WO1996026716A1 - Elcatonin controlled release microsphere formulation for treatment of osteoporosis - Google Patents

Abstract

This invention provides elcatonin- and elcatonin analog-comprising controlled release microsphere formulations, methods for their preparation and their utility in the treatment of osteopathies, for example, osteoporosis.

Description

ELCATONIN CONTROLLED RELEASE MICROSPHERE FORMULATION FOR TREATMENT OF OSTEOPOROSIS

Field of the Invention The present invention relates to pharmaceutical microsphere compositions having controlled release characteristics where the active agent is a water-soluble polypeptide which is a calcium regulatory hormone or, more particularly, an elcatonin, or analog thereof, useful for the treatment of osteoporosis.

Background of the Invention Calcitonins of natural origin are 32-amino acid peptide hormones involved in the regulation of calcium metabolism. Calcitonin, together with parathyroid hormone, participate in the regulation of bone metabolism. Under normal conditions, there exists a balance between osteolytic resorption and osteoblastic formation of new bone. Calcitonin appears to oppose the osteolytic activity of parathyroid hormone, acting directly to inhibit bone resorption. Calcitonin may also enhance new bone formation by stimulation of osteoblasts.

Bone resorption causes a release of calcium and alkaline phosphatase into the circulation, and correlates with the appearance of urinary hydroxyproline, resulting from the breakdown of collagen-containing bone matrix. According to physiological mechanisms, elevated serum calcium levels promote the secretion of calcitonin, which has a hypocalcemic effect. In normal individuals, bone resorption is minimal, and exogenous calcitonin has no hypocalcemic effect.

Many diseases in man, including not only those associated with bone resorption, but those related to other disorders, including malignancy, are marked by hypercalcemia, the persistence of which can be life-threatening. Exogenous calcitonin has proved to be a valuable therapeutic agent in treating these disorders. Calcitonin therapy is thus effective in diminishing hypercalcemia in patients with hyperparathyroidism, idiopathic hypercalcemia of infancy. Vitamin D intoxication, and osteolytic bone metastases. It similarly diminishes the hypercalcemia that accompanies malignancies with or without metastasis, and that of multiple myeloma.

Calcitonin is also effective in treating disorders wherein bone turnover or resorption is accelerated but changes in serum calcium levels are not always detected. One important disease of this type is osteoporosis, particularly postmenopausal type, wherein there is a progressive loss of bone mass. Patients with osteoporosis exhibit increased levels of serum calcium, phosphate, alkaline phosphatase and osteocalcin, as well as the fasting urinary excretion of hydroxyproline and calcium. Measurement of serum osteocalcin levels and direct bone density measurements are sensitive and thus useful in detecting osteoporosis.

Osteocalcin, a 49-amino acid peptide chain, is synthesized by osteoblasts in bone formation. Elevated serum osteocalcin levels correlate with metabolic bone diseases characterized by increased bone turnover, e.g., Paget's disease, osteoporosis, etc. Patients receiving treatment with calcitonin exhibit marked decreases in serum osteocalcin level together with decreases in other serum and urinary biochemical parameters. The efficacy of calcitonin in osteoporosis appears to be determined by its ability to increase total body calcium.

Paget's disease (osteitis deformans) is a disorder characterized by excessive resorption of bone accompanied by the imbalanced formation of new (pagetic) bone which lacks the characteristic architecture of normal bone. Calcitonin reduces the elevated serum levels of osteocalcin and alkaline phosphatase and urinary calcium and hydroxyproline seen in individuals with this disease. Benefits of calcitonin therapy in Paget's disease are indicated by radiologic evidence of bone remodeling, correlated with a reduced number of osteoclasts seen in bone biopsies, consistent with a decrease in bone resorption. Calcitonin also provides relief from the pain and tenderness associated with the disease.

Calcitonins are found in a variety of vertebrate species including mammals, birds and fish. The hormone is secreted by the C cells, which are localized in the thyroid gland of mammals and in the ultimobranchial glands in the lower vertebrates.

Human calcitonin (hCT) has the following amino acid sequence:

1 2 3 4 5 6 7 8 9 10 11 12 13

Cys— Gly- Asn- Leu- Ser- Thr- Cys- Mer- Leu- Gly- Thr- Tyr- T r-

14 15 16 17 18 19 20 21 22 23 24 26 26 Gin- Asp- Phe- Asn- Lys- Phe- His- Thr- Phe- Pro- Gin- Thr- Ala-

27 28 29 30 31 32 lie- Gly- Val- Gly- Ala- Pro- NH₂

Calcitonins of certain non-human species appear to be more potent in humans than human calcitonin. Calcitonins that are ultimobranchial in origin, such as salmon, eel and avian, are more potent than thyroidal calcitonins, such as human or porcine hormones. Salmon, eel, porcine and human calcitonins are currently in clinical use for the treatment of Paget's disease, osteoporosis and the hypercalcemia of malignancy. In spite of their higher potency, however, the calcitonins from other species, such as the ultimobranchial calcitonins, are not entirely satisfactory for human clinical use, primarily because the variable, poorly conserved middle portion of non-human calcitonins acts as an immunogen in vivo. The resulting antibody production can therefore limit their usefulness (Basava et al., U.S. Patent No. 5, 175, 146, December 29, 1992).

After administration to man by subcutaneous injection, all the natural calcitonins have a relatively short half life because, in spite of species differences which act to retard proteolysis by plasma enzymes, they are subject to rapid renal and tissue clearance as well. Also, because the activity of natural calcitonins depends on an intact disulfide bond between the cysteine groups at positions 1 and 7, the reduction of this unstable bond in vivo rapidly converts biologically active peptides to an inactive form (Basava et al., U.S. Patent No. 5,175, 146, December 29, 1992).

It would be useful to obtain a calcitonin-type peptide in a form which would render the compound to be more effective in clinical use because of greater stability in vivo, higher potency and/or longer viability.

Elcatonin [l-butanoicacid-26-L-asparticacid-27-L-valine-29-L-alanine-1 ,7-dicarbacalcitonin

(salmon) is a synthetic analog of the calcium-regulating hormone peptide calcitonin. Elcatonin exhibits calcium-regulating activity and is reported to be more potent and more stable than calcitonin due to the absence of disulfide linkages. Elcatonin is a 31 amino acid peptide with an o- aminosuberic acid bridge at the first and seventh amino acid positions: O

II

C (CH₂) ₄ CH₂

I I

Ser-Asn-Leu-Ser-Thr-NH-CH-CO-Val-Leu-Gly-Lys- Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-

Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH₂

The major difference between elcatonin and eel calcitonin is the replacement of cysteines at the first and seventh amino acid positions with σ-aminosuberic acid. It is believed that removal of the disulfide linkage in this calcitonin-type compound renders the compound more stable in serum, liver or kidney. When compared with known, naturally-occurring calcitonins, elcatonin and elcatonin analogs have the same or higher biological activity. Elcatonin is used for osteogenic hypercalcemia, Paget's disease, and improvement of pain in osteoporosis. Controlled release systems deliver drugs at a predetermined rate for an extended time period. It has long been appreciated that the continuous release of certain drugs over an extended period following a single administration could have significant practical advantages in clinical practice, and compositions have already been developed to provide extended release of a number of clinically useful drugs.

In particular, it is known that for many drugs suitable implantable devices for providing extended drug release may be obtained by encapsulating the drug in a biodegradable polymer or by dispersing the drug in a matrix of such a polymer so that the drug is released as the degradation of the polymer matrix proceeds.

However, the preparation of polymer formulations by known techniques such as mixing of the molten components, grinding, heat homogenation techniques such as compression and extrusion may result in a substantial, often nearly complete, loss of biological activity of an encapsulated bioactive protein ingredient. Large polypeptides are particularly susceptible to physical and chemical denaturation and consequent loss of biological potency from exposure to excessive heat, solvents, and shear forces. For this reason, incorporation of polypeptides in polymers has, until now, required either compromise in the degree of uniformity of the polypeptide-polymer dispersion, or has resulted in substantial loss of the biological potency of the polypeptide, or both. For example, a polymer-interferon formulation formed by heated mixing and extrusion under mild conditions retained less than 1 % of the original biological activity of the interferon [Eppstein et al. (1990) U.S. patent 4,962,0911. To compensate for the loss in biological activity during manufacturing processes, a large excess of polypeptide must be incorporated in the formulation, adding to the expense of producing and utilizing drug encapsulating polymer formulations. Accordingly, there is a need for a polymer formulation which provides controlled and continuous delivery of a polypeptide and which can be manufactured without significant loss of biological activity.

Suitable biodegradable polymers for use in sustained release formulations are known, and include polyesters, which gradually become degraded by hydrolysis when placed in an aqueous, physiological-type environment. Particular polyesters which have been used are the polyϋactic/glycolic acid) (PLG) polymers.

The use of PLG polymers for the long term delivery of proteins has become an important area of research in drug therapeutic applications. Drug-delivery research with PLG was largely confined to drugs that are relatively hydrophobic, for example, steroids [Wise et al., (1973) Contraception 8:227-234] and to small peptides [Hutchinson et al. (1 985) Biochem. Soc. Trans. 13:520-523; Sanders et al. (1986) J. Pharm. Sci. 75:365-370]. Drugs comprising polypeptides or proteins which are relatively hydrophilic and of relatively high molecular weight present an additional degree of difficulty in the preparation of controlled release microsphere formulations. For example, many such proteins are insoluble in PLG and their release through the polymer phase of the microsphere via diffusion is minimal and, instead, is determined by the chemical characteristic of the PLG.

The release mechanism generally involves movement of the polypeptide through a complex porous path in the polymer matrix. If the polymer erodes, this will affect the pore structure and accelerate the release. Factors influencing release rates include protein particle size and loading, protein solubility and molecular weight, polymer composition and molecular weight, and the dimensions and shape of the matrix [Bawa et al. (1985) J. Controlled Release 1 :259; Hsu et al. (1985) J. Biomed. Mater. Res. 19:445; Ogawa et al. (1988) Chem. Pharm. Bull. 36: 1502; Satzman et al. (1989) Biophy. J. 55: 1631. Polymer systems are now being used in animal studies to release proteins, including insulin, growth factors, angiogenesis inhibitors, etc. [Brown et al. (1986) Diabetes 35:692; Silverstein et al. (187) Science 237:291 ]. The first Food and Drug Administration

(FDA)-approved system for controlled release of a peptide, the Lupron Depot (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate, and lasting 30 days) was recently introduced for the treatment of prostate cancer. Other polymeric systems for releasing similar drugs are also under evaluation for treating endometriosis and other conditions [Sanders et al. (184) J. Pharm. Sci. 73:1294; Hutchinson et al. (1989) in Drug Carrier Systems,

Roerdink and Kroon, eds., Wiley, New York, pp. 1 1 1 -130].

The release characteristics for a bioactive agent from a controlled release microsphere formulation may be continuous or discontinuous. For example, the release of a polypeptide from microspheres is often preceded by a significant induction period, during which no polypeptide is released, or is biphasic, and comprises an initial period during which some polypeptide is released, a second period during which little or no polypeptide is released, and a third period during which most of the remainder of the polypeptide is released. Alternatively, the polypeptide may be released initially in an accelerated "burst effect," followed by a gradual extended release pattern.

PLG polymers and related materials incorporating a bioactive agent and demonstrating controlled release properties are described in many U.S. patents. Microencapsulation of calcitonin- type bioagents has been mentioned, for example, in U.S. Patent Nos.: 5, 143,661 , September 1 , 1992, Lawter et al.; 5, 134, 122, July 28, 1992, Orsolini; 5,066,436, November 1 9, 1991 , Komen et al.; 5,004,602, April 2, 1991 , Hutchinson; 5,000,886, March 19, 1991 , Lawter et al., 4,962,091 , October 9, 1990, Eppstein et al.; 4,897,268, January 30, 1990, Tice et al.; 4,801 ,739, January 31 , 1989, Franz et al.; and 4,767,628, August 30, 1988, Hutchinson, as well as WO 91 /12882, published September s, 1991 , Wantier et al., and GB 2,209,937, issued July 3, 1991 , Orsolini et al. None of these patents discloses the in vivo benefit or effectiveness of calcitonin- or elcatonin-containing controlled release microspheres in the treatment of osteoporosis or other related diseases.

Summary of the Invention

This invention provides controlled release microsphere formulations for the delivery of elcatonin or an elcatonin analog into the system of an animal at a continuous rate over an extended period of time. This invention contemplates not only the administration of one type of microsphere but also the administration of a mixture of at least two different types of microspheres. A composition comprising a mixture of controlled release microspheres is obtained by encapsulating quantities of the active ingredient in different polymer excipients which biodegrade at varying rates. An effective amount of controlled release microspheres may be administered to the animal parenterally (e.g., intravenously, intramuscularly, subcutaneously, intranasally, intraperitoneally, or by inhalation or implantation).

For example, a quantity of these controlled release microspheres is of such a polymer excipient that the active ingredient is released quickly after administration into an animal and, for the major part, is delivered within an initial period. A second quantity of the controlled release microspheres is of such a formulation that delivery of the encapsulated ingredient begins as the first quantity's delivery begins to decline. Variations of these polymer excipients can be prepared to obtain a desired release profile for the encapsulated bioactive agent.

This invention thus provides a method of delivering elcatonin or an elcatonin analog that is a preselected, continuous rate of delivery over an extended period of time, for example, for a duration of from approximately one week to approximately six months or more.

This invention also provides methods of preparing controlled release microspheres comprising elcatonin or elcatonin analogs using organic and aqueous phase separation methods, i.e., by the oil-in-oil and the oil-in-water techniques. Depending on the technique used for the preparation of the controlled release microspheres, release of the active ingredient from the microsphere proceeds at a rate predetermined in part by the rate of degradation of the specific polymeric formulation.

It is an object of this invention to prepare a variation of the controlled release microspheres such that the delivery period is shortened or increased. In one aspect of this invention, it was possible to design a delivery system having a controlled release profile of specific duration by using a polymer for the encapsulation process having a molecular weight smaller than taught by the art.

It is another object of this invention to provide elcatonin- or elcatonin analog-containing controlled release microspheres for use in the treatment of osteopathies, in particular, osteopathies manifesting abnormal serum osteocalcin levels.

It is a particular object of this invention to provide elcatonin-containing controlled release microspheres for use in the treatment of osteoporosis.

It is an additional object of this invention to provide elcatonin-containing controlled release microspheres to deliver an effective amount of elcatonin that causes a reduction in the level of serum osteocalcin.

It is a further object of this invention to provide elcatonin-containing controlled release microspheres having a biological potency similar or superior to that of non-encapsulated elcatonin in therapeutic applications.

Brief Description of the Drawings Figure 1 presents in vitro cumulative release of elcatonin from PLG, 50:50 (molecular weight of approximately 60,000 daltons), microspheres prepared by the oil-in-water technique.

Detailed Description of the Invention

The following definitions are provided in order to provide clarity as to the intent or scope of their usage in the specification and claims.

The term elcatonin or bulk elcatonin as used herein refers to elcatonin which is not associated with microsphere formulations and which may be administered as a bioactive agent in a pharmaceutically acceptable composition for therapeutic applications.

The term elcatonin analog as used herein refers to synthetic hypocalcemic peptides having the chemical structure ( CH₂) 5

-CO—Ser-Asn-Leu-Ser-Thr-NHCHCO - Val-Leu- Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-A₂₅-A₂₆-Gly-A₂₈- Gly-Thr-Pro-NH₂

wherein A₂₆ is Asp or Asn, A_2β is Val or Thr, and A_2B is Ala or Ser.

These peptides comprise analogs of elcatonin having amino acid substitutions and deletions which act to improve potency, prolong duration of the hormonal effect, enhance receptor binding and/or increase bioavailability. Such elcatonin analogs, as well as elcatonin, are less expensive and more easily synthesized than native calcitonin and have improved resistance to inactivation and degradation [Sakakibara et al. (1978) U.S. Patent No. 4,086,221 ].

The terms elcatonin controlled release microspheres and elcatonin-containing controlled release microspheres are used interchangeably herein and refer to controlled release microspheres comprising elcatonin as the active ingredient.

The terms controlled release microsphere formulation and controlled release microsphere composition are used interchangeably herein and refer to the microspheres or mixture of microspheres of the instant invention administered to an animal.

The term released elcatonin as used herein refers to elcatonin that is released with time from elcatonin-comprising controlled release microspheres.

The term hydrophilic peptide as used herein refers to peptides and other optional pharmaceutically acceptable components which are at least "very slightly soluble" by the definition given in the United States Pharmacopeia, XX, page 1 121 , i.e., having water solubilities of at least 0.1 -1 .0 mg/ml.

The term osteocalcin as used herein refers to the bone GLA protein (BGP), the most abundant of the non-collagen proteins of bone. Osteocalcin circulates in the blood and can be measured, for example, by radioimmunoassays. The terms polvdactic/olvcolic acid) polymer, polvdactide/olvcolide) polymer, po D.L-lactide- co-olvcolide) polymer or PLG. as used herein, are interchangeable and refer to polymer compositions comprising polylactic acid and polyglycolic acid, or salt derivatives thereof, used to prepare pharmaceutical compositions comprising microspheres encapsulating bioactive agents.

The terms controlled release, release profile, or release characteristics, as used herein, refer to the rate at which the active ingredient is released from microspheres. In general, release rates are determined by the design of the system and may be nearly independent of environmental conditions such as pH. These systems can deliver drugs for extended time periods (days to months).

The term biodegradable is used herein to mean that the polymer degrades when administered to a living organism by hydrolysis or as a result of enzymatically catalyzed degradation or by a combination of the two.

The term biocompatible as used herein refers to compatibility with living tissue. For example, a biocompatible polymer is a polymer that would not cause side effects detrimental to a host animal.

The terms bioaoent. bioactive aoent. bioactive compound, active peptide or active ingredient as used herein refer to a compound, synthetic or naturally occurring, that has a specific biochemical activity or a specific regulatory function.

The term effective amount as used herein refers to the quantity of active ingredient necessary to effect in an animal a change in a specific biochemical parameter. For example, an effective amount of elcatonin, as used herein, is the quantity of elcatonin which will produce a measurable and beneficial change in a biochemical parameter, e.g., a decrease in serum osteocalcin level. Further, an effective amount of elcatonin controlled release microspheres is the quantity of microspheres that must be administered to an animal in order to deliver continuously an effective amount of elcatonin such that a resultant, beneficial, biochemical or physiological effect is observed, e.g., a decrease in serum osteocalcin level.

The term initial period as used herein refers to the period of time immediately following administration of controlled release microspheres to an animal. The initial period spans a duration of no more than eight days, preferably one to two days, and more preferably about 12 hours.

The term animal as used herein refers to a mammal and, in particular, to a human. The term oil-in-oil technique as used herein refers to an organic phase separation method known in the art for the preparation of microspheres comprising a pharmaceutical agent incorporated in a biocompatible and biodegradable polymer matrix. Specific methods using the oil- in-oil technique are disclosed, for example, in Mathiowitz et al. (1988) J. Appl. Polym. Sci. 35:755- 774 and in Example 1 of the specification.

The term oil-in-water technique as used herein refers to an aqueous phase separation method known in the art for the preparation of microspheres comprising a pharmaceutical agent incorporated in a biocompatible and biodegradable polymer matrix. Specific methods using the oil- in-water technique are disclosed, for example, in Fong etal. (1986) J. Controlled Release 3: 1 19-130 and in Example 1 of the specification.

Guidelines and procedures for the synthesis of elcatonin as well as for analogs of elcatonin are detailed, for example, in Sakakibara et al., U.S. Patent No. 4,086,221 , issued 1978 and Morikawa et al. (1976) Experientia 32: 1 104-1 106.

Elcatonin and bioactive elcatonin analogs may be prescribed for osteopathy requiring calcium, such as Paget's disease, osteoporosis, osteomalacia, fracture, fibrous dysplasia of the bone or rachitis caused by corticosterone therapy or inactivation after menopause or external injury, and is especially suited to therapy in combination with calcium or phosphorous. Hypercalcemia associated with, for example, bone cancers, immobilization, hyperparathyroidism, adrenal insufficiency, milk-alkali syndrome, thyrotoxicosis, sarcoidosis, etc., may also be treated with elcatonin and its bioactive analogs.

The present invention is intended to be used in all diseases classified as osteoporosis, particularly post-menopausal osteoporosis, senile osteoporosis, idiopathic osteoporosis, immobilization osteoporosis, post-partum osteoporosis, juvenile osteoporosis, and osteoporosis secondary to gonadal insufficiency, malnutrition, hyperprolactinemia, prolactinoma, disorders of the gastrointestinal tract, liver, or kidneys, and osteoporosis that is a sequela of prior osteomalacia, chronic acidosis, thyrotoxicosis, hyperparathyroidism, glucocorticoid excess or chronic disorders involving the bone marrow, and heritable forms of osteoporosis such as osteogenesis imperfecta and its variants, and other heritable disorders of connective tissue.

Administration of elcatonin for therapeutic use presents two main problems. One problem is that aqueous solutions of elcatonin have been shown to lose activity with time due to heat and light instability. Inclusions of monocarboxylic acids or salts in aqueous solutions of elcatonin were shown to render the aqueous elcatonin solution more stable with time. [Yamada et al. (1990) U.S. Patent No. 4,977, 139.] The second problem is that elcatonin must be repeatedly administered parenterally. The third problem relates to the relatively short in vivo half life of elcatonin and the necessity for daily injections which, at best, is inconvenient and has the potential for undesirable side effects.

Microcapsules prepared with a biodegradable encapsulating polymer according to the current invention provide the ideal delivery system for elcatonin and related analogs. Implanted or injected subcutaneousiy or intramuscularly, the polymer portion of the microcapsule will biodegrade and bioerode, resulting in the release of the peptide into the body for periods ranging from several hours to several months.

If the capsules are to be administered by injection they may first be suspended in some non- toxic suspending vehicle. The exact make up of these injectable microcapsule suspensions will depend upon the amount of drug to be administered, the suspending capacity of the suspending agent and on the volume of solution which can be injected at a particular site or in a particular subject.

The compositions of this invention exhibit controlled release of the encapsulated substance over extended periods of time. This time period may range from approximately one week to approximately one year depending on the composition of the encapsulating excipient, its molecular weight, the diameter of the capsule, and the presence of a stabilizing agent or a polymer hydrolysis modifying agent in the core. Preferably, the release time will be about one to six months.

Many different methods and materials useful in the preparation of microencapsulated pharmaceutical agents are known to those skilled in the art as evidenced by the above-referenced patents and publications.

In brief, the procedure involves dissolving a biocompatible and biodegradable polymer, such as polγdactide/glycolide) or other similar polyester type polymer, in a halogenated hydrocarbon solvent, e.g., methylene chloride or other C,-C₄ halogenated alkane; dispersing the bioactive compound, in solid or aqueous form, in the polymer-solvent solution; adding a non-solvent (or coacervation agent), an organic liquid which is not miscible with the polymer, to cause phase- separation whereby the polymer is deposited on the dispersed bioactive substance; and adding a hardening solvent, e.g., alkanes such as heptane and cyclohexane, volatile silicone fluids, fatty acid esters, etc., to extract polymer solvent from the dispersion and to form microspheres suspended in the hardening solvent. While the composition of matter employing the above-described polyesters, organic solvents, non-solvents (coacervation agents), hardening agents and the processes by which the controlled release microspheres are produced are applicable to certain of a variety of bioagents (e.g., steroids and hydrophobic drugs), they are not immediately applicable to all hydrophilic peptides. For some polypeptides, for example, elcatonin, known procedures for the preparation of oil-in-oil and oil-in-water microspheres do not allow sufficient daily release of the bioactive compound for expeditious therapeutic use of the elcatonin-comprising controlled release microspheres.

Current methods of preparing microspheres include the oil-in-oil (o/o) technique [Mathiowitz et al. (1988) J. Appl. Polym. Sci. 35:755-774 and Wang et al. (1991 ) J. Controlled Release 17:23-

32] and the oil-in-water (o/w) technique [Fong et al. (1986) J. Controlled Release 3:1 19-130 and Wang et al. (1991 ) supra] . This invention comprises controlled release microspheres prepared by both the oil-in-oil and oil-in-water techniques. It is preferred that the PLG polymer used in both techniques be a copolymer having a 50:50 lactide to glγcolide ratio although, as is known and well characterized in the art, this ratio may be readily varied (between approximately 75:25 and approximately 25:75) to obtain a specific release profile. In specific embodiments of the invention using the oil-in-oil technique, a PLG polymer having a molecular weight of between approximately 5 and approximately 150 kD, and preferably between approximately 25 and approximately 100 kD, and more preferably between approximately 50 and approximately 75 kD, was utilized for the microencapsulation process. In other embodiments of the invention using the oil-in-water technique, however, the molecular weight of the PLG polymer used was between approximately 5 and approximately 150 kD, preferably between approximately 5 and approximately 75 kD, and more preferably between approximately 5 and approximately 20 kD. Although the prior art teaches the use of PLG having a molecular weight of approximately 25 kD or greater, the instant invention used PLG polymers of lower molecular weights, e.g., between approximately 9 and approximately 12 kD, and enabled the preparation of microspheres delivering sufficient elcatonin for a desired duration.

For the purposes of this invention the molecular weight of a particular polymer is determined as a function of its intrinsic viscosity as measured in a capillary viscometer using chloroform at 30°C or in gel permeation chromatography using chloroform at 35°C. The intrinsic viscosities of PLG polymers suitable for use in this invention range from about 0.2 dl/g to about 1 .5 dl/g and are preferably in the range of about 0.23 dl/g to about 0.70 dl/g. There appears to be a direct correlation between inherent viscosity and molecular weight.

Microspheres prepared by the oil-in-oil technique or microspheres prepared by the oil-in- water technique may be utilized for the controlled release of bioactive agent. It is preferred, however, that mixtures of oil-in-oil microspheres and oil-in-water microspheres be used for increased flexibility in designing the drug delivery schedule, i.e., to more accurately and more predictably control and regulate the release kinetics of the encapsulated bioactive agent.

The controlled release microspheres may range in diameter from about 0.1 to 1000 microns, preferably between 10-500 μm, and more preferably between 25-100 μm for the oil-in-oil preparation of microspheres or between 250-500 μm for oil-in-water prepared microspheres.

The present invention is well-suited to the controlled delivery of elcatonin and elcatonin analogs. The amount of active ingredient incorporated in the PLG polymer matrix using the oil-in-oil technique may vary between 0.01 and 40.0 weight %, and preferably between 0.05 and 40.0 weight % of the polymer used for encapsulation. With the oil-in-water technique, the amount of active compound incorporated in the polymer matrix may vary between 0.001 and 40.0 weight %, and preferably between 0.01 and 40.0 weight %.

The amount of peptide placed in a particular formulation depends not only on the desired daily dose but also on the number of days that dose level is to be maintained. While this amount can be calculated empirically, the actual dose delivered is a function of the degradation characteristics of the encapsulating polymer.

Optionally, certain chemicals, e.g., citric acid, sodium chloride, sodium carbonate, etc., which affect the rate of polymer hydrolysis may be dissolved in the aqueous solution containing the polypeptide before it is encapsulated by the polymer excipient. These chemicals are called polymer hydrolysis modifying agents. When present, these compounds may increase or decrease the rate at which the drug is released from the microcapsules. This effect is independent of a particular polymer composition or microcapsule size. When present, the hydrolysis modifying agent will be added in an amount between 0.1 and 20% by weight of the polymer but preferably it will be present in the amount of 5 to 10%.

Many polypeptides benefit from the presence of small quantities of stabilizers, buffers, salts and the like. Water-soluble components which may be useful in the practice of this invention include, but are not limited to, stabilizers, carbohydrates, buffers, salts, surfactants and plasticizers. Examples of suitable stabilizers include human serum albumin (HSA), gelatin, dextrose, and other carbohydrates. Examples of other carbohydrates suitable for incorporation in this invention include sucrose, maltose, mannose, glucose, fructose, lactose, sorbitol and glycerol. Suitable surfactants include Tween (e.g. Tween-20, Tween-80), Pluronic^® polyols such as Pluronic^® L101 , L121 and F127. Among the suitable plasticizers are the polyethylene glycols, glycerides and ethylcellulose. In a particular aspect of the invention, a biodegradable and biocompatible controlled release microsphere formulation having a particle size between approximately 30 and approximately 100 microns, was prepared by the oil-in-oil technique so as to possess specific characteristics, i.e., to encapsulate elcatonin or an elcatonin analog in a quantity sufficient for the desired duration of therapy and, furthermore, to provide kinetics for the release of elcatonin or its analog from the microspheres placed into an animal such that a given amount, preferably between about 45% and about 85%, and more preferably between about 50% and about 75%, of the amount of active ingredient in the microsphere is released initially ("burst effect"), preferably within approximately two days and more preferably within 24 hours, with the remainder of the active compound being released slowly over a period of up to approximately three to six months, preferably up to approximately one to three months, and more preferably up to approximately one to four weeks.

A specific embodiment of this aspect of the invention discloses the preparation of a controlled release microsphere formulation by the oil-in-oil technique which permitted elcatonin to be loaded at levels that were preferably between 75% and 90% of theory, and specifically between 85% and 89% of the theoretical content. (The theoretical content is calculated to be 1 .175 μg of elcatonin per mg of microspheres.) This elcatonin-containing controlled release microsphere formulation exhibited in vitro release kinetics which gave an initial release ("burst effect") comprising 73.1 % of the elcatonin in the microspheres within 12 hours with the remainder released slowly for approximately one to four weeks. The controlled release microspheres prepared by the oil-in-oil technique that gave these release characteristics had a particle size that measured between approximately 30 and approximately 100 microns in diameter.

In another particular aspect of the invention, a biodegradable and biocompatible, controlled release microsphere formulation, having a particle size between approximately 1 to approximately 750 microns and preferably between approximately 250 to approximately 500 microns, was prepared by the oil-in-water technique to possess specific characteristics, i.e., to encapsulate elcatonin or an elcatonin analog in a quantity sufficient for the desired duration of therapy and, furthermore, to provide kinetics for the release of elcatonin or its analog from the microspheres placed into an animal such that very little, less than approximately 5%, and preferably none, of the elcatonin of the microspheres is released initially (small or no "burst effect") and that the elcatonin content is released gradually within approximately a twelve month period, and preferably within approximately a one to six month period.

A specific embodiment of this aspect of the invention discloses the preparation of a controlled release microsphere formulation using PLG polymer having a molecular weight of approximately 60 kD prepared by the oil-in-water technique which allowed elcatonin to be loaded at a level of approximately 40% and preferably at levels between 35% and 75% of the theoretical content (the theoretical content is calculated to be 0.97 μg of elcatonin per mg of microspheres). This elcatonin-containing controlled release microsphere formulation exhibited in vitro release kinetics which did not give an initial "burst effect" release of elcatonin and, instead, gave a continuous rate of release for about three months (Figure 1 ).

In an alternate embodiment of this aspect of the invention, a different molecular weight PLG polymer was utilized for the microencapsulation process by the oil-in-water technique. PLG polymer having a molecular weight of approximately 9,400 was used in this alternate formulation. This alternate microsphere formulation gave essentially the same particle size of microspheres (approximately 250 to approximately 500 microns) and essentially the same elcatonin content

(approximately 40% of the theoretical content in oil-in-water microsphere preparations) as were obtained using the higher molecular weight polymers of approximately 60,000 daltons. However, in contrast to the formulation prepared with PLG polymer of approximately 60,000 daltons, this microsphere formulation exhibited faster in vitro release characteristics. Whereas the 60,000 dalton polymer formulation gave controlled release of elcatonin for approximately three months, this formulation prepared with the approximately 10,000 dalton polymer released the elcatonin from the microspheres in approximately two months.

The compositions of this invention will contain individually the active polypeptides in varying amounts depending on the biological effect desired. For example, treatment of osteoporosis using elcatonin-containing controlled release microsphere formulations of the invention may require a dosage level different from that used to treat hypercalcemia associated with osteopathies. Similarly, a particular pathological condition may necessitate different and specific dosages depending on whether elcatonin or an elcatonin analog is the active ingredient of the controlled release microspheres.

Dosage levels may also vary depending upon the species and the size of the animal. For example, rats were administered elcatonin in the range of from about 0.001 lU/kg/day to about 100 lU/kg/day, and preferably from about 0.05 lU/kg/day to about 1 5 lU/kg/day as treatment for osteoporosis. It is expected that elcatonin analogs would be used at similar dosage levels. The recommended therapeutic dosage levels of elcatonin for human subjects range from approximately 10 to 300 IU per administration, and preferably from approximately 40 to approximately 80 IU per administration. These human dosage values can be reevaluated readily in clinical studies using art known methodologies for specific therapeutic indications. Controlled release systems provide advantages over conventional drug therapies. For example, after ingestion or injection of standard dosage forms, the blood level of the drug rises, peaks, and then declines. Since each drug has a therapeutic range above which it is toxic and below which it is ineffective, oscillating drug levels may cause alternating periods of ineffectiveness and toxicity. A controlled release preparation maintains the drug in the desired therapeutic range by a single administration. Other potential advantages of controlled release systems include: (i) localized delivery of the drug to a particular body compartment, thereby lowering the systemic drug level; (ii) preservation of medications that are rapidly destroyed by the body (this is particularly important for biologically sensitive molecules such as proteins); (iii) reduced need for follow-up care; (iv) increased comfort; and (v) improved compliance.

It will be appreciated by those of ordinary skill in the art that the objects of this invention can be achieved without the expense of undue experimentation using well known variants, modifications, or equivalents of the methods and techniques described herein. The skilled artisan will also appreciate that alternative means, other than those specifically described, are available in the art to achieve the functional features of the molecules described herein and how to employ those alternatives to achieve functional equivalents of the molecules of the present invention. It is intended that the present invention include those variants, modifications, alternatives, and equivalents which are appreciated by the skilled artisan and encompassed by the spirit and scope of the present disclosure.

The following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications can be made to the methods and formulations described herein while not departing from the spirit and scope of the present invention.

EXAMPLES

Example 1 . Preparation of Microspheres.

(a) Oil-in-oil (o/o) emulsion technique.

Microspheres were prepared with the oil-in-oil (o/o) emulsion technique (also known as the coacervation technique) as follows:

(1 ) Methylene chloride <CH₂CI₂) (600 ml), silicone fluid 500 (30 ml) and Span 85 (2 ml) were mixed in a beaker with a high shear homogenizer at a low speed until a homogenous solution was obtained. (2) Poly (DL-lactide/glycolide) or PLG, a 50:50 copolymer, having a molecular weight of approximately 60 kD (Birmingham Polymers, Inc., Lot #051 -68-1 ), (0.5g) was dissolved in 4 ml of CH₂CI₂.

(3) Elcatonin (585 μg) was suspended in the PLG/CH₂CI₂ solution of step (2) by using an ultrasonic probe with a micro-tip. Ultrasonic irradiation was applied intermittently to give a uniform suspension in the PLG/CH₂CI₂ solution. Elcatonin (carbacalcitonin), Lot #ZG-287, obtained from Bachem Cat #PCAL38, had an activity level of 4402 lU/mg in the presence of acetate ion and water or 5143 lU/mg in the absence of acetate ion or water and had a net activity of 4202/5143 = 0.856.

(4) The solution from step (1 ) was mixed with a homogenizer at high speed and the polymer suspension from step (3) was added dropwise and slowly through an 18 gauge needle.

(5) After the polymer suspension had been added entirely, 35 ml of petroleum ether were added slowly. Homogenization was continued at high speed for 5 minutes and then at low speed for 5 minutes.

(6) The homogenizer was turned off and the solution was stirred rapidly with a magnetic stirrer. After fifteen minutes, 35 ml of petroleum ether were added slowly. After sixty minutes, an additional 35 ml of petroleum ether were added and stirring was continued for fifteen minutes.

(7) Petroleum ether (40-50 ml) was added and the microsphere dispersion was filtered through a 0.45 μm filter (Millipore Durapore). The microspheres were washed with excess petroleum ether to remove silicone fluid and methylene chloride.

(8) Microspheres were collected on filter paper and dried overnight under a vacuum.

(b) Oil-in-water (o/w) emulsion technique.

Microspheres were prepared with the oil-in-water (o/w) technique as follows:

(1 ) Polyvinyl alcohol (PVA) of molecular weight 8,000-10,000 (0.4 g) was dissolved in 100 ml of distilled water. (2) Poly (DL-lactide/glycolide) or PLG, a 50:50 copolymer having a molecular weight of approximately 60 kD or 9,400 daltons (Birmingham Polymers, Inc., Lot #051 -68-1 ) was dissolved in 2 ml of methylene chloride (CH₂CI₂).

(3) Elcatonin (485 μg) was suspended in the solution from step 2 with 30 seconds of sonication with a probe micro-tip in an ice bath. A few drops of CH₂CI₂ were used to wash all of the elcatonin into the copolymer solution.

(4) The elcatonin/copolymer suspension from step 3 was taken up into a 2 ml syringe with an 18 gauge needle. This suspension was added dropwise and slowly into 50 ml of the aqueous PVA solution in a 100 ml round bottom flask stirred with a magnetic stirrer at room temperature. Stirring was continued for 25 minutes.

(5) The flask was attached to a rotary evaporator (Buchi Model R Rotavapor). The evaporator was operated under house vacuum at a slow rotation speed and at a temperature of 35°C.

(6) The microsphere dispersion was filtered through a 0.45 μm filter (Millipore). Microspheres were washed with about 10 ml of water.

(7) The microspheres were dried at room temperature in a vacuum desiccator.

Example 2. Characterization of Polymer and Microspheres.

(a) Molecular weight and particle size.

Polymer molecular weights were determined by gel permeation chromatography (GPC) using a 7.8 mm ID x 30 cm column packed with Ultrastyragel^® ( < 10 μm, mixed bed resin) with methylene chloride as eluent. Polystyrene standards were used for calibration. Particle sizes of microspheres were determined by scanning electron microscopy (SEM, Hitachi S-570, Tokyo, Japan). Details of these measurement techniques are further described in Wang et al. (1990) Biomaterials 1 1 :679- 685.

(b) Elcatonin content in microspheres.

Elcatonin microspheres (50 mg) were dissolved in 0.5 ml CH₂CI₂ and filtered through a 0.45 μ filter which retains elcatonin. For microspheres prepared with the oil-in-oil emulsion technique, the filter was washed with

0.5 ml of 0.1 N HCI and the HCI solution was subjected to HPLC analysis for elcatonin content. The content for a particular batch of elcatonin microspheres gave 49.8 μg/50 mg which is about 85% of the theoretical content (theoretical content is calculated to be 1 .175 μg/mg). Typical contents are 75-90% of theory.

For microspheres prepared with the oil-in-water emulsion technique, the filter was washed with 3 ml of CH₂CI₂; the elcatonin retained on the filter was dissolved in 1 ml of 0.001 N HCI; and the HCI solution was subjected to HPLC analysis for elcatonin content. The content for a particular batch of elcatonin microspheres gave 19.3 μg/50 mg which is about 40% of the theoretical content (theoretical content is calculated to be 0.97 μg/mg). Typical contents are 35-50% of theory.

Similar results were obtained for microspheres prepared using PLG polymer having a molecular weight of 9,400 daltons.

(c) In vitro elcatonin release studies.

To assess the release characteristics of the elcatonin microspheres, a weighed quantity of elcatonin microspheres was placed into a volume of phosphate buffered saline (PBS) at 37°C and aliquots of the solution were removed at specific sampling times for assay, e.g., each day for the first four days and every other day for the remainder of the study. In vitro protein release studies were performed in duplicate. A typical procedure was as follows:

For microspheres prepared with the oil-in-oil emulsion technique, 62.3 mg of elcatonin microspheres were added to 1 ml of PBS at 37°C. After incubation for 12 hours, the aliquots of the incubation medium were removed and assayed by HPLC for elcatonin content. Content analysis indicated that 52% of the elcatonin content had been released.

The rapid initial release is known as the "burst effect" in which there is rapid release of peptide from the surface of microspheres. This is common for microspheres prepared by the oil-in- oil technique. The remaining peptide content (48% in this case) would be released slowly over approximately three to four weeks as the PLG copolymer biodegrades.

Elcatonin controlled release microspheres prepared by the oil-in-oil technique were also subjected to in vitro release studies under different conditions. 51 .0 mg of microparticles were placed in a microcentrifuge tube. The release medium was 0.5 ml of 0.001 N HCI containing 1 % Prionex^®. The release study was carried out at room temperature. At each sampling point the release medium was removed and replaced with 0.5 ml of fresh medium. 73% of the elcatonin content was released at day 1 and an additional 16.1 % was released at day 7. For microspheres prepared from the 60,000 dalton polymer with the oil-in-water emulsion technique, 50 mg of elcatonin microspheres were added to 1 ml of PBS at 37 °C. After incubation for 2, 6 and 24 hours, the entire incubation medium was removed and replaced with 1 .0 ml of PBS. After 24, hours, identical sampling was performed at regular time intervals over 29 days. The incubation medium samples were assayed for protein with the bicinchoninic acid (BCA) method using a BCA kit from Pierce, Rockford IL, to determine elcatonin in released samples. 35% of the elcatonin was released at day 29. The release profile is presented in Figure 1 .

Further in vitro release studies of elcatonin from microspheres of 60,000 dalton PLG polymer were performed by assaying residual elcatonin content at various time intervals. Approximately 10% was released in 17 days, 25% in 23 days, and 48% in 28 days. These results suggest that elcatonin is released continuously from these oil-in-water particles for approximately three months.

Elcatonin controlled release microspheres were also prepared from low molecular weight 50: 50 PLG polymer using the oil-in-water technique. The polymer molecular weight was determined by gel permeation chromatography to be approximately 9,400 daltons. The in vitro release profile was determined by placing 69.9 mg of the microspheres having an elcatonin content of 0.03% w/w in a test tube to which 0.01 N HCI containing 1 % Prionex^® was added. The centrifuge tube was placed in an incubator shaker at 37 °C. At the sampling time the entire release medium was replaced with fresh medium.

The amount of elcatonin released at 21 days was estimated from the residual content of elcatonin in the microspheres. Approximately 33.2% of the elcatonin was released in 21 days.

Elcatonin is released continuously from these oil-in-water microspheres prepared from low molecular weight (9,400 dalton) polymer for approximately two months. Oil-in-water microspheres prepared from higher molecular weight (60,000 dalton) polymer exhibited a longer duration of elcatonin release of approximately three months.

Example 3. Effectiveness of Elcatonin in Controlled Release Microsphere Formulations for the

Treatment of Osteoporosis Induced bv Ovariectomv in the Rat.

(a) Experimental osteoporosis in rats. An art recognized model for osteoporosis has been developed in ovariectomized rats. See, for example, Okumura et al. (1987) Bone 8:351 -355; and Hayashi et al. (1989) Bone 10:25-28.

Ovariectomized Spraque Dawleγ rats were obtained from Harlan Spraque Dawley, Inc., P.O. Box 29176, Indianapolis, IN 46229-0176. The animals were approximately twelve weeks of age at the start of the study. (b) Preparation of mixtures of the controlled release microsphere formulations.

The controlled release microspheres evaluated for treatment of osteoporosis were prepared by two different techniques: the oil-in-oil technique (o/o) [Example 1 (a)] and the oil-in-water technique (o/w) [Example Kb)]. A mixture of both types of microspheres, (o/o) and (o/w) was administered to rats. Both the (o/o) and (o/w) microsphere formulations were prepared with elcatonin (elcatonin controlled release microsphere formulation) and without elcatonin (placebo controlled release microsphere formulation).

(c) Treatment of model osteoporosis induced in rats, (i) Overall design of the evaluation study. Controlled release microsphere formulations comprising elcatonin were evaluated as a treatment for osteoporosis in rats. The study encompassed four groups of bilateral ovariectomized rats: a placebo control group, two groups receiving elcatonin controlled release microsphere formulations at different dose levels, and a group treated with bulk elcatonin administered as bolus injections.

Table 1 presents schematically the assignment of animals in each of the four groups.

TABLE 1 ANIMAL STUDY NUMBERS

Group Test Articles in Vehicle*/ Cage Color No. of Animal

Treatment Regimen Code Animals/Sex Study No.

Placebo Controlled Release F1

Microsphere Formulation F2 F3

1 Single Subcutaneous Dose White 7 Females F4 on Day 1 F5 F6 F7

Elcatonin Controlled Release F8

Microsphere Formulation F9 F10

2 Dose level 1 Yellow 7 Females F1 1 F12

Single Subcutaneous Dose F13 on Day 1 F14

Elcatonin Controlled Release F1 5

Microsphere Formulation F16 F17

3 Dose level 2 Green 7 Females F18 F19

Single Subcutaneous Dose F20 on Day 1 F21

Bulk Elcatonin Drug F22

Substance F23

F24

4 Bolus Subcutaneous Dose: Red 7 Females F25 3 times per week for 4 weeks F26 F27 F28

Vehicle: Saline + carboxγmethγl cellulose

Table 2 indicates the dosage level of the elcatonin administered to rats in each experimental group and also describes the mixture of microspheres administered to rats in groups 1 -3. TABLE 2

STUDY DESIGN

Test Articles Weight of Total Elcatonin Dose

Group in Vehicle/Treatment Microspheres Dose Volume

Regimen (mg/kg) (μg/kg) (ml/kg)

Placebo Controlled Release Microsphere Formulation 7.0 (o/o)

1 + 0 N.A.

Single Subcutaneous Dose 147.0 (o/w) on Day 1

Elcatonin Controlled Release Microsphere Formulation 3.5 (o/o)

+ 28 N.A.

2 Dose level 1 73.5 (o/w)

Single Subcutaneous Dose on Day 1

Elcatonin Controlled Release Microsphere Formulation 7.0 (o/o)

+ 56 N.A.

3 Dose level 2 147 (o/w)

Single Subcutaneous Dose on Day 1

Bulk Elcatonin Drug Substance*

N.A. 14 1 .0

4 Bolus Subcutaneous Dose: 3 times per week for 4 weeks

* Vehicle: Saline

(o/o) = microspheres prepared by the oil-in-oil technique.

(o/w) = microspheres prepared by the oil-in-water technique.

(ii) Use of elcatonin controlled release microspheres in the treatment of osteoporosis in rats.

(a) Study design of elcatonin treatment.

Twenty-eight rats were divided into four groups as shown in Table 2. The control group (Group 1 ) was given placebo controlled release microspheres; Groups 2 and 3 were given elcatonin controlled release microspheres and Group 4 was given elcatonin directly (not in controlled release microspheres). Rats were lightly anesthetized with metaphane in a bell jar within a fume hood and then administered an intramuscular injection of pentobarbital. In rats receiving elcatonin directly and not through microspheres, the initial dose of elcatonin was administered while the rats were under pentobarbital anesthesia. These rats in Group 4 were administered 1 .0 ml/kg of a physiological saline solution of 7 IU elcatonin/ml subcutaneously in the abdominal area on Mondays, Wednesdays and Fridays of each week during the study. Rats in Groups 1 , 2, and 3 were administered the elcatonin controlled release or placebo controlled release microsphere formulations by single subcutaneous instillation on the first day of the study. The rats were anesthetized with metaphane and pentobarbital, the dorsal hair between the scapulae was clipped and the skin cleaned with 70% ethanol. A small incision was made in the dorsal skin perpendicular to and between the scapulae with surgical scissors and the dorsal skin lifted with scissors and a blunt probe. The controlled release formulations were poured from the weighing paper quantitatively into the subdural pocket and the wound closed immediately with sterile wound clips. The surgical wounds healed normally without evidence of inflammation or infection.

Plastic tubes were prepared to contain 70 micrograms of accurately weighed bulk elcatonin per tube. The tubes were stored at -20°C until used. The entire content of one tube was quantitatively diluted with sterile physiological saline to a final volume of 60 ml on each day of dose administration using plastic pipettes and containers. Seventy micrograms of elcatonin at an activity of 6 lU/μg is 420 IU elcatonin which, when diluted to 60 ml, yields a solution of 7 IU elcatonin/ml. Rats in Group 4 received 1 .0 ml/kg (7.0 lU/kg) of these solutions at each dose administration. Rats in Group 1 received 7.0 mg/kg of an oil/oil and 147 mg/kg of an oil/water placebo controlled release microsphere formulation. Rats in Group 2 received 3.5 mg/kg of a 0.1 % elcatonin oil/oil and 73.5 mg/kg of a 0.033% elcatonin oil/water controlled release microsphere formulation. Rats in Group 3 received 7.0 mg/kg of the 0.1 % elcatonin oil/oil and 147 mg/kg of the 0.033% elcatonin oil/water controlled release microsphere formulations. Therefore, the rats in Group 2 received 3.5 μg (21 IU) elcatonin/kg from the oil/oil formulation and 24.5 μg (147 IU) elcatonin/kg from the oil/water formulation. Rats in Group 3 received exactly twice the Group 2 dose, i.e., 7.0 μg (42 IU) elcatonin/kg from the oil/oil and 49 μg (294 IU) elcatonin/kg from the oil/water formulation. Rats in Group 4 received 1 .17 μg (7 IU) elcatonin (not in microsphere formulation)/kg three times per week for the duration of the study. Total doses of elcatonin were 0, 28, 56, and 28 μg/kg (0, 168,

336, and 168 lU/kg) in Groups 1 , 2, 3, and 4, respectively.

During the study, the animals were examined daily. Any clinical signs of toxicity, including physical or behavioral abnormalities, were recorded. Detailed clinical observations were performed weekly and on the day of scheduled euthanasia, and included, but were not limited to, evaluations of the skin and fur, eyes and mucous membranes, respiratory, somatomotor activity and general behavior. In addition to scheduled clinical observations, the animals were observed for overt toxic effects between one-half hour and two hours following dosing. Body weights were recorded once per week and on the day of scheduled euthanasia.

(b) Effectiveness of elcatonin treatment.

The elcatonin-comprising controlled release microspheres of the invention were found to be effective in the treatment of induced osteoporosis in rats as indicated in Table 3.

TABLE 3

Total Theoretical Serum Osteocalcin

Test Elcatonin Total ng/ml

Group Articles Administered Elcatonin

(μg/kg) Released* Day -2 Day 7 Day 14 Day 21 Day 28 by Day 28

(μg/kg)

1 Placebo microspheres 0 0 140±7 130±6 122 ±13 103±17 107±18

2 Elcatonin microspheres 28 15.3 135±12 127±12 107±17 100 ±9 94±17

3 Elcatonin microspheres 56 30.6 132±9 107±14 98±15 81 ±14 77±13

4 Elcatonin injections for 14 14 137±8 132±15 139 ±7 102±14 84 ±6 4 weeks

^* Theoretical Total Elcatonin Released = estimation calculated from in vitro rate of release from the oil-in-oil and oil-in-water controlled release microspheres.

Control rats which received controlled release microspheres without elcatonin showed a decrease of approximately 23% in the serum osteocalcin level at Day 28 (the Day 28 level subtracted from the Day -2 level). Rats receiving elcatonin-containing controlled release microspheres (Groups 2 and 3) obtained a total amount of elcatonin in the microspheres of 28 μg/kg (Group 2) and 56 μg/kg (Group 3), respectively. Rats with the lower dosage of elcatonin microspheres showed a decrease in osteocalcin level at Day 28 of approximately 30%, while a decrease of approximately 42% was observed for the higher dosage of elcatonin microspheres. Rats treated directly with elcatonin not contained in microspheres (Group 4) showed a 39% decrease in serum osteocalcin levels at Day 28.

The results presented in Table 3 were analyzed using the statistical criteria, parametric and non-parametric. The statistical difference for each group versus the control (placebo) group at Day 28 was evaluated. A significant statistical difference was obtained between the high elcatonin microspheres dose (Group 3) and the control (Group 1 ) with P = 0.0037 and between the elcatonin injections (Group 4) and the placebo (Group 1 ) with P = 0.0191 . No significance was attached to values of P > 0.05.

Treatment with elcatonin-containing microspheres appeared to be at least as efficacious as the direct treatment with elcatonin (not contained in microspheres). For example, the amount of change in serum osteocalcin level between Day -2 and Day 28 can be used as an estimate of effectiveness of the elcatonin treatment. Administration of elcatonin directly (Group 4) showed a 39% decrease in osteocalcin level at Day 28 after direct treatment with elcatonin injections for a total dosage of 14 μg/kg (1 .17 μg/kg per injection and 3 injections per week for four weeks). In treatments with elcatonin-containing controlled release microspheres, similar decreases (e.g., 30% for Group 2 and 42% for Group 3) in serum osteocalcin levels at Day 28 were obtained. However, although rats in Group 2 received controlled release microspheres containing 28 μg/kg elcatonin, theoretically only approximately 15.3 μg/kg was released from the microspheres by Day 28. (It is estimated from in vitro release kinetics that approximately 100% of the elcatonin in oil-in-oil microspheres and approximately 48% of the oil-in-water microspheres are released by Day 28.) Thus, a dosage of approximately 14-15 μg/kg of elcatonin, whether administered directly or in microspheres (i.e., Group 4 or Group 2, respectively), produced a similar hypocalcemic effect in rat osteoporosis models.

To further compare the potency of unencapsulated elcatonin with elcatonin-containing controlled release microspheres, the effectiveness in lowering serum osteocalcin levels can be calculated as a function of effective elcatonin dosage. Table 4 presents a comparison of the estimated theoretical potency of unencapsulated elcatonin and elcatonin released from microspheres.

TABLE 4

Effective Elcatonin Dose μg/kg Serum

Test Osteocalcin Group Treatment Total Theoretical' Average Level Administered Amount Amount % Decrease Released Present at Day 28 by Day 28 Per Day*

Group 2 elcatonin 28 15.3 0.55 30 microspheres

Group 3 elcatonin 56 30.6 1 .09 42 microspheres

Group 4 elcatonin 14 14.0 1 .17 39 t Theoretical Amount Released = estimation calculated from in vitro rate of release from the oil- in-oil and oil-in-water controlled release microspheres.

* For Groups 2 and 3, the amount released by Day 28 was divided by 28 to obtain a daily average. For Group 4, 1.17 μg was injected three times a week for 4 weeks; the amount per injection was used as daily average.

Based on the theoretical release of elcatonin from the microspheres in Groups 2 and 3, calculated using the in vitro release kinetics of the two types of microspheres, it is estimated that an average daily dosage of 0.55 μg/kg of released elcatonin (Group 2) correlated with a 30% decrease in osteocalcin level by Day 28 and that an average daily dosage of 1 .09 μg/kg of released elcatonin (Group 3) correlated with a 42% decrease in osteocalcin level by Day 28. Similarly, for control Group 4 it can be estimated that after twelve doses of unencapsulated elcatonin (1 .17 μg/kg per dose, 3 doses per week for four weeks) a total of approximately 14 μg of elcatonin was administered and correlated with a 39% decrease in osteocalcin level at Day 28. These results suggest that elcatonin released from the controlled release microspheres of the invention exhibits an undiminished biological potency. Further inspection of the results in Table 4 additionally suggests that treatment with elcatonin-containing microspheres may perhaps be a superior or more potent and more effective treatment than treatment with multiple elcatonin injections, as a relatively similar effect on reduction of serum osteocalcin level was observed with a smaller but constant dose of elcatonin released from microspheres (approximately 0.55 μg/kg release daily in Group 2) than with multiple injections of elcatonin (1 .17 μg/kg dose per injection in Group 4). Example 4. LABORATORY PROCEDURES AND ASSAYS

(a) Necropsy.

All rats were euthanized by C0₂ inhalation and necropsied. At necropsy, the following parameters were evaluated and recorded: body weight; uterus weight; and confirmation that ovaries were missing. At sacrifice, the following were collected from each rat: 1 tibia, 1 femur; and 1 tail section. Bones were treated as follows: bones were stripped of soft tissue, placed into 40% ethanol/water, and stored at + 5°C for 24 hours. Bone were then transferred into 70% ethanol/water for 24 hours and then transferred again into 90% ethanol/water for storage.

(b) Serum samples. Blood samples (approximately 0.5 ml) were collected by inserting a microhematocrit capillary tube (Red Coded Tip, Fisher Scientific, Catalog Number 0266866) into the retro-orbital plexus of anesthetized animals. The drawn blood was allowed to coagulate for approximately 30 minutes; the clot was retracted; the tubes were stored at + 4°C for 15 minutes; and the serum was expelled into a microcentrifuge tube (USA Scientific, Catalog Number 14800500) and stored at -20°C .

(c) Urine samples.

Animals were provided with no food and 20 ml of water during urine collection. Animals were transferred to metabolism cages on Tuesdays at approximately noon. The urine collection vessel contained sufficient mineral oil to ensure that urine was not lost to evaporation. After the collection period, urine volume was accurately measured and recorded. The urine was centrifuged and the supernatant was poured into test tubes which were frozen and stored at -20°C until analyzed.

(d) Osteocalcin assay.

The serum osteocalcin level was measured by the procedure described in Price and Nishimoto (1980) Proc. Natl. Acad. Sci. USA 77:2234-2238.

(e) Measurement of urinary collagen crosslinks. The level of urinary collagen crosslinks was measured as described in Tordjman et al. (1994)

Bone and Mineral 26: 155-167.

(f) Bone densitometrv.

Rats were euthanized by exsanguination under isofluran anesthesia. For each left femur, bone mineral content (g) and bone area (cm²) were measured by dual energy X-ray absorptiometry using an ultra-high resolution mode (Hologic, QDR-1000), and bone mineral density (BMD) (g/cm²) was calculated. (g) Histomorpholoov

Bone histology and histomorphometry were carried out as described in Yamamura et al. (1994) Bone and Mineral 24:33-42.

(h) Syntheses of elcatonin and elcatonin analogs. Elcatonin is a synthetic, 31 -amino acid analog of the naturally occurring eel calcitonin hormone.

It has neither first nor seventh amino acid L-cysteine and the L-cysteine is replaced by alpha- aminosuberic acid. Elcatonin was synthesized by Morikawa et al. in 1976 [Experientia 32: 1 104- 1 1061.

Elcatonin analogs are synthetic hypocalcemic peptides which are similar in chemical structure and in biological properties to elcatonin as clinically useful agents. Elcatonin analogs have the chemical structure

( CH₂) ₅

—CO — Ser-Asn-Leu-Ser-Thr-NHCHCO - Val-Leu- Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-A₂₅-A₂₆-Gly-A₂₈- Gly-Thr-Pro-NH₂ wherein A₂₆ is Asp or Asn, A_2β is Val or Thr, and A₂₈ is Ala or Ser.

Methods for the synthesis of these synthetic elcatonin analogs are provided by Basava et al. (1992)

U.S. Patent No. 5, 175, 146.

Claims

What is claimed is:

1 . A controlled release microsphere composition capable of delivering continuously an effective amount of elcatonin or an elcatonin analog to an animal over an extended period of time, comprising a biodegradable and biocompatible poly(D,L-lactide-co-glycolide) polymer excipient encapsulating said elcatonin or elcatonin analog to form controlled release microspheres, said controlled release microsphere composition having a delivery profile such that the release of said elcatonin or elcatonin analog from said controlled release microspheres continues for said extended period of time and such that said released elcatonin or elcatonin analog retains at least about 75% of the biological activity which it possessed prior to encapsulation in said controlled release microspheres.

2. The controlled release microsphere composition of claim 1 wherein said controlled release microsphere composition comprises a mixture of at least two biodegradable and biocompatible poly(D,L-lactide-co-glycolide) polymer excipients, each excipient encapsulating said elcatonin or elcatonin analog to form at least first and second controlled release microspheres and each excipient having a different rate of release of said elcatonin or elcatonin analog therefrom, said controlled release microsphere composition having a delivery profile such that the release of said elcatonin or elcatonin analog from said second controlled release microspheres begins as the release of said elcatonin or elcatonin analog from said first controlled release microsphere declines.

3. The controlled release microsphere composition of claim 1 wherein said poly(D,L-lactide-co- glycolide) polymer excipient has a mole ratio of lactide to glycolide of approximately 50:50 and a molecular weight between about 50,000 and about 75,000 daltons or between about 5,000 and about 20,000 daltons.

4. The controlled release microsphere composition of claim 2 wherein said first and second poly(D,L-lactide-co-glycolide) polymer excipient have a mole ratio of lactide to glycolide of approximately 50:50 and wherein said first poly(D,L-lactide-co-glycolide) polymer excipient has a molecular weight between about 50,000 and about 75,000 daltons and said second poly(D,L- lactide-co-glycolide) polymer excipient has a molecular weight between about 5,000 and about 20,000 daltons.

5. The controlled release microsphere composition of claim 1 wherein said poly(D,L-lactide-co- giycolide) polymer excipient is prepared by an organic phase separation method (oil-in-oil technique) or by an aqueous phase separation method (oil-in-water technique).

6. The controlled release microsphere composition of claim 2 wherein said first poly(D,L-lactide-co- glycolide) polymer excipient is prepared by an organic phase separation method (oil-in-oil technique) and wherein said second poly(D,L-lactide-co-glycolide) polymer excipient is prepared by an aqueous phase separation method (oil-in-water technique).

7. The controlled release microsphere composition of claim 1 wherein said poly(D,L-lactide-co- glγcolide) polymer excipient further comprises an additional species of biologically active compound.

8. The controlled release microsphere of claim 1 wherein said animal is a human.

9. A use for claim 1 in the treatment of osteoporosis.

10. A use for claim 1 in reducing elevated serum osteocalcin levels.

1 1 . A method of preparing a composition for delivering continuously an effective amount of elcatonin or elcatonin analog to an animal over an extended period of time, comprising the step of encapsulating said elcatonin or elcatonin analog in a biodegradable and biocompatible poly(D,L-lactide-co-glycolide) polymer excipient to form controlled release microspheres having a delivery profile such that the release of said elcatonin or elcatonin analog from said controlled release microspheres continues for said extended period of time, and such that said released elcatonin or elcatonin analog retains at least about 75% of the biological activity which it possessed prior to encapsulation in said controlled release microspheres.

12. The method of claim 1 1 wherein said step of encapsulating said elcatonin or elcatonin analog proceeds in at least two separate biodegradable and biocompatible polγ(D,L-lactide- co-glycolide) polymer excipients to form at least first and second controlled release microspheres, each of said microspheres having a different rate of release of said elcatonin or elcatonin analog therefrom, such that a mixture of at least said first and second controlled release microspheres forms said composition capable of releasing an effective amount of said elcatonin or elcatonin analog and having a delivery profile such that the release of said elcatonin or elcatonin analog from said second controlled release microspheres begins as the release of said elcatonin or elcatonin analog from said first controlled release microsphere declines.

13. The method of claim 1 1 further comprising the step of administering an effective amount of said composition to the animal.

14. The method of claim 1 1 wherein said poly(D,L-lactide-co-glycolide) polymer excipient has a mole ratio of lactide to glycolide of approximately 50:50 and a molecular weight between about 50,000 and about 75,000 daltons or between about 5,000 and about 20,000 daltons.

15. The method of claim 12 wherein both said first and second polγ(D,L-lactide-co-glycolide) polymer excipients have a mole ratio of lactide to glycolide of approximately 50:50 and wherein said first poly(D,L-lactide-co-glycolide) polymer excipient has a molecular weight between about 50,000 and about 75,000 daltons and said second poly(D,L-lactide-co- glycolide) polymer excipient has a molecular weight between about 5,000 and about 20,000 daltons.

16. The method of claim 1 1 wherein said poly(D,L-lactide-co-glycolide) polymer excipient is prepared by an organic phase separation method (oil-in-oil technique) or by an aqueous phase separation method (oil-in-water technique).

17. The method of claim 12 wherein said first poly(D,L-lactide-co-glycolide) polymer excipient is prepared by an organic phase separation method (oil-in-oil technique) and wherein said second poly(D,L-lactide-co-glycolide) polymer excipient is prepared by an aqueous phase separation method (oil-in-water technique).

18. The method of claim 1 1 wherein said poly(D,L-lactide-co-glycolide) polymer excipient further comprises an additional species of biologically active compound.

19. A method of treatment for osteoporosis comprising the step of parenterally administering into an animal having osteoporosis as manifested by an elevated serum osteocalcin level an effective amount of a controlled release microsphere composition capable of delivering continuously an effective amount of elcatonin or elcatonin analog to said animal over an extended period of time, such that said elevated serum osteocalcin level is reduced.

20. A method of treatment for an osteopathic disorder comprising the step of parenterally administering into an animal having an osteopathic disorder manifested by an elevated serum osteocalcin level an effective amount of a controlled release microsphere composition capable of delivering continuously an effective amount of elcatonin or elcatonin analog to said animal over an extended period of time, such that said elevated serum osteocalcin level is reduced.

21 . The method of claim 19 wherein said animal is a human.

22. The method of claim 20 wherein said animal is a human.