Title:  Biodegradable pH/thermosensitive hydrogels for sustained delivery of biologically active agents

United States Patent:  6,451,346

Issued:  September 17, 2002

Inventors:  Shah; Subodh (Newbury Park, CA); Dai; Weiguo (Winnetka, CA)

Assignee:  Amgen Inc (Thousand Oaks, CA)

Appl. No.:  221178

Filed:  December 23, 1998

The present invention relates generally to the development of pharmaceutical compositions which provide for sustained release of biologically active polypeptides. More specifically, the invention relates to the use of pH/thermosensitive biodegradable hydrogels, consisting of a A-B di block or A-B-A tri block copolymer of poly(d,l- or l-lactic acid) (PLA) or poly(lactide-co-glycolide) (PLGA) (block A) and polyethylene glycol (PEG) (block B), with ionizable functional groups on one or both ends of the polymer chains, for the sustained delivery of biologically active agents.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms shall have the following meaning:

"Reverse thermal gelation temperature" is defined as meaning the temperature below which a copolymer is soluble in water and above which the block copolymer solution forms a semi-solid, i.e. gels, emulsions, dispersions and suspensions.

"LCST", or lower critical solution temperature, is defined as meaning the temperature at which a block copolymer undergoes reverse thermal gelation (solution to gel to solution). For purposes of the present invention, the term "LSCT" can be used interchangeably with "reverse thermal gelation temperature".

"Depot" is defined as meaning a drug delivery liquid which, following injection into a warm blooded animal, has formed a gel upon having the temperature raised to or above the LCST.

"Biodegradable" is defined as meaning that the block copolymer will erode or degrade in vivo to form smaller non-toxic components.

"Parenteral administration" is defined as meaning any route of administration other than the alimentary canal, including, for example, subcutaneous and intramuscular.

For purposes of the present invention, the terms thermosensitive, thermoreversible, and thermoresponsive can be used interchangeably.

The present invention involves utilization of block copolymers having biodegradable hydrophobic ("A") block segments and hydrophilic ("B") block segments. The block copolymers are di block, e.g., A-B, or tri block copolymers, e.g., A-B-A or B-A-B, type block copolymers.

Biodegradable hydrophobic A block segments contemplated for use include poly(.alpha.-hydroxy acid) members derived from or selected from the group consisting of homopolymers and copolymers of poly(lactide)s (d,l- or l-forms), poly(glycolide)s, polyanhydrides, polyesters, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate, blends and copolymers thereof.

The term "PLGA" as used herein is intended to refer to a polymer of lactic acid alone, a polymer of glycolic acid alone, a mixture of such polymers, a copolymer of glycolic acid and lactic acid, a mixture of such copolymers, or a mixture of such polymers and copolymers. Preferably, the biodegradable A block polymer will be polylactide-co-glycolide (PLGA) The PLGA may be non-ionic, e.g., hydroxy-terminated, or may be ionic, e.g., carboxy-terminated. As relates to the ionic polymers, the ionizable functional groups may be on either one or both ends of the polymer chain, and terminal ionizable groups contemplated for use include any ionizable group having a pK_a 3-8, e.g., carboxylic acids, amines, sulfonic acids, ammonium salts.

The range of molecular weights contemplated for the A block polymers to be used in the present processes can be readily determined by a person skilled in the art based upon such factors the desired polymer degradation rate. Typically, the range of molecular weight for the A block will be 1000 to 20,000 Daltons.

Hydrophilic B block segments contemplated for use include polyethylene glycols having average molecular weights of between about 500 and 10,000. These hydrophilic segments may also contain ionizable groups, if for example, B-A-B type copolymers are used.

The copolymer compositions for the tri block copolymers of the present invention are specially regulated to assure retention of the desired water-solubility and gelling properties, i.e., the ratios must be such that the block copolymers possess water solubility at temperatures below the LCST, and such that there is instant gelation under physiological conditions (i.e. pH 7.0 and 37^oC.) so as to minimize the initial burst of drug. In the hydrogels of the present invention the hydrophobic A block makes up 20% to 80% by weight of the copolymer and the hydrophilic B block makes up 80% to 20% of the copolymer.

The concentration at which the block copolymers of the present-invention remain soluble below the LCST is typically up to about 60% by weight, with 10%-30% preferred. The concentration utilized will depend upon the copolymer composition actually used, as well as whether or not a gel or emulsion is desired.

The pH/thermosensitive hydrogels of the present invention comprise ionic block copolymers such that the resultant hydrogels exhibit pH-responsive gelation/de-gelation in addition to the reverse thermal gelation properties (see FIG. 1). The hydrogels may comprise non-ionic block copolymers mixed or "blended" with ionic block copolymers and the weight ratios of ionic block copolymer to non-ionic block copolymer in the blends can be adjusted such that the resultant hydrogels possess the desirable rate of degradation, de-gelation and rate of clearance from the injection site. Because this new class of hydrogels provide for an improved rate of clearance of the hydrogel from the injection site, they are more commercially practicable than those hydrogels and compositions previously described in that they.

The biodegradable, pH/thermosensitive block copolymers of the present invention can be prepared in a two-step procedure which utilizes thermal condensation. In step 1, thermosensitive, hydroxy-terminated A-B-A block copolymers of PLGA/PLA (block A) and PEG (block B) are synthesized by mixing either homopolymer of poly lactide (PLA) or copolymer of poly lactide-co-gycolide (PLGA) with polyethylene glycol (PEG) and allowing di-hydroxy PEG to react with PLGA or PLA at 160^oC. under reduced pressure. Different weight ratios of PLGA and PEG were used for thermal condensation to obtain a series of block copolymers with desirable copolymer composition and block lengths. Copolymer composition and relative block lengths were confirmed by ¹ H-NMR spectroscopy. In step 2, the thermosensitive, hydroxy-terminated A-B-A block copolymers are further reacted with, e.g., succinic anhydride, to obtain A-B-A block copolymers with succinic acid groups at one or both ends of the polymer chain, thus providing hydrogels which exhibit thermosensitive/pH-responsive gelation. This two-step procedure is graphically depicted in FIG. 2, Scheme 1.

The biodegradable, ionic block copolymers of the present invention can also be synthesized by single step condensation of PLGA with activated PEG. This procedure is graphically depicted in FIG. 2, Scheme 2.

Alternatively, the thermosensitive, non-ionic block copolymers could be synthesized in a melt process which involves ring opening polymerization of A block using B block as the initiator. In a typical experiment, the A-B-A tri block copolymer is prepared by stannous octoate catalyzed ring-opening polymerization of d,l-dilactide (or PLGA) using .alpha.,.omega.-dihydroxy-terminated PEG as the initiator The mole ratio of B block to d,l-dilactide (or PLGA) is used to control the lengths of the A blocks, and provide a series of polymers with increasing A block contents and hydrophobicities. The relative A and B block lengths can, be confirmed by ¹ H-NMR spectroscopy

The process used to mix the copolymers with a biologically active agent and/or other materials involves dissolving the A-B-A tri block copolymers in an aqueous solution, followed by addition of the biologically active agent (in solution, suspension or powder), followed by thorough mixing to assure a homogeneous distribution of the biologically active agent throughout the copolymer Alternatively, the process can involve dissolving the A-B-A tri block copolymer in a biologically active agent-containing solution. In either case, the process is conducted at a temperature lower than the gelation temperature of the copolymer and the material is implanted into the body as a solution which then gels into a depot in the body. In the compositions of the present invention, the biologically active agent will generally have a concentration in the range of 0 to 200 mg/mL.

Buffers contemplated for use in the preparation of the biologically active agent-containing hydrogels are buffers which are all well known by those of ordinary skill in the art and include sodium acetate, Tris, sodium phosphate, MOPS, PIPES, MES and potassium phosphate, in the range of 25 mM to 500 mM and in the pH range of 4.0 to 8.5.

It is also envisioned that other excipients, e.g., various sugars (glucose, sucrose), salts (NaCl, ZnCl) or surfactants, may be included in the biologically active agent-containing hydrogels of the present invention in order to alter the LCST or rate of gelation of the gels. The ability to alter the rate of gelation and/or LCST is important and an otherwise non-useful hydrogel may be made useful by addition of such excipients.

As used herein, biologically active agents refers to recombinant or naturally occurring proteins, whether human or animal, useful for prophylactic, therapeutic or diagnostic application. The biologically active agent can be natural, synthetic, semi-synthetic or derivatives thereof. In addition, biologically active agents of the present invention can be perceptible. A wide range of biologically active agents are contemplated. These include but are not limited to hormones, cytokines, hematopoietic factors, growth factors, antiobesity factors, trophic factors, anti-inflammatory factors, small molecules and enzymes (see also U.S. Pat. No 4,695,463 for additional examples of useful biologically active agents). One skilled in the art will readily be able to adapt a desired biologically active agent to the compositions of present invention.

Proteins contemplated for use would include but are not limited to interferon consensus (see, U.S. Pat. Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 hereby incorporated by reference including drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference including drawings), granulocyte-colony stimulating factors (see, U.S. Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185, hereby incorporated by reference including drawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including drawings), and leptin (OB protein) (see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 hereby incorporated by reference including figures).

The type of leptin used for the present preparations may be selected from those described in PCT International Publication Number WO 96/05309, as cited above and herein incorporated by reference in its entirety. FIG. 3 of that publication (as cited therein SEQ ID NO: 4) depicts the full deduced amino acid sequence derived for human leptin (referred to as the human "OB" protein). The amino acids are numbered from 1 to 167. A signal sequence cleavage site is located after amino acid 21 (Ala) so that the mature protein extends from amino acid 22 (Val) to amino acid 167 (Cys). For the present disclosure, a different numbering is used herein, where the amino acid position 1 is the valine residue which is at the beginning of the mature protein. The amino acid sequence for mature, recombinant methionyl human leptin is presented herein as SEQ ID NO: 1, where the first amino acid of the mature protein is valine (at position 1) and a methionyl residue is located at position -1 (not included in the sequence below).

SEQ ID NO: 1

V P I Q K V Q D D T K T L I K T I V T R I N D I S H T Q S V S S K Q K V T G L D F I P G L H P I L T L S K M D Q T L A V Y Q Q I L T S M P S R N V I Q I S N D L E N L R D L L H V L A F S K S C H L P W A S G L E T L D S L G G V L E A S G Y S T E V V A L S R L Q G S L Q D M L W Q L D L S P G C

However, as with any of the present leptin moieties, the methionyl residue at position -1 may be absent.

Alternatively, one may use a natural variant of human leptin, which has 145 amino acids and, as compared to rmetHu-leptin of SEQ ID NO: 1, has a glutamine absent at position 28.

Generally, the leptin moiety for human pharmaceutical use herein will be capable of therapeutic use in humans (see also, animal leptins, below). Thus, one may empirically test activity to determine which leptin moieties may be used. As set forth in WO96/05309, leptin protein in its native form, or fragments (such as enzyme cleavage products) or other truncated forms and analogs may all retain biological activity. Any of such forms may be used as a leptin moiety for the present preparations, although such altered forms should be tested to determine desired characteristics. See also, PCT International Publication Numbers WO 96/40912, WO 97/06816, 97/18833, WO 97/38014, WO 98/08512and WO 98/28427, herein incorporated by reference in their entireties.

One may prepare an analog of recombinant human leptin by altering amino acid residues in the recombinant human sequence, such as substituting the amino acids which diverge from the murine sequence. Murine leptin is substantially homologous to human leptin, particularly as a mature protein and, further, particularly at the N-terminus. Because the recombinant human protein has biological activity in mice, such an analog would likely be active in humans. For example, in the amino acid sequence of native human leptin as presented in SEQ ID NO: 1, one may substitute with another amino acid one or more of the amino acids at positions 32, 35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145. One may select the amino acid at the corresponding position of the murine protein (see Zhang et al., 1994, supra) or another amino acid.

One may further prepare "consensus" molecules based on the rat OB protein sequence. Murakami et al., Biochem. Biophys. Res. Comm., 209:944-52 (1995) herein incorporated by reference. Rat OB protein differs from human OB protein at the following positions (using the numbering of SEQ ID NO: 1): 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118, 136, 138 and 145. One may substitute with another amino acid one or more of the amino acids at these divergent positions. The positions underlined are those in which the murine OB protein as well as the rat OB protein are divergent from the human OB protein and, thus, are particularly suitable for alteration. At one or more of the positions, one may substitute an amino acid from the corresponding rat OB protein, or another amino acid.

The positions from both rat and murine OB protein which diverge from the mature human OB protein are 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145. An OB protein according to SEQ ID NO: 1 having one or more of the above amino acids replaced with another amino acid, such as the amino acid found in the corresponding rat or murine sequence, may also be effective.

In addition, the amino acids found in rhesus monkey OB protein which diverge from the mature human OB protein are (with identities noted in parentheses in one letter amino acid abbreviation): 8 (S), 35 (R), 48 (V), 53 (Q), 60 (I), 66 (I), 67 (N), 68 (L), 89 (L), 100 (L), 108 (E), 112 (D) and 118 (L). Since the recombinant human OB protein is active in cynomolgus monkeys, a human OB protein according to SEQ ID NO: 1 having one or more of the rhesus monkey divergent amino acids replaced with another amino acid, such as the amino acids in parentheses, may be effective. It should be noted that certain rhesus divergent amino acids are also those found in the above murine and rat species (positions 35, 68, 89, 100, 108 and 118). Thus, one may prepare a murine/rat/rhesus/human consensus molecule (using the numbering of SEQ ID NO: 1) having one or more of the amino acids replaced by another amino acid at positions: 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142 and 145. The positions underlined are those in which all three species are divergent from human OB protein. A particularly preferred human leptin analog is one wherein the amino acids at position 100 (Trp) or 138 (Trp), and more preferably, both positions are substituted with another amino acid, preferably Gln.

Other analogs may be prepared by deleting a part of the protein amino acid sequence. For example, the mature protein lacks a leader sequence (-22 to -1). One may prepare the following truncated forms of human OB protein molecules (using the numbering of SEQ ID NO 1):

(i) amino acids 98-146;

(ii) amino acids 1-99 and (connected to) 112-146;

(iii) amino acids 1-99 and (connected to) 112-146 having one or more of amino acids 100-111 sequentially placed between amino acids 99 and 112.

In addition, the truncated forms may also have altered one or more of the amino acids which are divergent (in the murine, rat or rhesus OB protein) from human OB protein. Furthermore, any alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.

It is desirable to have such protein containing sustained-release compositions as such compositions could serve to enhance the effectiveness of either exogenously administered or endogenous protein, or could be used, for example, to reduce or eliminate the need for exogenous protein administration.

Moreover, because the materials utilized in the present invention are biocompatible and biodegradable, use of the protein compositions of the present invention help prevent adverse injection site reactions sometimes associated with injections of various proteins such as leptin.

In addition, biologically active agents can also include insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, interferons (alpha, beta, gamma), tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF-bp), interleukin-1 receptor antagonist (IL-1ra), brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte derived growth factor (MGDF), novel erythropoiesis stimulating protein, keratinocyte growth factor (KGF), thrombopoietin, platelet-derived growth factor (PGDF), colony simulating growth factors (CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptokinase and kallikrein. The term proteins, as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof.

Also included are those polyptides with amino acid substitutions which are "conservative" according to acidity, charge, hydrophobicity, polarity, size or any other characteristic known to those skilled in the art. See generally, Creighton, Proteins, W. H. Freeman and Company, N.Y., (1984) 498 pp. plus index, passim. One may make changes in selected amino acids so long as such changes preserve the overall folding or activity of the protein. Small amino terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain, may also be present. See, in general, Ford et al., Protein Expression and Purification 2:95-107 (1991), which is herein incorporated by reference. Polypeptides or analogs thereof may also contain one or more amino acid analogs, such as peptidomimetics.

In general, comprehended by the invention are pharmaceutical compositions comprising effective amounts of chemically modified protein, or derivative products, together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers needed for administration. (See PCT 97/01331 hereby incorporated by reference.) The optimal pharmaceutical formulation for a desired biologically active agent will be determined by one skilled in the art depending upon the route of administration and desired dosage. Exemplary pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton, Pa., pgs 1435-1712 (1990)).

The pharmaceutical compositions of the present invention are administered as a liquid via intramuscular or subcutaneous route and undergo a phase change wherein a gel is formed within the body, since the body temperature will be above the gelation temperature of the material. The release rates and duration for the particular biologically active agents will be a function of, inter alia, hydrogel density and the molecular weight of the agent.

Therapeutic uses of the compositions of the present invention depend on the biologically active agent used. One skilled in the art will readily be able to adapt a desired biologically active agent to the present invention for its intended therapeutic uses. Therapeutic uses for such agents are set forth in greater detail in the following publications hereby incorporated by reference including drawings. Therapeutic uses include but are not limited to uses for proteins like interferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293, hereby incorporated by reference including drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference including drawings), granulocyte-colony stimulating factors (see, U.S. Pat. Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT Publication No. 94/17185, hereby incorporated by reference including drawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including drawings), novel erythropoiesis stimulating protein (PCT Publication No. 94/09257, hereby incorporated by reference including drawings), and the OB protein (see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 hereby incorporated by reference including figures). In addition, the present compositions may also be used for manufacture of one or more medicaments for treatment or amelioration of the conditions the biologically active agent is intended to treat.

In the sustained-release compositions of the present invention, an effective, amount of active ingredient will be utilized. As used herein, sustained release refers to the gradual release of active ingredient from the polymer matrix, over an extended period of time. The sustained release can be continuous or discontinuous, linear or non linear, and this can be accomplished using one or more polymer compositions, drug loadings, selection of excipients, or other modifications. The sustained release will result in biologically effective serum levels of the active agent (typically above endogenous levels) for a period of time longer than that observed with direct administration of the active agent. Typically, a sustained release of the active agent will be for a period off days to weeks, depending upon the desired therapeutic effect.

Claim 1 of 7 Claims

What is claimed is:

1. A pharmaceutical composition for the sustained administration of an effective amount of a protein, comprising an injectable biodegradable polymeric matrix into which said protein has been incorporated, said polymeric matrix having reverse thermal gelation properties and pH-responsive gelation/de-gelation properties; wherein said polymeric matrix is a biodegradable block copolymer comprising:

(a) 55% to 80% by weight of a hydrophobic A polymer block comprising poly lactide-co-glycolide (PLGA) having an average molecular weight of between 1000-20,000; and

(b) 20% to 45% by weight of a hydrophilic B polymer block comprising a polyethylene glycol having an average molecular weight of between 500-10,000; and wherein said biodegradable block copolymer has ionizable functional groups on one or both ends of the polymer chains.
 

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