radioactive, etc.) to a substrate to be detected.

In the examples that follow, we use the following nonmenclature for our polyamides: since the backbone is a polyamide, the letters PA will apply; the letter designating the coupling group will follow, M for maleimide and S for N-hydroxysuccinimide; a hyphen will separate the alphabetic code from the approximate molecular weight. Thus, a polyamide identified as PAM-3800 is a polyamide bis(maleimide) having a molecular weight of about 3800 Daltons.

DESIGN AND SYNTHESIS OF POLYAMIDES

In the following examples the polyamide condensation products are characterized in three ways. Size exclusion chromatographic (SEC) analysis is completed using a Superose™ 12 column and 50 mM phosphate, pH 6.5, mobile phase delivered at a flow rate of 0.4 mL/min. with detection at 220 nm; this analysis confirms that polymerized products were formed and permits approximation of molecular weights and the range of molecular weights of the components in the product mixture. Thin-layer chromatographic (TLC) analysis permits separation and characterization of end-group functionality of the components in the product mixture. The structure of each component is assigned on the basis of relative migration (Rf) and reactivity toward ninhydrin spray reagent. Under the TLC conditions, 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 diaminohexane/PEG/ethyl acetate in methanol/phosphate buffer.

Equivalent weights are converted to molecular weights based on the average number of amines per molecule. Alternatively, the NMR spectrum can be used to estimate the molecular weights of the polyamides, as follows: the first step is to divide the structure of the polyamide into end groups and repeating units. Then the molecular weight of each part is calculated. Next 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.

There are two succinate residues in the end groups of the polyamide derivative: therefore, the resonance at about 2.53 ppm and the one at 2.92 ppm each results from four hydrogens. The average area response is (2.197 + 2.605)/2 or 2.401 units per four hydrogens on each succinate. Similarly, the two internal methylene groups of the butanediamine residue in the repeating unit contain four hydrogens. The observation that the integrated area of the latter resonance (16.034 units) is larger than that of the four- hydrogen response for either type of succinate hydrogen indicates that there are multiple butanediamine residues within the repeat units in the polymer. We can estimate the value of the multiple by ratioing the integrated areas: 16.034/2.401 or approximately 7. Thus, there are seven repeat groups in the polyamide. 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.

Synthesis of PAS-2400 Example 1(&)

Polycondensation of Ethylene glycol bis(methoxycarbonylmethyl) and 1.4-diaminobutane. Ethylene glycol bis(methoxycarbonylmethyl) ether (EDE), which has an ether group oc 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. In general, the polycondensations were completed as follows. For the solution method. 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. In the melt method, 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. Analysis of the reaction mixtures by SEC confirmed that polymerized products were formed. TLC analysis (stationary phase: silica gel; eluent: 2- propanol / NH4OH / H2O, 7:1:2, by volume) of the product showed three spots having Rf values of 0.1, 0.4, and 0.7, respectively. The structures of the corresponding polyamides were assigned on the basis of reactivity toward ninhydrin and base as oc ,ω-diaminopolyamide (designated I in the figure), a -amino-ω-esterpolyamide (designated II), and oc ,ω-diesterpolyamide (designated HI), respectively (Figure 1). In addition, product m is ninhydrin-negative while products I and π are ninhydrin-positive, indicating the products has at least one primary amine group. Finally, products II and DI can be hydrolyzed with dilute aqueous NaOH, whereas I cannot, indicating products π and in contain at least one ester group.

The yield of these products depends on the molar ratio of DAB to EDE. A molar ratio of one gives I as the major product. A molar ratio of

DAB/EDE greater than one gives polyamide II as the major product. In contrast, HI became the major product with a molar ratio of DAB/EDE of less than one.

The results of polycondensation of EDE and DAB are summarized in Figure 2. The experimental data indicate that oc -amino, ω-ester polyamide π having a molecular weight (MW) of about 2,400 Dalton is best produced by the solution method at 30°C. oc ,ω-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. oc ,ω-Diester-polyamides HI 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 m is obtained as the major product. Example 1(b)

Conversion of polyamide in to an activated cross-linking agent. Crude diester m (Figure 1), obtained by condensation of EDE and DAB, was hydrolyzed with dilute sodium hydroxide to the corresponding di- acid. After hydrolysis, the reaction mixture was treated with AG50W-X8 resin (BioRad) to remove sodium ion and by-products I and π. The di-acid was obtained in a pure state as judged by TLC. The di-acid was treated with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in chloroform to convert it to the corresponding polyamide bis(N- hydroxysuccinimide ester) (designated PAS-2400).

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. The reaction mixture was stirred at room temperature under nitrogen, and the reaction was monitored by size exclusion chrmoatography 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 solution was stirred at 5°C under nitrogen overnight and then dialyzed against lactated Ringer's solution to give the final product. Experimental data are summarized in Figures 3, 4, and 5. Note: In the figures NHS-PA 6 is an alternate designation for PAS-2400.

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. However, competitive hydrolysis of the polymerization agent is also fast. As the solution pH is increased, higher yields of high molecular weight polymers are obtained. For example, 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.

P5O 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. Thus, the excellent oxygen-binding function of DCLHb is maintained in these polymers. RP- HPLC analysis (Figure 5) indicates than both β- and oc - chains are modified. However, 3-chains apparently are more extensively modified than are oc -chains.

Thus, 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). However, increases in chain length using hydrocarbon components would reduce the aqueous solubility of the protein.

With this in mind, we synthesized polyamides from diester EDE and each of two longer diamines: ethylene glycol bis(3-aminopropyl) ether (EGBE; MW 176) and diethylene glycol bis(3-aminopropyl) ether (DGBE; MW 220). See Figure 6. The SEC retention times of each of the polyamides suggested these products had higher molecular weights, but the products were waxy and had low melting points. Purification of such products by crystallization is extremely difficult. To minimize these shortcomings we combined three concepts to select appropriate activated esters for the synthesis of longer polyamides. First, we identified components that are di-acids having β-ether links; these di-acids are easily converted to activated diesters. Our initial di-acid of choice was diglycolic acid. Second, we converted one end of this di-acid to an amide by reacting two equivalents of di-acid with one equivalent of diamine; this generated a new and longer di-acid that we can use as a component for longer polyamides. Our first di-acid of choice was 1,4- (carboxymethoxyacetamido) butane, which we used as the activated methyl diester BMDAB (MW 348). Insertion of the methylene (hydrocarbon) groups reduced the flexibility of the molecule sufficiently to render it a crystalline solid and retention of the ether link preserved the solubility in water. Third, we increased the length of the diamine component in a way that will maintain water solubility; thus, we used ethylene glycol bis(3- aminopropyl) ether (EGBE) and diethylene glycol bis(3-aminopropyl) ether (DGBE) as the diamine components in polyamide synthesis.

The activated diester building block, BMDAB, was obtained in two steps (Figure 7). DAB (1,4-diaminobutane) 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 HCl or HCl in dioxane solution. The advantage of using HCl 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.

In contrast to the polycondensation of EDE and DAB by the solution method, which gives the α-methylester-ω-aminopolyamide as the major product when a molar ratio of EDE to DAB of 1 was used, the polycondensation of equimolar quantities of BMDAB and EGBE or DGBE or of molar ratios of BMDAB to DGBE of greater than 1 (Figure 8) gave mixtures containing substantial amounts of three products: an α-ester-ω- amine (reactive to ninhydrin; hydrolyzed by base); an α,ω-diamine (reactive to ninhydrin); and a diester (unreactive to ninhydrin). Unfortunately, the presence of large amounts of other products made the purification of a desired product tedious. However, we found that the use of an excess of diamine (e.g., a molar ratio of diamine to BMDAB of 1.3) gave the α,ω- bisamine polyamide as the major product containing only very small amounts of the monoamine by-product. This latter procedure is therefore preferred to produce the polyamide backbone of the polymerization reagents. Example 4

Conversion to Activated Polymerization Agents: Polyamide bisfN- hydroxysuccinimide ester.

The first attempt to synthesize polyamide bis(N-hydroxysuccinimide) ester (designated PAS-3070) was a three step synthesis from BMDAB and EGBE (Figure 9). First, 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 (Norit™ 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. Second, conversion of the white product to the corresponding bis(2-carboxy- ethylcarbonyl) polyamide (Figure 9, 3a) was completed by reaction of (2a) with succinic anhydride in DMF (a small amount of methanol was added to enhance to the solubility of 2a). The reaction produced a yellow product mixture containing bis(2-carboxy-ethylcarbonyl)polyamide (3a) as the major product and two minor by-products: the methyl ester of (2a) and an un¬ known by-product containing a free amino group as indicated by TLC. Therefore, 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. 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. For example, 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. Furthermore, recrystallization of bis(2-carboxyethyl- carbonyl) polyamide (3a) in methanol/acetone converts some of the di-acid to the corresponding methyl ester(s). To avoid these drawbacks, our preferred synthetic strategy is as follows. First, steps 1 and 2 (Figure 9) 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. In addition, the use of methanol as crystallization solvent for 3 was excluded to avoid the formation of methyl ester of 3. Example 5

Synthesis of PAS-4200.

PAS-4200 (Figure 9, 4b) was prepared using the integrated approach above.

Example 6

Conversion to Activated Polymerization Agents: Polyamide Bis(maleimidopropionate) .

Synthesis of polyamide bis(maleimidopropionate) (designated PAM- 4080) was completed as a "one-pot" two-step synthesis which is summarized in Figure 10. Crude polyamide bisamine (Figure 10,- 2B) was obtained by heating BMDAB and DGBE in refluxing methanol for 24 hours followed by decolorizing with Norit A and was immediately treated with N- hydroxysuccinimido-3-maleimidopropionate (SMP, Figure 10, 5) to give 6b. The first attempt to carry out the latter step by mixing 2b and 5 in a molar ratio of 1:2 in chloroform in the presence of triethylamine gave a higher molecular weight product. Since SMP is a bifimctional cross-linking reagent, it could polymerize 2b under these conditions. Increasing the SMP/2b molar ratio to 4.5 and the slow addition of 2b in chloroform containing triethylamine to a solution of SMP in chloroform eliminated the polymerization of 2b by SMP. Thus, 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. In contract to PAS derivatives, PAM derivatives are stable in water.

Example 7

Polymerization of DCLHb with Polyamide Polymerization Reagents. 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 8.0 with 1 molar HEPES pH 9.0 and a solution of 1 M N-acetyl-L-cysteine, pH 9.0 (molar ratio NAC/DCLHb of 5) was added. The solution was stirred at 5 °C under nitrogen overnight and then dialyzed against lactated Ringer's solution to give the final product. Experimental results are summarized in Figures 11 to 18.

Polymerization of DCLHb with the activated ester PAS derivatives may be summarized as follows, (a) The degree of polymerization and the yield of oligomers increased with the molar ratio of PAS used, (b) Concurrent with increases in the molar ratio of PAS used, the elution time of DCLHb monomer decreased, suggesting that decoration of DCLHb by PAS is occurring, (c) Polymerization was fast; it was complete within 2 to 3 hours, (d) The SEC profiles of polymeric product obtained by employing five equivalents of PAS-3070 and three equivalents of PAS-4200 are very similar. This also demonstrates that longer reagents facilitate polymerization of DCLHb. (e) Four equivalents of PAS-3070 and 2.5 equivalents of PAS- 4200 gave the best product mixtures under these experimental conditions, (f) PAS derivatives do not affect the P50 values of DCLHb: the P50 values of polymerized product are in the range of 29 to 36 mm Hg. (g) RP-HPLC analyses (Figure 13) indicate that both β and era chains are modified by PAS, but era chains to a lesser extent than β chains.

DCLHb polymerization by PAM derivatives may also be summarized, (a) As was true of the PAS derivatives, the yield of oligomer increased with the number of molar equivalents of PAM used, (b) Elution times of the monomer decreased with the number of molar equivalents of PAM used; thus, decoration of DCLHb by PAM is likely, (c) Two equivalents of PAM give the best product mixtures, (d) RP-HPLC profiles (Figure 15) suggest that reagent reacted specifically. A specific β' peak, which could be a modified β peak, was detected at all ratios of PAM tested. Specific reaction with the β subunits was also supported by the decrease in titrable thiol residues. Reagent PAM is expected to bind specifically to cysteine-B93 residues, and about 65% and 90% of thiol groups are modified when 1 and 2 equivalents of PAM are used, respectively, (e) 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. (f) αα-Chains are also modified, but much less extensively than the β 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 5g/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.

Example 8 In vitro exposure of isolated mammalian cells. 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.

No toxicity was observed at the lower doses and moderate toxicity was observed at the highest dose. Accordingly, the polyamides of the present invention are expected to be nontoxic when administered as conjugates of therapeutically useful substrates.

Example 9

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 as controls. All animals were monitored for seventy two hours for signs of overt toxicity; none were observed. The animals 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.

Example 10

Compatibility with human erythrocytes. To determine the biocompatibility of PAM-4080 with human erythrocytes, the stock polyamide solution was diluted five-fold with lactated Ringer's solution. A volume of this preparation was mixed with an equal volume of heparinized human blood, vortexed gently, and placed in an incubator (37°C) overnight. After an incubation period of 16 hours, a 100-μL aliquot of the supernatant was removed from the top of the test sample; care was taken not to disturb the sedimented red cells below. The aliquot was mixed with 5000 μL of SEC mobile phase, filtered through a 0.2 μm pore-size filter and injected on a Superose™ 12 column for SEC analysis for native hemoglobin. The experimental data indicated that less than 0.1 % hemolysis had occurred. This amount of hemolysis was considered negligible.

Example 11

Compatibility with human monocytes. The potential of PAM-4080 for causing white blood cell activation was evaluated. The stock polyamide solution was diluted five-fold with lactated Ringer's solution. 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. After an incubation period of 16 hours, cytokines were analyzed by pipetting an aliquot of the sample into microtiter wells and quantitation by ELISA. The concentrations of Tumor Necrosis Factor (TNFα.), Interleukin-lβ and Interleukin-6 determined were no different from those found by exposure of human monocytes to lactated Ringer's solution. Thus, PAM-4080 is compatible with human monocytes.

Example 12 Compatibility of PA-DCLHb with human monocytes. The potential of polyamide decorated and polymerized DCLHb (PA-DCLHb) to induce cytokine production by human monocytes was evaluated. 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 solutions were made by mixing a volume of each test and control article with an equal volume of peripheral blood mononuclear cell preparation. After incubation of each resulting test and control solution at 37 °C for about 16 hours, an aliquot of each sample was transferred into separate wells of microtiter plates and the concentrations of Tumor Necrosis Factor (TNFα), Interleukin-lβ and Interleukin-6 were quantitated by E ISA. The concentrations of each cytokine determined are shown in the Table below. The experimental data indicate that induction of TNFa, EL-1, and IL-6 are low and in some cases comparable to Ringers. In summary, PA-DCLHb appears to be very compatible with human monocytes.

Example 13 Cvtokine Induction bv PAS-DCLHb.

Samples of six PAS-DCLHb product mixtures were submitted for cytokine testing. The products selected were prepared by polymerization of 3 g/dL DCLHb in 0. IM HEPES buffer at pH 7.0. Each sample was diluted to a DCLHb concentration of about 1 g/dL and passed through an END-X™ endotoxin-removing filter. The filtrate was tested for cytokine induction using the method described in Example 12.

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. However, the PAS-Hb products, irrespective of the extent of decoration or polymerization, all yield low TNF-α and IL-lβ responses. None of the samples show an IL-6 response.

As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. All such variations are intended to be included in the scope of the following claims.

Claims (12)

CLAIMS:

1. A water-soluble, substantially nonimmunogenic polyamide selected from the formulas I, H, and HI: I Y-A-X-Y π Z-B-X-Z

(a) where terminus Y is H or a carboxyl coupling group;

(b) where terminus Z is H or a coupling group attached to an amine group; and

(c) where X is a polyamide selected from: (B-A)_n, (A- B)_n, (AA)_n and branched polyamides formed by linking (B-A)_n, (A-B)_n or (AA)_n to a central polyacid, polyamine or polyamino acid; and

(d) where A is a oc ,ω-di-acid; B is a oc ,ω-diamine; AA is a oc ,ω-amino acid; n is the number of amide repeat units in the polyamide; and

(e) where the acid subunits of the amide repeat units are (i) organic acids having fifteen or fewer atoms in the chain and having one or more heteroatoms O, S, P or N present as substituents on or atoms in the chain, or (ii) two or more of such organic acids bridged by water-soluble organic diamines; and

(f) where the amine subunits of the amide repeat units are organic, water-soluble amines having at least one primary amine group and having fifteen or fewer atoms in the chain and having one or more heteroatoms O, S, P or N present as substituents on or atoms in the chain; and

(g) where n is from 2 to about 100.

2. Two or more polyamides of Claim 1 linked by a central polyacid, polyamine or polyamino acid to form branched, water-soluble polyamides.

3. A polyamide of Claim 1 reacted with a substrate having a diagnostic or therapeutic biological activity selected from the group consisting of proteins including enzymes, haptens, and antibodies; polypeptides; polynucleotides; steroids; and carbohydrates; wherein the product of said reaction is water-soluble, substantially nonimmunogenic and retains a diagnostically or therapeutically useful amount of the substrate's biological activity.

4. A polyamide of Claim 1 decorating a substrate having a diagnostic or therapeutic biological activity selected from the group consisting of proteins including enzymes, haptens, and antibodies; polypeptides; polynucleotides including probes; and carbohydrates; wherein the product of said decorating is water-soluble, substantially nonimmunogenic, and retains a diagnostically or therapeutically useful amount of the substrate's biological activity.

5. A polyamide of Claim 1 cross-linking two or more substrates having a diagnostic or therapeutic biological activity selected from the group consisting of proteins including enzymes, haptens, and antibodies; polypeptides; polynucleotides including probes; and carbohydrates; wherein the product of said cross-linking is water-soluble, substantially nonimmunogenic and retains a diagnostically or therapeutically useful amount of the substrate's biological activity.

6. A polyamide of Claim 1 polymerizing three or more substrates having a diagnostic or therapeutic biological activity selected from the group consisting of proteins including enzymes, haptens, and antibodies; polypeptides; polynucleotides including probes; and carbohydrates; wherein the product of said polymerizing is water-soluble, substantially nonimmunogenic and retains a diagnostically or therapeutically useful amount of the substrate's biological activity.

7. A polyamide of Claim 1 decorating a product of Claim 6.

8. A polyamide of Claim 1 decorating a product of Claim 5.

9. A product of Claim 6 wherein said substrates are hemoglobin molecules and wherein said polyamide is bis(maleimidoglycyl) polyamide having a molecular weight of about 4080 daltons.

10. A product of Claim 6 wherein said substrates are hemoglobin molecules and wherein said polyamide is bis(N-oxa-succinimidyl) polyamide having a molecular weight of about 240 daltons.

11. Water-soluble, nonimmunogenic branched or straight chain polyamides having molecular weights of about 300 to about 20,000 grams per mole; comprising from 1 to about 100 amide repeat units where each repeat unit comprises: (a) a water-soluble organic acid subunit having at least one carboxylate group and fifteen or fewer atoms separating the amide functionalities in the polyamide;

(b) covalently linked as an amide to;

(c) 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.

12. A polyamide of Claim 1 where terminus Y and terminus Z are independently activated by reacting said polyamide with bi or polyfunctional protein reagents selected from the group consisting of dialdehydes, N- hydroxysuccimmide esters, functionalized acetals, bis-maleimides, bifunctional imino esters, diepoxides, and dicarboxylic acid clorides.