A biologically active conjugate is disclosed comprising a biopolymer and a therapeutic agent joined by a disulfide bond. The conjugate, when formulated in a pharmaceutical composition with a suitable carrier, has improved in vivo stability and activity, and can be targeted to a variety of cells, tissues and organs.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention features a biopolymer-therapeutic agent conjugate in which the biopolymer and therapeutic agent are joined by a disulfide bond. The biologically active conjugate of this invention is useful as a drug delivery vehicle for the in vivo delivery of the therapeutic proteins to specific cells, organs or tissues in a subject. Drug delivery specificity is achieved by appropriate selection of the structure and molecular weight of the biopolymer.

The chemistry used to prepare the conjugates permits the site-specific reaction between the biopolymer and the therapeutic agent. The therapeutic agent contains a reactive thiol group, which can be present in an unmodified version of the therapeutic agent, as in the case of cysteine for example. Alternatively, the thiol group can be introduced into a modified version of a therapeutic agent that does not normally contain a reactive thiol group.

In one embodiment, the therapeutic agent can be reacted, through the reactive thiol group, with a chemically modified version of the biopolymer. This reaction typically occurs at a pH in the range of from about 6.0 to about 10. The biopolymer is activated and modified by reaction with an activating agent, such as a carbodiimide, and reacted with an organic disulfide compound. The organic disulfide compound contains a terminal group, such as an amino group or a hydroxyl group, which is reactive with the carboxylic acid group of the biopolymer in the presence of the activating agent. The reaction of the biopolymer, activating agent and organic disulfide compound occurs at a pH of from about 2.0 to 8.0.

In another embodiment, the therapeutic agent can be reacted, again through the thiol group, with the reducing end of the biopolymer. The biopolymer is first reacted with an organic disulfide compound containing a terminal group, such as an amino group or a hydroxyl group, which is reactive with the terminal carboxyl group of the biopolymer. The reaction of the biopolymer and organic disulfide compound occurs over a wide pH range, typically at a pH of from about 2.0 to 9.0.

In one aspect, the reaction of the biopolymer and therapeutic agent results in the attachment of the biopolymer to the therapeutic agent through a disulfide bond. The linking group or spacer, which can be a lower alkyl, separates the biopolymer from the therapeutic agent. The linking or spacer is a residue resulting from the cleavage of the organic disulfide compound by the reactive thiol of the therapeutic agent.

Typical biopolymers include any of the polyanionic polysaccharides, such as hyaluronic acid and any of its hyaluronate salts, such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate, carboxymethyl cellulose, carboxymethyl amylose, chondroitin-6-sulfate, dermatin sulfate, heparin, and heparin sulfate, as well as polyacrylic acid, polycarbophil, carboxymethyl chitosan, poly-.alpha.-glutamic acid, poly-.gamma.-glutamic acid, carrageenan, and sodium alginate. The common feature of the biopolymers of this invention is that they are biocompatible, as that term is defined herein, they contain carboxylic acid functionality, and they can be modified to react with an organic disulfide compound. Such modification can occur, for instance, by reaction of the biopolymer with a suitable activating agent, such as a carbodiimide, to render the carboxylic group vulnerable to nucleophilic attack by, for instance, an amine or a hydroxyl. Alternatively, the modification can occur at the terminal or end group of the biopolymer by reduction of a terminal carbonyl group using a Schiff base.

In a preferred embodiment, the biopolymer is hyaluronic acid having a molecular weight in the range of from about 7.5.times.10.sup.2 daltons to about 1.times.10.sup.7 daltons. The hyaluronic acid is preferably activated by reaction with an activating agent to render it vulnerable to nucleophilic attack. Suitable activating agents for this purpose include carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide.

The organic disulfide compound can be virtually any organic compound having a disulfide bond. Preferably, the disulfide bond is positioned at one end of an alkyl chain, while the other end of the chain terminates in a group reactive with the carbonyl group of the biopolymer. Preferably, the group that reacts with the biopolymer is an amino, carboxyl or hydroxyl group, but most preferably an amino group. In addition to being capable of reacting with the biopolymer, the organic disulfide compound is also capable of reacting with the active thiol group of the therapeutic agent. Preferred organic disulfide compounds include, in general, the nitro-pyridines, thio-pyridines, substituted S-phenyl disulphides, S-sulfonate derivatives, 9-anthrymethyl thioesters, S-carboxymethyl derivatives and nitro-thiobenzoic acid derivatives. More preferably, the organic disulfide compound is a thio-nitro-pyridine, and most preferably 3-nitro-2-pyridinesulfenyl-ethylamine.

The therapeutic agent is preferably one or more of the following: small organic molecules, proteins, nucleic acids, antibodies, peptides, amino acids, lipids, polysaccharides, cell growth factors, and enzymes. More preferably, the therapeutic agent is native or recombinant colony-stimulating factor ("CSF"), an amino acid or glucocerebrosidase. The therapeutic agent should contain a reactive thiol group to react with the modified biopolymer. The reactive thiol group can either be inherently part of the therapeutic agent, as in the case of cysteine, or the reactive thiol group can be introduced into the therapeutic molecule using known techniques. For example, a free thiol group can be introduced into a recombinant therapeutic protein molecule for conjugation and modification. Furthermore, some therapeutic drugs, such as Captopril--a drug used to treat hypertension--inherently contain a free sulfhydryl group as shown in the structure below -- see Original Patent.

The amino groups of therapeutic agents can be conveniently converted into thiols by reaction with Traut's Reagent (aminothiolane).

The therapeutic agent is selected for the particular indication that is to be treated, and the biopolymer is selected, both as to its type and molecular weight, for its ability to target a particular organ, cell or tissue. For instance, a therapeutic agent for treating Gaucher's Disease, a serious liver ailment, is the enzyme glucocerebrosidase. Glucocerebrosidase can be targeted to the liver by forming a conjugate with an appropriately sized hyaluronic acid molecule.

The biologically active conjugate of the present invention provides for improved stability of the therapeutic agent as compared to the use of the unconjugated or unmodified therapeutic agent, or the use of other carriers or conjugated compounds, such as polyethylene glycol ("PEG") or lipids. The improved stability results in increased residence time in the body of a subject and increased circulation time in the blood stream. The conjugates of this invention also display improved targeting to specific tissues, organs and cells. Improved targeting is achieved through the selection of specific types and molecular weights of the biopolymers.

In a further aspect, the invention involves the attachment of a biopolymer onto the surface of a substrate by means of a disulfide linkage. The substrate can be a polymeric material, a ceramic or a metal. Preferably, the substrate is part of a medical device or instrument, such as a stent, graft, suture, catheter, tubing or guidewire. The substrate is modified to contain an amino group, which can then be converted into a thiol group. The substrate can then be reacted with the biopolymer modified with the organic disulfide compound to immobilize the biopolymer onto the substrate.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein, including published patent applications, and issued or granted patents, are hereby incorporated by reference in their entireties. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

The biologically active biopolymer-therapeutic agent conjugates of the present invention can be prepared by using a variety of chemical preparatory methods. An important feature of the conjugates of this invention is that the linkage between the therapeutic agent and biopolymer contains a disulfide bond. The disulfide bond is formed by the reaction of the therapeutic agent containing an active thiol with the biopolymer, which has also been modified to contain a disulfide group by reaction with an organic disulfide compound. The procedure for preparing the biopolymer-therapeutic agent conjugates of this invention is described in more detail below.

Prior to the preparation of the conjugate, it is necessary to first select an appropriate biopolymer, and to modify the biopolymer so that it can react with the therapeutic agent and form a disulfide bond. The biopolymer is selected from biocompatible polymers that contain a carbonyl group. The term "biocompatible", as used herein, is intended to denote a substance that has no medically unacceptable toxic or injurious effects on biological function, or which is tolerated by the body. Examples of acceptable biopolymers include the polyanionic polysaccharides, such as hyaluronic acid and any of its hyaluronate salts, such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate, carboxymethyl cellulose ("CMC"), carboxymethyl amylose, carboxymethyl chitosan, chondroitin-6-sulfate, dermatin sulfate, heparin, and heparin sulfate, as well as poly-.alpha.-glutamic acid, poly-.gamma.-glutamic acid, carrageenan, and sodium alginate. The term "polyanionic polysaccharide", as used herein, is intended to mean polysaccharides containing more than one negatively charged group, e.g. carboxyl groups at pH values above about a pH of 4.0.

Biopolymers suitable for a particular application are selected from this group of candidate biopolymers on the basis of their ability to target particular tissues, organs or cells, and their in vivo stability, i.e. the in vivo residence time in the circulatory system, or specific tissues, cells or organs. In a preferred embodiment, the biopolymer is hyaluronic acid having a molecular weight in the range of from about 7.5.times.10.sup.2 daltons to about 1.times.10.sup.7 daltons.

These biopolymers can be "activated" by reacting the biopolymer with a suitable activating agent to render the carboxylic group on the biopolymer vulnerable to nucleophilic attack. Suitable activating agents include carbodiimides, and preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide. The reaction between the biopolymer and activating agent occurs in an aqueous medium, preferably at a pH of from about 2.0 to about 8.0, and more preferably a pH of from about 4.0 to about 5.1. Activation of the biopolymer can be useful if the therapeutic agent is linked to the intermediate carboxylic acid groups of the biopolymer.

The activated biopolymer is reacted with an organic disulfide compound. Suitable organic disulfide compounds can be selected from a wide range of molecules, including the nitro-pyridines, thio-pyridines, substituted S-phenyl disulfides, S-sulfonate derivatives, 9-anthrymethyl thioesters, S-carboxymethyl derivatives and nitro-thiobenzoic acid derivatives, and preferably the thio-nitro-pyridines. A particularly preferred organic disulfide compound is 3-nitro-2-pyridinesulfenyl-ethylamine.

In one embodiment, the organic disulfide compound is a compound of general formula R-L-S--S-M where R is an amino, hydroxyl or carbonyl group, L, if present, is a spacer, preferably a lower normal or iso-substituted alkyl group, and more preferably an ethyl group, each S is a sulfur atom, and M is an organic moiety. The spacer, L, contains a terminal group that is reactive with the activated biopolymer. Preferably, the terminal group is an amino, carboxyl or hydroxyl group, but most preferably an amino group. In addition to being capable of reacting with the biopolymer, the organic disulfide compound is also capable of reacting with the active thiol group of the therapeutic agent.

The preparation of the preferred organic disulfide compound of the present invention, 3-nitro-2-pyridinesulfenyl-ethylamine, can be illustrated as follows -- see Original Patent.

As shown above, benzyl-3-nitro-2-pyridyl-sulfide is reacted with dichloroethane and sulfuryl chloride to prepare 3-nitro-2-pyridinesulfenyl chloride. The 3-nitro-2-pyridinesulfenyl chloride is reacted with 2-aminoethanethiol and formic acid to prepare 3-nitro-2-pyridinesulfenyl-ethylamine as a precipitated product.

The activated biopolymer can then be reacted with the organic disulfide compound as shown in the following reaction scheme: G-COOH+R-L-S--S-M.fwdarw.G-COR-L-S--S-M where G is a biopolymer with a pendant carboxyl group, R is preferably an amino group, L, if present, is a spacer, preferably a lower alkyl group, each S is a sulfur atom, and M is an organic moiety. Preferably, the organic disulfide compound is 3-nitro-2-pyridinesulfenyl-ethylamine ("NEA"), and the reaction of NEA and hyaluronic acid, the preferred biopolymer, can be illustrated as shown below, where "EDC" designates 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and "HOBt" designates hydroxybenzotriazole -- see Original Patent.

Alternatively, the biopolymer can be reacted with the organic disulfide compound as shown in the following reaction scheme: G-CHO+R-L-S--S-M.fwdarw.G-C--R-L-S--S-M where G, R, L S and M are as defined above. Preferably, the organic disulfide compound is 3-nitro-2-pyridinesulfenyl-ethylamine ("NEA"), and the reaction of NEA and hyaluronic acid, the preferred biopolymer, can be illustrated as shown below, where NaCNBH.sub.3 is sodium cyanoborohydride -- see Original Patent.

In the reaction scheme illustrated above, the biopolymer terminal ring opens as a result of a mutarotation equilibrium which occurs naturally in carbohydrates. This forms a terminal aldehyde group, which is the only aldehyde group in the molecule and can form a Schiff base. The aldehyde reacts with the terminal amino group of the organic disulfide compound. The addition of the sodium cyanoborohydride is a well known reaction to reduce the resulting Schiff base. Other reagents which are known to be able to reduce Schiff bases include sodium borohydride, lithium borohydride, lithium cyanoborohydride, sodium aluminum hydride, lithium aluminum hydride, tetrabutyl ammonium cyanobororhydride, sodium amalgam, potassium graphite, and catalytic hydrogenation over platinum or nickel.

As illustrated above, this embodiment results in the attachment of the organic disulfide compound to the reducing end of the biopolymer. This permits the reaction of one mole of organic disulfide compound per mole of biopolymer in a quantitatively controlled manner, which can be result in higher yields, and more precise drug targeting and delivery.

The attachment of the organic disulfide compound need not be restricted to aldehydes inherent in the biopolymer. One could introduce an aldehyde to the biopolymer by a reduction/oxidation sequence as described, for example, by Raja, et al., Analytical Biochemistry 139: 168-177, 171 (1984). Alternatively, one could attach an aldehyde to the biopolymer by modifying an existing functional group of the biopolymer, such as a hydroxyl or carboxyl group. Methods for accomplishing this are well known in the chemical arts. Once the aldehyde is introduced or attached to the biopolymer, the organic disulfide compound may be reacted with the biopolymer as described herein.

The biopolymer-organic disulfide complex is then reacted with a therapeutic agent of choice. The therapeutic agent is selected based on the particular disease state to be treated, and the organ, tissue or cell to be targeted. Suitable therapeutic agents include small organic molecules, proteins, nucleic acids, antibodies, peptides, amino acids, lipids, polysaccharides, cell growth factors, and enzymes. More preferably, the therapeutic agent is native or recombinant colony stimulating factor, an amino acid or glucocerebrosidase.

Glucocerebrosidase is an enzyme which is used to treat a liver condition known as Gaucher's Disease. When glucocerebrosidase is selected as the therapeutic agent, it is advantageous to also select hyaluronic acid, having an appropriate molecular weight, to target the therapeutic agent to liver cells.

The reaction of the therapeutic agent and the HA-NEA complex can be illustrated as shown below -- see Original Patent.

As shown in the above reaction scheme, the therapeutic agent of choice contains an active thiol (--SH) group, that reacts with the HA-NEA conjugate, displacing the thio-nitro-pyridine residue. The therapeutic agent (shown above as the solid circle) is attached to the hyaluronic acid by a disulfide bond and an amine-terminated ethyl chain (spacer). The reaction occurs at a neutral to basic pH in the range of from about 6-10.

The biologically active conjugates of this invention can be formulated as pharmaceutical compositions for medical diagnosis or treatment, together with appropriate pharmaceutically acceptable carriers and, optionally, other therapeutic or diagnostic agents, using well known formulation protocols. Administration of the pharmaceutical composition can be accomplished using an appropriate vehicle, such as tablets, implants, injectable solutions, and the like. Acceptable carriers include buffering agents and adjuvants. The precise amount of the biologically active conjugate used in the pharmaceutical composition can be determined based on the nature of the condition to be treated, and the potency of the therapeutic agent used. This invention contemplates both local administration and time release modes of administration. As used in this application, the term "subject" is intended to denote a human or non-human mammal, including, but not limited to, a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, rat and mouse.

The process of the present invention can also be employed to modify the surface of a medical device or instrument. A biopolymer, such as hyaluronic acid, can be immobilized onto the surface of a substrate which has been modified to contain, for instance, exposed amino groups, which can be reacted with Traut's reagent and then HA-NEA as shown below -- see Original Patent.

The aminated surface, prepared, for instance, by cold plasma deposition of an allyl amine, is treated with a reagent, such as Traut's reagent, to convert the amino groups into free thiol groups. The derivatized surface is then reacted with HA-NEA to immobilize HA to the surface by a di sulfide bond. The advantage of this approach is the specificity of the reaction for the free sulfhydryl group between the surface and the activated disulfide in the biopolymer. Under these reaction conditions, the activated biopolymer can only react with the surface and not with other biopolymer molecules, thereby creating a modified surface having a well defined biopolymer thickness. By contrast, the use of exogenously added activating agents, such as glutaraldehyde and carbodiimide, to achieve similar results, can result in interpolymer covalent bond formation that can cause uncontrolled increases in biopolymer coating thicknesses. Another advantage is the use of mild reaction conditions, such as the use of an aqueous solvent, ambient temperatures, and a pH in the range of from about 6-10.

This surface modification approach can be used to modify the surface characteristics of stents, to prevent platelet activation and aggregation, or catheter surfaces, to inhibit cell adhesion. An additional advantage of this approach is that the HA will only react with the surface, and not with itself, so the thickness and composition of the HA layer can be readily controlled.

From the above description, one skilled in the art can readily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
 

Claim 1 of 23 Claims

1. A biologically active conjugate of a biopolymer and a therapeutic agent comprising a compound of formula; or a pharmaceutically acceptable salt thereof: G-C(O)--R-L-SS--B wherein G-C(O)-- is a biopolymer comprising at least one carbonyl group, --C(O)--, on the biopolymer backbone bound to R, and R is an amino group or an oxygen atom, or G-C--R-L-SS--B Wherein G-C-- is a biopolymer having a methylene group, C, bound to R, and R is an imino or amino group; and wherein L is lower alkyl spacer, B is a therapeutic agent, and each S is a sulfur atom.

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