Title:  Sustained release hydrophobic bioactive PLGA microspheres

United States Patent:  6,855,331

Issued:  February 15, 2005

Inventors:  Vook; Noelle Christine (Schaumburg, IL); Jacob; Elliott (Silver Spring, MD); Setterstrom; Jean A. (Alpharetta, GA); van Hamont; John (West Point, NY); Vaughan; William (Silver Spring, MD); Duong; Ha (Montclair, CA)

Assignee:  The United States of America as represented by the Secretary of the Army (Washington, DC)

Appl. No.:  165975

Filed:  June 10, 2002

Abstract

A controlled release microcapsulate pharmaceutical formulation for burst-free, sustained, programmable release of hydrophobic bioactive agent over a duration from 24 hours to 100 days comprising: and a blend of end-capped uncapped biocompatible, biodegradable poly(lactide/glycolide).

Description of the Invention

FIELD OF THE INVENTION

This invention relates to providing novel pharmaceutical compositions for local delivery and burst-free programmable, sustained release of hydrophobic drugs from biocompatible, biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres. The resulting product is administered locally into soft tissues by sub-cutaneous or intra-muscular injection where it will locally sustain release the drug.

This invention relates generally to providing novel blend of end-capped and uncapped providing novel biocompatible and biodegradable PLGA microspheres for burst-free programmable sustained release of hydrophobic biologically active agents which degrade over a period of up to 100 days in an aqueous physiological environment. The active agents contemplated within the scope of this invention includes the delivery of poly-peptide antibiotics, antimalarials, antituberculosis drugs, anesthetics, analgesics, anticancer agents, antiparasitic agents, antibacterial agents, antifungal agents, antiinflammatory agents, immunosuppressive agents, immunostimulatory agents, dentinal desensitizers, odor masking agents, nutritional agents, antioxidants, and insulins.

The invention also relates especially to providing novel pharmaceutical compositions for sustained release of compounds for treating cancer, inflammatory and/or autoimmune disorders from PLGA microspheres.

BACKGROUND OF THE INVENTION

This invention is particularly effective for the localized delivery of chemotherapeutic hydrophobic anticancer agents, inclusive of paclitaxel(taxol)doxorubicin, 5-fluorouracil, campthothecin, cisplatin, and metronidazole, their corresponding derivatives and functionally equivalents, and combinations thereof from PLGA microspheres.

One-third of all individuals in the United States of America (U.S.) alone will develop cancer.

Although the five-year survival rate has risen dramatically to nearly fifty percent as a resulting progress in early diagnosis and the therapy, cancer still remains second only to cardiac disease as a cause of death in the U.S. twenty percent of Americans die from cancer, half due to lung, breast, and colon-rectal cancer.

Breast cancer is the second leading cause of death in women in the U.S. Approximately 135,000 women are diagnosed with and 42,000 women die from breast cancer annually (1). Breast cancer treatment plans include a combination of surgery, radiotherapy, and chemotherapy (CT). The general treatment plan for stage I and II breast cancer is conservative surgery and radiotherapy (2,3,4,5). The general treatment plan for stage III and IV breast cancer is a combination of surgery, radiotherapy, and systemic CT using chemotherapeutic agents such as taxol (6,7,8,9,10).

Taxol treatment is recommended for the treatment of breast cancer when CT for metastatic disease has failed or when disease relapse has occurred within 6 months of adjuvant CT. Advantages of taxol treatment include: (1) lack of cardiotoxicity; (2) a mechanism of action, the stabilization of microtubules, which targets a large percentage of tumor cells, as opposed to normal cells; and (3) inhibition of angiogensis (11,12). Taxol is systemically administered intravenously (i.v.), primarily as a bolus administration. Systemic CT using taxol, as well as other chemotherapeutic drugs, is highly effective, in terms of additional years of life gained as a result of therapy; however, there are many problems associated with this treatment regimen.

CT drugs are high by cytotoxic (13), and typically, large doses of CT drugs are needed to produce an optimal therapeutic response(13, 14); therefore, CT drugs have a low therapeutic index(13). Side effects commonly seen with taxol CT include: nausea, vomiting, fever, weight changes, musculoskeletal pain, neuropathy, general malaise, immune dysfunction, and the development of tumor resistance to taxol (9,15,16).

An additional side effect seen in patients treated with taxol is a severe hypersensitivity reaction due to Cremophor EL, taxol's solubilizing agent (17). This side effect is controlled via patient pre-medication using a combination of corticosteroids, antihistamines, and histamine receptor antagonists. Often, patients become so ill during therapy that they are removed from treatment regimens or that drug dosages are lowered. The consequence of these regimen changes is fluctuating drug levels, which equates to decreased efficacy.

The problems associated with systemic taxol treatment signal the need for the development of a drug delivery system which offers a safer and a more effective means of administering toxic agents, such as taxol, to breast cancer patients, as well as to other cancer patients.

Delivery systems based on prolonged exposure to taxol have been investigated as a means to overcome the problems associated with bolus administration of taxol. These systems include infusional administration of taxol over 1,3,24, or 96 hours and administration of taxol via polymeric carrier vehicle. Infusional data suggests that cytotoxicity may be enhanced due to the increased exposure of cycling cells and has shown, in vitro, 4.4 fold less resistance in multi-drug resistant MCP-7 human breast carcinoma cells exposed to taxol for 24 hours as compared to 3 hours (16). A 96-hour taxol infusion study in patients with metastatic breast cancer showed that this infusion schedule: (1) was better tolerated than bolus administration of taxol, as evidenced by mild side effects, such as nausea and myalgia; (2) did not cause significant hypersensitivity reactions despite the omission of corticosteroid pre-treatment; (3) did not result in any cardiac, renal, or hepatic toxicity, and (4) resulted in major objective responses in 7/26 patients (26.9%), with a 6 month median response duration (16). In this trial, the predominant toxic side effect was granulocytopenia which resulted in taxol dose reduction in 3/26 patients (11.54%) and in hospitalization of 4/26 patients (15.38%).

Taxol infusion regiments offer significant advantages over bolus administration of taxol in terms of systemic toxicity, efficacy, and resistance; however, immune dysfunction still appears to be the major limiting factor in the success of this treatment regimen.

Certain chemotherapeutics such as paclitaxel (taxol) and camptothecin, which are efficacious when administered systemically must be delivered at very high dosages in order to avoid toxicity due to poor bioavailability. For example, paclitaxel (taxol) has been used systemically with efficacy in treating several human tumors, including ovarian, breast, and non-small cell lung cancer. However, maintenance of sufficient systemic levels of the drug for treatment of tumors has been associated with severe, in some cases "life-threatening" toxicity, as reported by Sarosy and Reed, J. Nat. Med. Assoc. 85(6):427-431 (1993). Paclitaxel is a high molecular weight (854), highly lipophilic deterpenoid isolated from the western yew, Taxus brevifolia, which is insoluble in water. It is normally administered intravenously by dilution into saline of the drug dissolved or suspended in polyoxyethylated castor oil. This carrier has been reported to induce an anaphylactic reaction in a number of patients (Sarosy and Reed (1993) so alternative carriers have been proposed, such as a mixed micellar formulation for parenteral administration, described by Alkan-Onyuksel, et al., Pharm. Res. 11(2), 206-212 (1994). There is also extensive non-renal clearance, with indications that the drug is removed and stored peripherally. Pharmacokinetic evidence from clinical trials (Rowinsky, E. K., et al., Cancer Res. 49:4640-4647 (1989) and animal studies (Klecker, R. W., Proc. Am. Cancer Res. 6.43:381 (1993) indicates that paclitaxel penetrates the intact blood-brain barrier poorly, if at all, and that there is no increased survival from systemic intraperitoneal injections of paclitaxel into rats with intracranial gliomas. Paclitaxol has been administered in a polymeric matrix for inhibition of scar formation in the eye, as reported by Jampel, et al., Opthalmic Surg. 22, 676-680 (1991), but has not been administered locally to inhibit tumor growth.

SUMMARY OF THE INVENTION

This invention provides a method and novel pharmaceutical compositions for local delivery and burst-free programmable sustained release of hydrophobic drugs from biocompatible, biodegradble poly (DL-lactide-co-glycolide) (PLGA) microspheres. The hydrophobic drugs are released over a time period while at the same time preserving its bioactivity and bioavailability.

It is therefore an object of the present invention to provide a chemotherapeutic composition and method of use thereof which provides for effective long term release of chemotherapeutic agents that are not stable or soluble in aqueous solutions or which have limited bioavailability in vivo for treatment of solid tumors.

It is a further object of the present invention to provide a composition and method of use for the treatment of solid tumors with chemotherapeutic agents that avoids high systemic levels of the agent and associated toxicities.

DETAILED DESCRIPTION OF THE INVENTION

The novel compositions described herein are formulated of chemotherapeutic agent, such as paclitaxel (taxol), doxorubicin, 5-fluorouracil, campthothecin, cisplatin, metronidazole campthothecin and combinations, derivatives, or functional equivalents thereof, which are not water soluble and has poor bioavailability in vivo encapsulated into a biocompatible/biodegradable polymeric matrix or in combination with hydrophillic agents for use especially in the treatment of breast cancer.

The polymer bound taxol formulations of this invention represent another strategy to achieve prolonged exposure to taxol which appears to be more advantageous than infusion regiments. Of greatest utility are the biodegradable polymers, which include the poly(lactide-co-glycolide)(PLGA) copolymer. A variety of biodegradable polymer bound taxol formulations have been developed and have been shown to inhibit tumor growth and angiogenesis in animal models with minimal systemic toxicity; however, the release kinetics of taxol in these systems, which range from 10-25% of the drug released in approximately 50 days, are, most likely, not optimal for clinical use (18,17,11,12,19,20,21,22). The advantages of biodegradable polymers as a carrier for taxol include: (1) complete biodegradation, requiring no follow-up surgery to remove the drug carrier when the drug supply is exhausted (23,24); (2) tissue biocompatibility (25); 3) ease of administration via s.c. ir u,n, ubhectuib (23,26,27); (4) controlled, sustained release of the encapsulated drug upon hydrolysis of the polymer(27,23,28,24);(5) minimization or elimination of systemic toxicity, such as neutropenia 23,24,26,27); and (6) the convenience of the biodegradable polymer system itself, in terms of versatility (23,29,24,26).

Surprisingly, our data indicates that we have developed a series of taxol/PLGA formulations which exhibit different and highly desirable rates of sustained, controlled release of taxol. Due to the sustained release characteristics of these microspheres, taxol/PLGA microsphere formulations are intended as a one-time treatment which sustain releases taxol from a subcutaneous (s.c) or intramuscular (i.m.) depot. Taxol/PLGA microspheres having a core load of 10%, 20%, 40%, and 50% were prepared via solvent evaporation and were characterized via scanning electron microscopy (SEM), particle sizing, and high performance liquid chromatography (HPLC). Microsphere morphology of these formulations showed intact spheres with an average diameter range of 5.69-7.75 um (FIGS. 1,3,5,7). Taxol core loading efficiency of all formulations ranged form 91.9%-95.48%. In vitro release of taxol into 37 degrees C PMS/albumin (pH 7.4; 0.4% albumin) over time was calculated based on the residual amount of taxol in the microsphere pellet. Results showed: (1)40.19% taxol release in 10 days using a 10% core load formulation; (2) 71.58% taxol release in 6 days using a 20% core load formulation (FIG. 4); (3) 48.09% taxol release in 6 days using a 40% core load formulation (FIG. 6); and (4) 39.84% taxol release in 6 days using a 50% core load formulation (FIG. 8).

A preliminary toxicity study using placebo PLGA microspheres and taxol/PLGA microspheres (20% core load formulation) was performed using C57/black, 6-8 week old, intact female mice. Animals were randomized into 12 groups of 4, ear notched, and weighed. Microencapsulated taxol and control polymer were individually resolubilized and injected either subcutaneously (s.c.) or intramuscularly (i.m. (inocula volume=50 ul). The right side of the animal received control polymer, and the left side of the animal received microencapsulated taxol. Each day, animals were examined for signs of toxicity. On days 2,4,6, and 8, animals were weighed, one from each group was sacrificed, and blood was collected for WBC count and for taxol quantitation via HPLC.

Treatment groups were as follows: (1) dose of 0.04 mg/kg using s.c. route; (2) dose of 0.4 mg/kg using s.c. route; (3) dose of 2 mg/kg using s.c. route; (4) dose of 4 mg/kg using s.c. route; (5) dose of 8 mg/kg using s.c. route; (6) dose of 16 mg/kg using s.c. route; (7) dose of 0.04 mg/kg using i.m. route; (8) dose of 0.4 mg/kg using i.m. route; (9) dose of 2 mg/kg using i.m. route; (10) dose of 4 mg/kg using i.m. route; (11) dose of 8 mg/kg using i.m. route; and (12) dose of 16 mg/kg using i.m. route. Dosage selection was based on the maximum tolerated systemic dose of taxol in mice, which is 16 mg/kg of taxol every 5 days over a 3 week period.

Results showed: (1) no signs of toxicity at the injection sites of any animal at any time; (2) no signs of weight loss rather, on average, the animals gained weight; and (3) no appreciable changes in white blood cell (WBC)count. Taxol concentration in serum samples was too low to be detectable/quantitable via our taxol extraction and HPLC methods. Additionally, problems experienced with the serum samples-were: (1) protein interference with the taxol peak; (2) moderate to gross hemolysis of the serum samples; and (3) the presence of additional peaks in the chromatogram, which may have been taxol metabolites. Further refinement of our methodologies for taxol extraction from serum and for trace HPLC analysis, which is currently in progress, will increase the sensitivity of taxol detection and quantitation and will eliminate these problems. Since no signs of toxicity were determined, this study is being repeated using taxol doses up to 150 mg/kg; however, this preliminary data suggests that depot administration of taxol via microspheres should not cause systemic toxicity.

Applicants have demonstrated that a s.c., or an i.m. injection of taxol encapsulated in a PLGA copolymer is equally as effective as, or perhaps better than, conventional systemic taxol therapy for human breast cancer treatment and does not cause the side effects commonly associated with conventional therapy.

Specific Embodiments

Most specifically, the embodiments of this invention are inclusive of the following items:

1. A controlled release microcapsule pharmaceutical composition of burst-free, sustained, programmable release of a hydrophobic bioactive agent over a duration of 24 hours to 100 days, comprising a hydrophobic bioactive agent and a blend of end-capped and uncapped biocompatible, biodegradable poly(lactide/glycolide).

2. The composition of Item 1 wherein the agent is released in an amount effective to inhibit growth of cancer cells.

3. The composition of Item 2 wherein the biodegradable poly(lactide/glycolide) has ratios ranging from 99/1 to 50/50.

4. The composition of Items 1, 2 or 3 wherein said copolymer has a molecular weight from 10 to 100 kDa.

5. The composition of Items 1, 2, 3 or 4 wherein the copolymer is a blend of hydrophobic end-capped polymer with terminal residues functionalized as esters and hydrophillic uncapped polymer with terminal residues existing as carboxylic acids.

6. The composition of Items 1, 2, 3, 4, or 5 wherein the agent is selected from the group consisting of paclitaxel, doxorubicin, 5-fluorouracil, camptothecin, cisplatin, metronidazole, and combinations thereof.

7. The compositions of Items 1, 2, 3, 4, 5 or 6 further comprising additional biologically active compounds selected from the group consisting of chemotherapeutics, antibiotics, antivirals, antinflammatories, cytokines, immunotoxins, anti-tumor antibodies, anti-angiogenic agents, anti-edema agents, radiosensitizers, and combinations thereof.

8. A method of administering to a patient in need of treatment a pharmaceutically-effective amount of a hydrophobic bioactive agent comprising administering the bioactive agent locally to an infectious area, wherein the agent is incorporated into and controlled released, burst-free, from a blend of end-capped and uncapped biocompatible, biodegradable poly(lactide/glycolide) over a period of 24 hours to 100 days.

9. The method of Item 8 wherein the bioactive agent is an anticancer agent.

10. The method of Item 9 wherein the anticancer agent is selected from the group consisting of paclitaxol, doxorubicin, 5-fluorouracil, camptothecin, cisplatin, metronidazole, and combinations thereof.

11. The method of Item 10 wherein the anticancer agent is paclitaxol.

12. The method of Item 8, 9, 10 or 11 wherein the bioactive agent is administered to the patient prior to the onset of infections.

13. The method of Item 8, 9, 10 or 11 wherein the bioactive agent is administered to the patient in need thereof post-infection.

14. The method of Item 8, 9, 10 or 11 wherein the bioactive agent is administered intra-muscularly or subcutaneously.

15. The method of Item 8 further comprising administering radiation in combination with the composition.

16. The method of Item 8 further comprising administering with the bioactive agent additional biologically active compounds selected from the group consisting of chemotherapeutics, antibiotics, antivirals, antiinflammatories, cytokines, immunotoxins, antitumor antibodies, anti-angiogenic agents, anti-edema agents, radiosensitizers, and combinations thereof.

17. The method of Item 8 wherein the composition is in the form of micro-implants and are administered by injection or infusion.

18. The method of Item 10 wherein the form of cancer being treated is selected from the group consisting of ovarian, breast, lung, prostatic, and melanoma, brain tumor cells, and cancer of the colon-rectum, esophagus, liver, pancreas, and kidney.

19. A method for inhibiting the proliferation of rapidly proliferating abnormal mammalian cells, said method comprising contacting said cells with a cell proliferating inhibiting amount of an anticancer agent which has been incorporated into and controlled released, burst-free, from a blend of end-capped and uncapped biocompatible, biodegradable poly(lactide/glycolide), for a programmable time sufficient to inhibit said proliferation.

Claim 1 of 13 Claims

What we claim is:

1. A method of preparing a controlled release microcapsule pharmaceutical composition of burst-free sustained, programmable release of a hydrophobic bioactive agent over a duration of 24 hours to 100 days, comprising the steps of:

a) mixing a hydrophobic bioagent with a blend of end-capped and uncapped biocompatible, biodegradable poly(lactide/glycolide) copolymer, wherein said end-capped polymer has terminal residues functionalized as esters and said uncapped polymer has terminal residues existing as carboxylic acids; and

b) performing a solvent evaporation process to form said microcapsules.

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