Therapeutics and methods for treating osteoarthritis

Abstract

Pharmaceutical compositions and methods of treating osteoarthritis comprising active pharmaceutical ingredient (API) encapsulated poly(lactic-co-glycolic acid) (PLGA) microspheres, wherein the composition comprises at least about 10% active active pharmaceutical ingredient (API) (wt%), a lactide:glycolide (L:G) ratio of 75:25, wherein the weight of the microspheres is between about 4 to about 15 kDa, and wherein the pharmaceutical composition is a spray dried dispersion (SDD).

Description

[0001] THERAPEUTICS AND METHODS FOR TREATING OSTEOARTHRITIS RELATED APPLICATIONS [1] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 550,582, filed on February 6, 2024, the entire teachings of which are incorporated herein by reference. BACKGROUND [2] Arthritis is an inflammatory disease characterized by inflammation of a joint, which term includes synovial tissue and membranes. There are many forms of arthritis, including without limitation, osteoarthritis (hypertrophic or degenerative arthritis), rheumatoid arthritis, arthritis due to infection (tuberculosis, Lyme disease, rheumatic fever, etc.), suppurative arthritis, juvenile arthritis, and gouty arthritis. Osteoarthritis is a degenerative joint disease in which cartilage and bone are primarily affected. Osteoarthritis is especially common among older people, and usually affects a joint on one side of the body. In osteoarthritis, the cartilage breaks down and wears away, causing pain, swelling, and loss of motion of the joint. [3] Approximately 80% of older adults, ages 55 years and older, have evidence of osteoarthritis on X-ray. Of these, an estimated 60% experience symptoms. It is estimated that 240 million adults worldwide have symptomatic osteoarthritis, including more than 30 million U.S. adults. There is no known cure for osteoarthritis, and consequently clinical efforts aimed at treating it are presently directed toward symptomatic relief of pain. Traditional remedies such as the application of heat for temporary, local pain relief are helpful for some patients, and suitable exercise and physical therapy programs can help in maintaining joint mobility. Joint replacement surgery may be advised in severe cases. [4] Despite the availability of a wide range of medications and treatment modalities for arthritis and inflammatory diseases in general, as described above, none has proved to be entirely satisfactory for osteoarthritis. Treatments and medications that focus on just alleviating pain can carry their own potentially harmful side effects. In particular, there remains a need for innovative treatments that target the underlying cause of osteoarthritis, and thereby help reduce, eliminate, or slow its progression (expressed symptomatically

[0002] 1  \\4154‐0697‐7112  v4      by bone erosion, cartilage erosion, inflammation, swelling, abnormal neovascularization, etc.). SUMMARY [5] The present disclosure relates to pharmaceutical compositions comprising active pharmaceutical ingredient (API) encapsulated poly(lactic-co-glycolic acid) (PLGA) microspheres, wherein the composition comprises at least about 10% active active pharmaceutical ingredient (API) (wt%), a lactide:glycolide (L:G) ratio of 75:25, wherein the weight of the microspheres is between about 4 to about 15 kDa, and wherein the pharmaceutical composition is a spray dried dispersion (SDD). The composition can comprise at least about 20% active API (wt%). The active API can be a calcium channel regulator. The calcium channel regulator can be amlodipine, bepridil, diltiazem, hypochloride, felodipine, gallopamil, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, devapamil, emopamil, or a combination thereof. The pharmaceutical composition can include a hydrochloride salt of the calcium channel regulator. The calcium channel regulator can be verapamil. The composition can include about 20% active API (wt%), and the active API can be verapamil. [6] The present disclosure also relates to methods for treating arthritis in a patient in need thereof, comprising administering to the patient an effective dose of a pharmaceutical composition described herein. The arthritis can be osteoarthritis. The administering can be achieved by injection into an intraarticular space of a joint of the patient. The joint can be a hip joint, a joint of a foot, a joint of an ankle, a knee joint, a joint of a hand, a joint of a spine, a shoulder joint, or a combination thereof. The patient can be administered a single injection. The single injection can include the API in solution and the API in microspheres. The injection can include between about 0.5 mg to about 2.0 mg of API in solution. The injection can include at least about 32 mg of API in microspheres. The composition used in the methods disclosed herein can further include a vehicle appropriate for injection into the human body. The vehicle can be physiological saline. The API can be released from the microspheres for a duration of at least three months. The duration of release can be between about three

[0003] 2  \\4154‐0697‐7112  v4      to about six months. The duration of release can be about six months. The method can include delivery of at least about 32 mg of API over about six months. The method can include a single injection of about 0.5-2.0 mg of verapamil in solution and the pharmaceutical composition comprising about 32 mg of verapamil encapsulated PLGA microspheres, into an intraarticular space of a joint of the patient, wherein the pharmaceutical composition is delivered over about six months. [7] The present disclosure also relates to pharmaceutical compositions and methods for treating osteoarthritis in a patient in need thereof comprising administering to the patient an effective dose of a pharmaceutically acceptable composition including a first therapeutic, wherein the first therapeutic includes one or more calcium channel regulators or pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof, and at least a first portion of the first therapeutic is encapsulated in microspheres. According to a further embodiment, the one or more calcium channel regulator includes one or more of amlodipine, bepridil, diltiazem hypochloride, felodipine, gallopamil, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, devapamil, and emopamil, and the number of distinct calcium channel regulators in the first therapeutic is one of exactly one, two, three, or four. According to a further embodiment, the first therapeutic includes a hydrochloride salt of the one or more calcium channel regulators. According to a further embodiment, the method further comprises injecting the composition into an intraarticular space of a joint of the patient. According to a further embodiment, the joint is one of a hip joint, a joint of a foot and / or an ankle, a knee joint, a joint of a hand, a joint of the spine, and a shoulder joint. According to a further embodiment, the method further comprises injecting the composition in the patient in a single dose of between 1.00 to 10.00 mL. According to a further embodiment, the composition includes 0.00001-3.0 mg / mL of the first therapeutic. According to a further embodiment, the composition further comprises a vehicle appropriate for injection into the body. According to a further embodiment, the vehicle includes physiological saline. According to a further embodiment, the vehicle further includes a second portion of the first therapeutic not encapsulated in the microspheres. According to a further embodiment, the method further comprises a ratio of the first portion to the second portion is between 0.250 and 4.000. According to a further embodiment, the method further comprises

[0004] 3  \\4154‐0697‐7112  v4      administering a second therapeutic, wherein the second therapeutic is not a calcium channel regulator. According to a further embodiment, the second therapeutic is hyaluronic acid, the second therapeutic is included in the composition, and further comprising injecting the composition into an intraarticular space of a joint of the patient. According to a further embodiment, between 85.0% and 95% of the microspheres are between 6.00 µm and 100.00 µm in diameter. According to a further embodiment, 90.0% of the microspheres are between 20.00 µm and 55.00 µm in diameter. According to a further embodiment, the microspheres are comprised of exactly one, two, or three of poly lactic / glycolic acid (PLGA), polylactic acid (PLA), and polyethylene glycol (PEG). According to a further embodiment, the method further comprises adding saline solution to a plurality of dried microspheres containing either first therapeutic that has been one of freeze dried and vacuum dried or a mixture of each. According to a further embodiment, the microspheres are comprised of poly lactic / glycolic acid (PLGA), the first portion is 32mg of first therapeutic, and the first therapeutic is released from the microspheres at rate of 0.050 and 0.080 mg / hr for a duration of between 3.0 – 6.0 months. [8] The present disclosure further relates to methods of treating arthritis (e.g., osteoarthritis) and pharmaceutical compositions comprising a plurality of microspheres, between 0.001 mg and 35 mg of a first therapeutic including one or more calcium channel regulators or pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof, at least a first portion of the first therapeutic is one of freeze dried and vacuum dried and the first portion is encapsulated in the microspheres, the calcium channel regulator includes one or more of amlodipine, bepridil, diltiazem hypochloride, felodipine, gallopamil, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, devapamil, and emopamil as a hydrochloride salt, between 85.0% and 95% of the microspheres are between 6.00 µm and 100.00 µm in diameter; and the microspheres are comprised of exactly one, two, or three of poly lactic / glycolic acid (PLGA), poly lactic acid (PLA), and poly ethylene glycol (PEG). [9] The present disclosure further relates to a kit comprising a first volume containing a plurality of microspheres, between 0.001 mg and 35 mg of a first therapeutic including one or more calcium channel regulators or pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof, at least a first portion of the first therapeutic is one of freeze dried and vacuum dried and the first

[0005] 4  \\4154‐0697‐7112  v4      portion is encapsulated in the microspheres, a second volume containing a second portion of the first therapeutic in a liquid state, and instructions for combining the first volume and the second volume.

[0010] The present disclosure further relates to a pharmaceutical composition comprising a plurality of spherical carriers formed of poly lactic / glycolic acid (PLGA), between 19.0% and 21.0% active loading of one or more of verapamil and pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof, where the PLGA has a L to G ratio of between 70:20 and 80:20, where the PLGA has a molecular weight of between 4.0 and 15.0 kDa. In further embodiments, the spherical carriers are microspheres. In further embodiments, the spherical carriers are spray dried dispersions (SDDs).

[0011] The present disclosure further relates to a pharmaceutical composition comprising a plurality of spherical carriers formed of poly lactic / glycolic acid (PLGA), between 10% and 30% (e.g., 20%) active loading of one or more active ingredients, where the active ingredients are one or more of verapamil and pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof, where the PLGA has a L to G ratio of between 50:50 and 75:25, and where the PLGA has a molecular weight of between 4.0 and 107.0 kDa (e.g., 4.0-15.0 kDa). In further embodiments, the pharmaceutical composition has a ratio of active ingredients to PLGA of 20:80. In further embodiments, the PLGA has an L to G ratio of 75:25. In further embodiments, the PLGA has a molecular weight of between 4.0 and 15.0 kDa. In further embodiments, the pharmaceutical composition delivers between 16.0 mg and 64.0 mg of active ingredient over 6 months when injected into a human joint. In further embodiments, the pharmaceutical composition delivers 32.0 mg of active ingredient over 6 months when injected into a human joint.

[0012] The present disclosure relates to pharmaceutical compositions of a therapeutic (e.g., calcium channel regulators, included encapsulated in microspheres), or a pharmaceutically acceptable salt, solvate, ester, amide, clathrate, stereoisomer, enantiomer, prodrug or analogs thereof, and use of these compositions for the treatment of osteoarthritis, including primary osteoarthritis and secondary osteoarthritis, and osteoarthritis of the hip, osteoarthritis of the foot and / or ankle, osteoarthritis of the knee, osteoarthritis of the hand, osteoarthritis of the spine, osteoarthritis of the shoulder, or cervical osteoarthritis, for example.

[0006] 5  \\4154‐0697‐7112  v4

[0013] In some embodiments, the therapeutic, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered as a pharmaceutical composition that further includes a pharmaceutically acceptable excipient.

[0014] In some embodiments, administration of the pharmaceutical composition to a human results in a peak plasma concentration of the therapeutic between 0.05 μM-10 μM (e.g., between 0.05 μM-5 μM).

[0015] In some embodiments, the condition is osteoarthritis.

[0016] In certain embodiments, the osteoarthritis is mild to moderate osteoarthritis.

[0017] In further embodiments, the osteoarthritis is moderate to severe osteoarthritis.

[0018] In other embodiments, the pharmaceutical composition is formulated for extended release. In still other embodiments, the pharmaceutical composition is formulated for immediate release. In further embodiments, pharmaceutical composition is formulated for a burst immediate release followed by a lower concentration extended release.

[0019] In some embodiments, the pharmaceutical composition is administered concurrently with one or more additional therapeutic agents for the treatment or prevention of the osteoarthritis.

[0020] In some embodiments, the therapeutic, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered as a pharmaceutical composition that further includes a pharmaceutically acceptable excipient.

[0021] In some embodiments, the peak plasma concentration of the therapeutic is maintained for up to 14 hours. In other embodiments, the peak plasma concentration of the therapeutic is maintained for up to 1 hour.

[0022] In some embodiments, administration of the pharmaceutical composition to a human results in a peak synovial fluid concentration of the therapeutic at the joint of injection, at a location proximate to the osteoarthritis, of between 0.05 μM-10 μM (e.g., between 0.05 μM-5 μM).

[0023] In some embodiments, the peak synovial fluid concentration at the joint of injection of the therapeutic is maintained for up to 14 hours. In other embodiments, a lower plateau synovial fluid concentration of the therapeutic is maintained for up to 20 days.

[0024] In other embodiments, the therapeutic is administered at a dose that is between 0.05 mg-5 mg / kg weight of the human.

[0025] In certain embodiments, the pharmaceutical composition is formulated for injection into, for example, the intraarticular space, the spinal lamellae, annulus fibrosus, and / or the

[0007] 6  \\4154‐0697‐7112  v4      nucleus pulposus.

[0026] In other embodiments, the pharmaceutical composition is formulated for extended release.

[0027] In still other embodiments, the pharmaceutical composition is formulated for immediate release.

[0028] As used herein, the term “delayed release” includes a pharmaceutical preparation, e.g., an intraarticular space injected formulation, which spreads into the synovial fluid and releases at a substantially controlled rate. In some embodiments, delayed release of the active agent (e.g., a therapeutic as described herein) results from the active agent being encapsulated in a microsphere.

[0029] The term an “effective amount” of an agent, as used herein, is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.

[0030] The terms “extended release” or “sustained release” interchangeably include a drug formulation that provides for gradual release of a drug over an extended period of time, e.g., 6-12 hours, 1 to 25 days or more, compared to an immediate release formulation of the same drug. Preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period that are within therapeutic levels and fall within a peak plasma concentration range that is between, for example, 0.05-10 μM, 0.1-10 μM, 0.1-5.0 μM, or 0.1-1 μM.

[0031] The term “immediate release” includes where the agent (e.g., therapeutic), as formulated in a unit dosage form, has a dissolution release profile under in vitro conditions in which at least 55%, 65%, 75%, 85%, or 95% of the agent is released within the first two hours of administration to, e.g., a human. Desirably, the agent formulated in a unit dosage has a dissolution release profile under in vitro conditions in which at least 50%, 65%, 75%, 85%, 90%, or 95% of the agent is released within the first 30 minutes, 45 minutes, or 60 minutes of administration.

[0032] The term “pharmaceutical composition,” as used herein, includes a composition containing a compound described herein (e.g., calcium channel regulators, included encapsulated in microspheres, or any pharmaceutically acceptable salt, solvate, or prodrug thereof), formulated with a pharmaceutically acceptable excipient, and typically manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.

[0008] 7  \\4154‐0697‐7112  v4

[0033] Pharmaceutical compositions can be formulated, for example, for intraarticular administration, for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for subcutaneous injection, intramuscular injection, intrathecal injection, or in any other formulation described herein.

[0034] A “pharmaceutically acceptable excipient,” as used herein, includes any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non- inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, maltose, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0035] The term “pharmaceutically acceptable prodrugs” as used herein, includes those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the disclosure.

[0036] The term “pharmaceutically acceptable salt,” as use herein, includes those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio, especially hydrochloride salts of the described therapeutics. Pharmaceutically acceptable salts are well known in

[0009] 8  \\4154‐0697‐7112  v4      the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds of the disclosure or separately by reacting the free base group with a suitable organic or inorganic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0037] The terms “pharmaceutically acceptable solvate” or “solvate,” as used herein, includes a compound of the disclosure wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the administered dose. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri- hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N’- dimethylformamide (DMF), N,N’-dimethylacetamide (DMAC), 1,3-dimethyl-2- imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2- pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”

[0038] The term “prevent,” as used herein, includes prophylactic treatment or treatment that prevents one or more symptoms or conditions of a disease, disorder, or conditions

[0010] 9  \\4154‐0697‐7112  v4      described herein (e.g., osteoarthritis). It is to be appreciated that osteoarthritis is a disease that can develop over the course of many years, with a significant amount of that time having little or no symptoms or pain. During the period of pre-symptomatic disease, gross changes are occurring in the joint. Treatment can be initiated, for example, prior to (“pre-exposure prophylaxis”) or following (“post-exposure prophylaxis”) a diagnosis of the disease, disorder, or conditions. Treatment that includes administration of a compound of the disclosure, or a pharmaceutical composition thereof, can be acute, short-term, or chronic. The doses administered may be varied during the course of preventive treatment.

[0039] The term “prodrug,” as used herein, includes compounds which are rapidly transformed in vivo to the parent compound of the above formula. Prodrugs also encompass bioequivalent compounds that, when administered to a human, lead to the in vivo formation of therapeutic. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, each of which is incorporated herein by reference. Preferably, prodrugs of the compounds of the present disclosure are pharmaceutically acceptable.

[0040] As used herein, and as well understood in the art, “treatment” includes an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e. not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. As used herein, the terms “treating” and “treatment” can also include delaying the onset of, impeding or reversing the progress of, or alleviating either the disease or condition to which the term applies, or one or more symptoms of such disease or condition.

[0041] The term “unit dosage forms” includes physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined

[0011] 10  \\4154‐0697‐7112  v4      quantity of active material calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients.

[0042] As used herein, the term “plasma concentration” includes the amount of therapeutic present in the plasma of a treated subject (e.g., as measured in a rabbit using an assay described below or in a human).

[0043] Various objects, features, aspects, and advantages of the present disclosure will become more apparent from the following detailed description of preferred embodiments of the disclosure, along with the accompanying drawings in which like numerals represent like components. The present disclosure may address one or more of the problems and deficiencies of the current technology discussed above. However, it is contemplated that the disclosure may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claims should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate various embodiments and together with the general description given above and the detailed description of the drawings given below, serve to explain the principles of the claimed subject matter. It is to be appreciated that the accompanying drawings are not necessarily to scale since the emphasis is instead placed on illustrating the principles of the disclosure. The present diclosure will now be described, by way of example, with reference to the accompanying drawings.

[0045] FIG. 1 is a table showing results of an experiment evidencing the efficacy of the therapeutic described herein.

[0046] FIG.2 is a table showing the measured thermal properties of verapamil HCl.

[0047] FIG.3 is a table showing the organic solubility of verapamil HCl and selected grades of PLGA.

[0048] FIG.4 is a graph showing the measured solubility of verapamil as a function of pH in PBS.

[0049] FIG.5 is a table showing the solubility of verapamil as a function of pH in PBS.

[0050] FIG.6 is a table showing the prototype verapamil SDD formulations for initial screening.

[0012] 11  \\4154‐0697‐7112  v4

[0051] FIG.7 is a set of six electron micrographs showing representative SEM images of verapamil SDDs.

[0052] FIG.8 is an electron micrograph of 10%A SDD with 50:50 L:G PLGA showing signs of strings and crystalline material.

[0053] FIG.9 is a table showing measured glass transition temperatures for verapamil SDDs and in-going polymers.

[0054] FIG.10 is a set of two representative thermograms.

[0055] FIG.11 is a graph showing PXRD diffractograms of verapamil SDDs;

[0056] FIGs.12A and 12B are two graphs that show initial release of verapamil from prototype SDDs as a function of pH.

[0057] FIG.13 is a table of initial release of verapamil from prototype SDDs as a function of pH.

[0058] FIG.14A is a graph showing extended sink dissolution of verapamil SDDs in PBS at pH 7.4 with 0.2% sodium hyaluronate, and FIGFIG.14B is a graph showing extended sink dissolution of verapamil SDDs in PBS at pH 7.4 without 0.2% sodium hyaluronate.

[0059] FIG.15A is a graph showing release projections for verapamil SDDs through 6 months in PBS at pH 7.4 with 0.2% sodium hyaluronate, and FIG.14B is a graph showing release projections for verapamil SDDs through 6 months PBS at pH 7.4 without 0.2% sodium hyaluronate.

[0060] FIGs.16A-16C are six graphs showing fits to experimental data used to generate release projections for verapamil SDDs through 6 months.

[0061] FIG.17 is a table showing composition of round 2 verapamil SDDs and one rational for selection.

[0062] FIG.18 is a photograph of round 2 verapamil SDDs.

[0063] FIG.19 shows a set of electron micrographs of round 2 verapamil SDDs.

[0064] FIG.20 is a table showing tabulated particle size distribution parameters of the second round SDDs.

[0065] FIG.21 is a table showing the measured glass transition temperatures for round 2 verapamil SDDs.

[0066] FIG.22 provides two graphs showing the impact of vortexing on initial burst in sink

[0013] 12  \\4154‐0697‐7112  v4      dissolution of 20%A, 75:35 L:G, 4-15 kDa PLGA SDD (round 1) in PBS at pH7.4 with (top) and without (bottom) 0.2% sodium hyaluronate (or right and left respectively if the page is rotated counterclockwise 90 degrees).

[0067] FIG.23 provides two graphs showing the extended sink dissolution of round 2 verapamil SDDs in PBS at pH 7.4 with (top) and without (bottom) 0.2% sodium hyaluronate (or right and left respectively if the page is rotated counterclockwise 90 degrees).

[0068] FIG.24 is two graphs showing release projections for round 2 verapamil SDDs through 6 months in PBS at pH 7.4 with (top) and without (bottom) 0.2% sodium hyaluronate; and

[0069] FIG.25 provides five graphs showing fits to experimental data used to generate release projections for round 2 verapamil SDDs through 6 months. DETAILED DESCRIPTION

[0070] The inventors have established clinical evidence for treatment of osteoarthritis (OA) with calcium channel blocker therapeutics, notably verapamil, encapsulated PLGA microspheres. This disclosure further describes embodiments of spray dried dispersion (SDD) formulations for providing extended release of verapamil in synovial fluid. Over 6 months, the pharmaceutical composition described herein can deliver between 16.0 mg and 64.0 mg, between 24.0 mg and 48.0 mg, or 32.0 mg of the therapeutic. The pharmaceutical composition described herein can deliver about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, about 50, about 52, about 54, about 56, about 58, about 60, about 62, or about 64 mg over 6 months. The pharmaceutical composition described herein can deliver less than 16, less than 18, less than 20, less than 22, less than 24, less than 26, less than 28, less than 30, less than 32, less than 34, less than 36, less than 38, less than 40, less than 42, less than 44, less than 46, less than 48, less than 50, less than 52, less than 54, less than 56, less than 58, less than 60, less than 62, or less than 64 mg over 6 months. The pharmaceutical composition described herein can deliver more than 16, more than 18, more than 20, more than 22, more than 24, more than 26, more than 28, more than 30,

[0014] 13  \\4154‐0697‐7112  v4      more than 32, more than 34, more than 36, more than 38, more than 40, more than 42, more than 44, more than 46, more than 48, more than 50, more than 52, more than 54, more than 56, more than 58, more than 60, more than 62, or more than 64 mg over 6 months.

[0071] The claims will be understood by reference to the following detailed description, which should be read in conjunction with the appended drawings. It is to be appreciated that the following detailed description of various embodiments is by way of example only and is not meant to limit, in any way, the scope of the present disclosure. In the summary above, in the following detailed description, in the claims below, and in the accompanying drawings, reference is made to particular features (including method steps) of the present disclosure. It is to be understood that the disclosure in this specification includes all possible combinations of such particular features, not just those explicitly described. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments, and in the disclosure generally. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and grammatical equivalents and variants thereof, as used herein, are intended to be open- ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. are used herein to mean that other components, ingredients, steps, etc. are optionally present. For example, an article “comprising” (or “which comprises”) components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

[0072] The term “at least” followed by a number is used herein to denote the start of a range

[0015] 14  \\4154‐0697‐7112  v4      beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example “at least 1” means 1 or more than 1. The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40% means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)-(a second number),” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm. .

[0073] The embodiments set forth the below represent the necessary information to enable those skilled in the art to practice the disclosure and illustrate the best mode of practicing the disclosure. For the measurements listed, embodiments including measurements plus or minus the measurement times 5%, 10%, 20%, 50% and 75% are also contemplated. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0074] The term “substantially” means that the property is within 80% of its desired value. In other embodiments, “substantially” means that the property is within 90% of its desired value. In other embodiments, “substantially” means that the property is within 95% of its desired value. In other embodiments, “substantially” means that the property is within 99% of its desired value. For example, the term “substantially complete” means that a process is at least 80% complete, for example. In other embodiments, the term “substantially complete” means that a process is at least 90% complete, for example. In other embodiments, the term “substantially complete” means that a process is at least 95% complete, for example. In other embodiments, the term “substantially complete” means that a process is at least 99% complete, for example.

[0016] 15  \\4154‐0697‐7112  v4

[0075] The term “substantially” includes a value is within about 10% of the indicated value. In certain embodiments, the value is within about 5% of the indicated value. In certain embodiments, the value is within about 2.5% of the indicated value. In certain embodiments, the value is within about 1% of the indicated value. In certain embodiments, the value is within about 0.5% of the indicated value.

[0076] The term “about” includes when value is within about 10% of the indicated value. In certain embodiments, the value is within about 5% of the indicated value. In certain embodiments, the value is within about 2.5% of the indicated value. In certain embodiments, the value is within about 1% of the indicated value. In certain embodiments, the value is within about 0.5% of the indicated value.

[0077] In addition, the embodiments disclosed herein do not require that all the advantageous features and all the advantages of any of the embodiments need to be incorporated into every embodiment of the disclosure.

[0078] The embodiments described herein utilize calcium-channel blockers or regulators, preferably at least partially encapsulated in microparticles (such as microspheres) as a part of a therapeutic for osteoarthritis.

[0079] Representative examples of calcium-channel blockers or regulators that may be used include amlodipine, bepridil, diltiazem hypochloride, felodipine, gallopamil, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, devapamil; and emopamil and mixtures thereof, and pharmaceutically acceptable salts, solvates, esters, amides, clathrates, stereoisomers, enantiomers, prodrugs or analogs thereof, as well as specific antibodies against the channels. As the name implies, calcium-channel blockers or regulators respectively block or regulate the movement of calcium ions through ion channels in the membranes of cells. A “specific antibody against the channel” means an antibody against an antigenic determinant of the ion-channel protein that is capable of blocking the function of the ion channel when the antibody binds to the antigenic determinant. A detailed description of a selection of the representative calcium-channel blockers or regulators, with noteworthy enantiomers and salts follow.

[0080] Verapamil has (+) and (-) / (R) and (S) enantiomers. (-)-Verapamil, or (S)-verapamil is also known as 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4- dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile that has S configuration. It is a conjugate base of a (S)-verapamil(1+). It is an enantiomer of a

[0017] 16  \\4154‐0697‐7112  v4      dexverapamil. (-)-Verapamil has a molecular formula of C27H38N2O4, and a chemical structure of:

[0081] Dexverapamil, , (3,4-dimethoxyphenyl)-5-{[2- (3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile that has R configuration. Dexverapamil is a conjugate base of a dexverapamil(1+), and is an enantiomer of (S)-verapamil. Dexverapamil has a molecular formula of C27H38N2O4, and has a chemical structure of:

[0082] Verapamil and also has (+) and (-) / (R) and (S) has a molecular formula of C27H39ClN2O4

[0083] Devapamil, also known as desmethoxyverapamil, is a phenylalkylamine (PAA) derivative and has (+) and (-) / (R) and (S) enantiomers.

[0084] (-)-Devapamil, is also known as (S)-devapamil and (2S)-2-(3,4-dimethoxyphenyl)-5-[2- (3-methoxyphenyl) ethyl-methylamino]- 2-propan- 2-ylpentanenitrile. (-)-Devapamil has a molecular formula of C26H36N2O3,and has a chemical structure of:

[0018] 17  \\4154‐0697‐7112  v4

[0085] (+)- - -2-(3,4-dimethoxyphenyl)-5-[2- (3-methoxyphenyl)ethyl-methylamino]-2-propan-2-ylpentanenitrile, has a molecular formula of C26H36N2O3, and has a chemical structure of:

[0086] Devapamil and also has (+) and (-) / (R) and (S) enantiomers.

[0087] Emopamil has (+) and (-) / (R) and (S) enantiomers. Emopamil also known as 5- [methyl(2-phenylethyl)amino]-2-phenyl-2-propan-2-ylpentanenitrile, has a Molecular Formula of C23H30N2

[0088] Emopamil hydrochloride and

[0019] 18  \\4154‐0697‐7112  v4      (2S)-5-[methyl(2-phenylethyl)amino]-2-phenyl-2-propan-2-ylpentanenitrile, hydrochloride, and has a molecular formula of C23H31ClN2.

[0089] Emopamil hydrochloride, (R), is also known as benzeneacetonitrile, alpha-(1- methylethyl)-alpha-(3-(methyl(2-phenylethyl)amino)propyl), - monohydrochloride, (R)-, and has a molecular formula of C23H31ClN2.

[0090] Nifedipine is a dihydropyridine calcium channel blocking agent also known as dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. Nifedipine has a molecular formula of C17H18N2O6, and a chemical structure of:

[0091] Nifedipine and has the molecular formula of C17H19ClN2O6.

[0092] Diltiazem is a 5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5- tetrahydro-1,5-benzothiazepin-3-yl acetate in which both stereocentres have S configuration. It is a conjugate base of a diltiazem(1+), and is an enantiomer of an ent- diltiazem. Diltiazem has a molecular formula of C22H26N2O4S, and has a chemical structure of:

[0020] 19  \\4154‐0697‐7112  v4

[0093] Ent-diltiazem is a -2-(4-methoxyphenyl)-4-oxo-2,3,4,5- tetrahydro-1,5-benzothiazepin-3-yl acetate in which both stereocentres have R configuration. It is a conjugate base of an ent-diltiazem(1+) and an enantiomer of a diltiazem. Ent-diltiazem has a molecular formula of C22H26N2O4S and has a chemical structure of:

[0094] Diltiazem diltiazem and has the molecular formula of

[0095] Microspheres: Microspheres are homogeneous, monolithic particles in the size range of about 1-300 μm. Administration of drugs in the form of microspheres provides the option to localizes the therapeutic (i.e., API) at the site of action and by prolonging drug release. Calcium channel blocker microspheres: The present disclosure conceives delivering

[0021] 20  \\4154‐0697‐7112  v4      microspheres loaded with calcium channel regulator into the joints of patients suffering from osteoarthritis. The inventors tested the efficacy of this method, and this novel method of delivering the therapeutic. Methods of Treatment

[0096] In one embodiment, a method for treating osteoarthritis comprises directly administering to the joint an effective amount of calcium-channel regulator microspheres. Administering the calcium-channel regulator microspheres is preferably accomplished by direct (intraarticular) injection of a composition comprising the calcium-channel regulator microspheres into an arthritic joint. The injection can be injection into the intraarticular space of a joint of the patient. The joint can be a hip joint, a joint of a foot and / or an ankle, a knee joint, a joint of a hand, a joint of the spine, and / or a shoulder joint. Intraarticular injection differs from other methods of administering calcium-channel regulator microspheres in that it allows biologically sufficient concentrations of calcium- channel regulator microspheres to be applied to the affected synovial tissue without the risk of producing the undesirable side-effects that can occur as the result of the higher concentrations of calcium-channel regulator microspheres by other administration techniques. Injection techniques are known to those skilled in the art. For example, a useful description for injecting the knee joint is given in “Viscosupplementation Under Fluoroscopic Control,” D. Waddell, D. Estey, D. C. Bricker, and A. Marsala, American Journal of Medicine in Sports, 4:237-241 and 249, 2001, which is incorporated by reference herein.

[0097] In one embodiment, an effective amount of calcium-channel regulator microspheres is administered to an osteoarthritic joint in a pharmaceutically acceptable composition. An “effective amount” is an amount that is sufficient to reduce any or all of the symptoms of osteoarthritis in the treated joint, such as inflammation, pain, stiffness and / or loss of function, and producing a minimal amount or none of the undesirable side effects resulting from an overdose of calcium-channel regulator microspheres, such as tissue death or injury, joint swelling, etc. What is an effective amount will vary depending on the ion channel, the method used for administration and the joint being treated. In some embodiments, a combination of multiple calcium channel regulators, may be effective.

[0098] According to one embodiment, an effective amount of calcium-channel regulator microspheres for treating osteoarthritis in accordance with the present disclosure using

[0022] 21  \\4154‐0697‐7112  v4      intraarticular injection may be in the range of 0.00001-10.0 mg, preferably dissolved or suspended in physiological saline or other vehicle appropriate for injection into the body. Preferred compositions comprise one or more calcium-channel regulator microspheres at a total concentration of 0.00001-2.5 mg / mL. Typically, 1-4 mL of the composition may be injected into the joint at one time. Administration of a large free dose of ion-channel regulator to a joint by intraarticular injection to synovial tissue may be likely to produce undesirable side effects resulting from the toxic effect of the ion-channel regulator on joint tissue at that level. An effective amount of ion-channel regulator used in the present disclosure may be an order of magnitude less than the amount of (+)-verapamil that is taught in as being effective for the treatment of rheumatoid arthritis by intraarticular injection. See, for example, Mak U.S. Pat. No. 6,190,691, col. 83, lines 35-54. As one specific example of the present disclosure, an effective amount of verapamil may be 0.02-0.8 mg when directly injected into an adult knee joint.

[0099] In another embodiment, calcium-channel regulator microspheres may be administered in combination with one or more other osteoarthritis treatment agents, either in separate compositions or in the same composition. Preferably, the other osteoarthritis treatment agent is in the form of an injectable composition, i.e. a composition that is suitable for being injected directly into the affected joint (intraarticular injection). The treatment method of the present disclosure can readily be customized to the individual patient's needs, and may be used instead of or in conjunction with other treatment modalities including but not limited to physical therapy, treatments that provide localized pain relief (heat, massage, application of liniments, etc.), and with other medications that help reduce disability, relieve pain, and improve the patient's quality of life.

[0100] In one embodiment, an effective dose of a pharmaceutical composition described herein is administered to a patient in need thereof to treat arthritis (e.g., osteoarthritis). The arthritis may be osteoarthritis. The administration may be a single injection including both API in solution and API encapsulated PLGA microspheres into an intraarticular space of a joint. In some embodiments, the single injection includes the API (e.g., verapamil) in solution and the API (e.g., verapamil) encapsulated microspheres. The API in solution can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, or 3.0 mg of API (e.g., verapamil). The API in solution can be between 0.2 and 2.0 mg of API, e.g., between 0.5 and 2.0 mg of API, between 0.5 and 1.5 mg of API, between 0.5 and 1.0 mg of API, between 0.5 and 0.75

[0023] 22  \\4154‐0697‐7112  v4      mg of API, between 0.2 and 1.5 mg of API, or between 0.2 and 1.0 mg of API, between 0.2 and 0.5 mg of API. The API (e.g., verapamil) encapsulated microspheres can contain about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, about 50, about 52, about 54, about 56, about 58, about 60, about 62, about 64, about 16 to about 64, about 16 to about 48, about 16 to about 32, or about 16 to about 24 mg of API. The single injection including API in solution and API encapsulated microspheres can deliver about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, or about 40 mg of API to a patient over about 6 months.

[0101] The single injection including API in solution and API in microspheres can release API over an extended duration (e.g., about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 7 months). The extended duration may be about 1 month to about 6 months, about 2 months to about 6 months, about 3 months to about 6 months, about 4 months to about 5 months, about 1 month to about 5 months, about 1 month to about 4 months, about 1 month to about 3 months, about 1 month to about 2 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 4 months to about 6 months, or about 4 months to about 5 months.

[0102] Accordingly, examples of treatments contemplated by the present disclosure include an intraarticular injection of a composition including calcium-channel regulator microspheres followed by another intraarticular injection of another osteoarthritis treatment agent, e.g. a viscosupplement, steroid or other injectable osteoarthritis treatment agent; an intraarticular injection of a calcium-channel regulator microspheres composition followed by oral or intravenous administration of another osteoarthritis treatment agent such as a non-steroidal anti-inflammatory drug; an intraarticular injection of a single composition comprising calcium-channel regulator microspheres and at least one viscosupplement, steroid or other injectable osteoarthritis treatment agent; and so forth.

[0103] A treatment composition according to one embodiment of the disclosure comprises calcium-channel regulator microspheres and one or more other osteoarthritis treatment agent(s). The individual concentrations of the calcium-channel regulator microspheres

[0024] 23  \\4154‐0697‐7112  v4      and the other osteoarthritis treatment agent(s) are sufficient to provide an effective amount of each ingredient to the affected joint. Preferably, the composition comprises calcium-channel regulator microspheres at a concentration of 0.00001-2.0 mg / mL and other osteoarthritis treatment agent(s) at a concentration of 0.01-25 mg / mL.

[0104] In one embodiment, the composition is suitable for intraarticular injection in accordance with the method of the present disclosure, and both the calcium-channel regulator microspheres and other osteoarthritis treatment agent are “injectable”. As used herein, the term “injectable” means any osteoarthritis treatment agent that is in a form suitable for intraarticular injection. In one embodiment, the injectable other osteoarthritis treatment agents may comprise at least one corticosteroid such as a glucocorticoid. As one specific non-limiting example, the composition of the present disclosure may comprise 1-25 mg / mL of the injectable steroid osteoarthritis treatment agent methylprednisolone acetate.

[0105] In another embodiment, the injectable other osteoarthritis treatment agent may comprise at least one viscosupplement. As used herein and in the art, the term “viscosupplement” refers to any substance that is used to restore and / or increase the cushioning and lubrication of arthritic synovial fluid by intraarticular injection. Preferred viscosupplements include hylan, hyaluronic acid and other hyaluronan (sodium hyaluronate) compounds, which are natural complex sugars of the glycosaminoglycan family. Hyaluronan is a long-chain polymer containing repeating disaccharide units of Na-glucoronate-N-acetylglucosamine. By way of example, commercially available hyaluronan viscosupplements include Synvisc®, Hyalgan®, Supartz®, and Orthovisc®. As one specific non-limiting example, the composition of the present disclosure may comprise 1-15 mg / mL of a hyaluronan compound.

[0106] Other osteoarthritis treatment agents comprising the composition of the present disclosure may also include those used in any modality of arthritis treatment, such as oral administration, intravenous administration, etc. Examples of other osteoarthritis treatment agents include, without limitation, non-steroidal anti-inflammatory drugs (NSAIDS) such as ibuprofen, naproxen, and COX-2 inhibitors; analgesics such as aspirin and acetaminophen; glycans, including glucosamines, e.g. glucosamine sulfate and glucosamine hydrochloride; and proteoglycans, such as chondroitin compounds, as well as various other known narcotics, steroids, antibiotics, immunomodulators, penicillamine, and the like.

[0107] The compositions of the present disclosure may also contain other materials such as

[0025] 24  \\4154‐0697‐7112  v4      fillers, stabilizers, coatings, coloring agents, preservatives, fragrances, and other additives known in the art. The compositions may be in liquid or gel form and may be provided in time-release formulations. Pharmaceutical Compositions

[0108] The methods described herein can also include the administration of pharmaceutically acceptable compositions that include the therapeutic, or a pharmaceutically acceptable salt, solvate, or prodrug thereof loaded in the microsphere and / or as a carrier for the microsphere. When employed as pharmaceuticals, any of the present compounds can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. While administration is preferably intraarticular, administration may be topical, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or by suppositories.

[0109] The present disclosure further relates to a pharmaceutical composition comprising a plurality of spherical carriers formed of poly lactic / glycolic acid (PLGA), between 10% and 30% (e.g., 20%) active loading of one or more active ingredients (e.g., verapamil). The pharmaceutical composition can comprise about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, between 10% and 25%, between 10% and 20%, between 10% and 15% active loading of one or more active ingredients (e.g., verapamil). The active ingredient can be, for example, one or more of verapamil and pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof. The PLGA can have a L to G ratio of between 50:50 and 75:25, for example 55:45, 60:40, 65:35, or 70:30. The PLGA can have a molecular weight of between 4.0 and 107.0 kDa, between 4.0 and 100 kDa, between 4.0 and 90 kDa, between 4.0 and 80 kDa, between 4.0 and 70 kDa, between 4.0 and 60 kDa, between 4.0 and 50 kDa, between 4.0 and 40 kDa, between 4.0 and 30 kDa, between 4.0 and 20 kDa, between 4.0 and 15 kDa, between 4.0 and 10 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, or about 20 kDa.

[0026] 25  \\4154‐0697‐7112  v4

[0110] This disclosure also includes pharmaceutical compositions which can contain one or more pharmaceutically acceptable carriers. In making the pharmaceutical compositions of the disclosure, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, and soft and hard gelatin capsules. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives.

[0111] The therapeutic agents of the disclosure can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier. The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington: The Science and Practice of Pharmacy, 22ndEd., Gennaro, Ed., Lippencott Williams & Wilkins (2012), a well-known reference text in this field, and in the USP / NF (United States Pharmacopeia and the National Formulary), each of which is incorporated by reference. In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

[0112] Examples of suitable excipients are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. Other exemplary excipients are described in Handbook of Pharmaceutical Excipients, 8thEdition, Sheskey et al., Eds., Pharmaceutical Press (2017), which is incorporated by reference.

[0027] 26  \\4154‐0697‐7112  v4

[0113] The methods described herein can include the administration of a therapeutic, or prodrugs or pharmaceutical compositions thereof, or other therapeutic agents. Exemplary therapeutics include those that block or down regulate the calcium ion channel (including amlodipine, bepridil, diltiazem hypochloride, felodipine, gallopamil, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, devapamil, and emopamil or pharmaceutically acceptable salts, solvates, esters, amides, clathrate, stereoisomers, enantiomers, prodrugs or analogs thereof).

[0114] The pharmaceutical compositions can be formulated so as to provide immediate, extended, or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

[0115] The compositions can be formulated in a unit dosage form, each dosage containing, e.g., 0.1-500 mg of the active ingredient. For example, the dosages can contain from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg,, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg,, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mg to about 150 mg, from about 40 mg to about 100 mg, from about 50 mg to about 100 mg of the active ingredient, from about 50 mg to about 300 mg, from about 50 mg to about 250 mg, from about 100 mg to about 300 mg, or , from about 100 mg to about 250 mg of the active ingredient. For preparing solid compositions such as tablets, the principal active ingredient is mixed with one or more pharmaceutical excipients to form a solid bulk formulation composition containing a homogeneous mixture of a compound of the present disclosure. When referring to these bulk formulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets and capsules. This solid bulk formulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active

[0028] 27  \\4154‐0697‐7112  v4      ingredient of the present disclosure.

[0116] Parenteral Administration: Within the scope of the present disclosure are also injections and parenteral depot systems from biodegradable polymers. These systems are injected or implanted into the joint, muscle, or subcutaneous tissue and release the incorporated drug over extended periods of time, ranging from several days to several months. Both the characteristics of the polymer and the structure of the device can control the release kinetics which can be either continuous or pulsatile. Polymer-based injections and parenteral depot systems can be classified as implants or microparticles. The former are cylindrical devices injected into the subcutaneous tissue whereas the latter are defined as spherical particles in the range of 10 – 100 µm. Extrusion, compression or injection molding are used to manufacture implants whereas for microparticles, the phase separation method, the spray-drying technique and the water-in-oil-in-water emulsion techniques are frequently employed. Biodegradable polymers to form microparticles include polyesters from lactic and / or glycolic acid, e.g. poly(glycolic acid) and poly(L- lactic acid) (PLG / PLA microspheres). Of particular interest are in situ forming depot systems, such as thermoplastic pastes and gelling systems formed by solidification, by cooling, or due to the sol-gel transition, cross-linking systems and organogels formed by amphiphilic lipids. Examples of thermosensitive polymers used in the aforementioned systems include, N-isopropylacrylamide, poloxamers (ethylene oxide and propylene oxide block copolymers, such as poloxamer 188 and 407), poly(N-vinyl caprolactam), poly(siloethylene glycol), polyphosphazenes derivatives and PLGA-PEG-PLGA.

[0117] Pharmaceutical compositions of the present disclosure comprise an effective amount of one or more calcium channel regulators, included encapsulated in microspheres dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one calcium channel regulators, included encapsulated in microspheres in solution or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by “Remington: The Science and Practice of Pharmacy,” 20th Edition (2000), which is incorporated herein by reference in its entirety. Moreover, for animal (for example, human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety

[0029] 28  \\4154‐0697‐7112  v4      and purity standards as required by FDA Office of Biological Standards.

[0118] In various embodiments, the compositions of the present disclosure further comprise cyclodextrin. Cyclodextrins are a general class of molecules composed of glucose units connected to form a series of oligosaccaride rings (See Challa et al., 2005, AAPS PharmSciTech 6:E329-E357). In nature, the enzymatic digestion of starch by cyclodextrin glycosyltransferase (CGTase) produces a mixture of cyclodextrins comprised of 6, 7 and 8 anhydroglucose units in the ring structure (α-, β-, and γ- cyclodextrin, respectively). Commercially, cyclodextrins are also produced from starch, but different, more specific enzymes are used. Cyclodextrins have been employed in formulations to facilitate the delivery of cisapride, chloramphenicol, dexamethasone, dextromethoraphan, diphenhydramine, hydrocortisone, itraconazole, and nitroglycerin (Welliver and McDonough, 2007, Sci World J, 7:364-371). In various embodiments, the cyclodextrin of the disclosure is hydroxypropyl-Beta-cyclodextrin, sulfobutylether-beta- cyclodextrin, alpha-dextrin or combinations thereof. In certain embodiments, cyclodextrin may be used as a solubilizing agent.

[0119] In various other embodiments, compositions of the present disclosure may comprise human serum albumin purified from plasma, or recombinant human serum albumin. In certain embodiments, human serum albumin may be used as a solubilizing agent. In other embodiments, the compositions of the disclosure may comprise propylene glycol. In other embodiments, the compositions of the disclosure may comprise perfluorooctyl bromide. In other embodiments, the compositions of the disclosure may comprise perfluorocarbon. In certain embodiments, perfluorocarbon may be used as a solubilizing agent.

[0120] In various embodiments, a preservative or stabilizer may be included in the composition or solution. For example, the prevention of the action of microorganisms may be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (for example, methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, EDTA, metabisulfite, benzyl alcohol, thimerosal or combinations thereof. Agents which may be included suitable for use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the composition is preferably sterile and must be fluid to facilitate easy injectability. Solutions are preferably stable under the conditions of

[0030] 29  \\4154‐0697‐7112  v4      manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Examples of stabilizers which may be included include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc. Appropriate stabilizers or preservatives may be selected according to the route of administration desired. A particle filter or microbe filter may be used and may be necessary according to the route of administration desired.

[0121] Administration of the disclosed compositions in a method of treatment may be achieved in a number of different ways, using methods known in the art. Such methods include, but are not limited to, topically administering solutions, suspensions, creams, pastes, oils, lotions, gels, foam, hydrogel, ointment, liposomes, emulsions, liquid crystal emulsions, and nano-emulsions.

[0122] The therapeutic and prophylactic methods of the disclosure thus encompass the use of pharmaceutical compositions of the disclosure. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. For example unit dose container may be such that a calcium channel regulators encapsulated in microspheres solution is contained in a crushable sealed ampoule which in turn is enclosed in protective covering on which pressure is applied to crush the ampoule which then releases a calcium channel regulators encapsulated in microspheres solution for percolation through a flint-type tip which capped the ampoule in protective covering. When such packaging configuration is employed, care is taken to leave as little as possible or ideally no headspace in ampoule for any volatile portion of the solution to escape and cause a change in solution composition over a period of shelf life.

[0123] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts, including mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the

[0031] 30  \\4154‐0697‐7112  v4      ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0124] Pharmaceutical compositions that are useful in the methods of the disclosure may be prepared, packaged, or sold in formulations suitable for ophthalmic, vaginal, topical, intranasal, buccal, or another route of administration.

[0125] A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. A unit dose is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0126] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0127] In addition to the active ingredient, a pharmaceutical composition of the disclosure may further comprise one or more additional pharmaceutically active agents. Non-limiting examples of such an additional pharmaceutically active agents are fluorouracil cream, imiquimod cream, ingenol mebutate gel, diclofenac sodium gel, topical retinoids, and tirbanibulin (Klisyri) ointment.

[0128] Controlled- or sustained-release formulations of a pharmaceutical composition of the disclosure may be made using conventional technology.

[0129] Formulations of a pharmaceutical composition suitable for topical administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules, crushable or otherwise, or in multi-dose containers containing a preservative. Formulations for topical administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles,

[0032] 31  \\4154‐0697‐7112  v4      solutions, suspensions, creams, pastes, oils, lotions, gels, foam, hydrogel, ointment, liposomes, emulsions, liquid crystal emulsions, nanoemulsions, implantable sustained- release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.

[0130] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile formulations may be prepared using a non-toxic acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other formulations that are useful include those which comprise the active ingredient in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0131] In some embodiments, the pharmaceutical compositions of the disclosure may be contained in a crushable ampule irrespective of the route of delivery to the patient.

[0132] It is contemplated that any embodiment discussed in this specification may be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure may be used to achieve methods of the disclosure.

[0133] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0033] 32  \\4154‐0697‐7112  v4      Dosing Regimens

[0134] The present methods for treating osteoarthritis are carried out by administering a therapeutic for a time and in an amount sufficient to result in decreased joint pain, joint stiffness, tenderness at or near joint, joint tenderness, loss of flexibility, grating sensation, bone spurs, and or swelling

[0135] The amount and frequency of administration of the compositions can vary depending on, for example, what is being administered, the state of the patient, and the manner of administration. In therapeutic applications, compositions can be administered to a patient suffering from osteoarthritis in an amount sufficient to relieve or least partially relieve the symptoms of the osteoarthritis and its complications. The dosage is likely to depend on such variables as the type and extent of progression of the osteoarthritis, the severity of the osteoarthritis, the age, weight and general condition of the particular patient, the relative biological efficacy of the composition selected, formulation of the excipient, the route of administration, and the judgment of the attending clinician. Effective doses can be extrapolated from dose- response curves derived from in vitro or animal model test system. An effective dose is a dose that produces a desirable clinical outcome by, for example, improving a sign or symptom of the osteoarthritis or slowing its progression.

[0136] The amount of therapeutic per dose can vary. For example, a subject can receive from about 0.1 μg / kg to about 10,000 μg / kg. Generally, the therapeutic is administered in an amount such that the peak plasma concentration ranges from 150 nM-250 μM.

[0137] Exemplary dosage amounts can fall between 0.1-5000 μg / kg, 100-1500 μg / kg, 100-350 μg / kg, 340-750 μg / kg, or 750-1000 μg / kg. Exemplary dosages can 0.25, 0.5, 0.75, 1º, or 2 mg / kg. In another embodiment, the administered dosage can range from 0.05-5 mmol of therapeutic (e.g., 0.089-3.9 mmol) or 0.1-50 μmol of therapeutic (e.g., 0.1-25 μmol or 0.4-20 μmol).

[0138] The plasma concentration of therapeutic can also be measured according to methods known in the art. Exemplary peak plasma concentrations of therapeutic can range from 0.05-10 μM, 0.1-10 μM, 0.1-5.0 μM, or 0.1-1 μM. Alternatively, the average plasma levels of therapeutic can range from 400-1200 μM (e.g., between 500-1000 μM) or between 50-250 μM (e.g., between 40-200 μM). In some embodiments where sustained release of the drug is desirable, the peak plasma concentrations (e.g., of therapeutic) may be maintained for 6-14 hours, e.g., for 6-12 or 6-10 hours. In other embodiments where

[0034] 33  \\4154‐0697‐7112  v4      immediate release of the drug is desirable, the peak plasma concentration (e.g., of therapeutic) may be maintained for, e.g., 30 minutes.

[0139] The frequency of treatment may also vary. The subject can be treated one or more times per day with therapeutic (e.g., once, twice, three, four or more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). Preferably, the pharmaceutical composition is administered 1 or 2 times per 24 hours. The time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten or more days. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).

[0140] Kits: Any of the pharmaceutical compositions of the disclosure described herein can be used together with a set of instructions, i.e., to form a kit. The kit may include instructions for use of the pharmaceutical compositions as a therapy as described herein, with the instructions being provided either as a hard copy, such as printed on paper, and / or as directions to access the instructions electronically, such as a web address or QR code to a website where the instructions are provided. For example, the instructions may provide dosing and therapeutic regimes for use of the compounds of the disclosure to reduce symptoms and / or underlying cause of the osteoarthritis.

[0141] While various embodiments of the present disclosure have been described in detail, it is apparent that various modifications and alterations of those embodiments will occur to and be readily apparent those skilled in the art. However, it is to be expressly understood that such modifications and alterations are within the scope and spirit of the present disclosure, as set forth in the claims. Further, the embodiments described herein are capable of being practiced or of being carried out in various other related ways. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the

[0035] 34  \\4154‐0697‐7112  v4      items listed thereafter and equivalents thereof as well as additional items, while only the terms “consisting of” and “consisting only of” are to be construed in the limitative sense. EXAMPLES Example 1: Injection of controlled release verapamil microspheres into osteoarthritic mouse model knee joints

[0142] A dose of controlled release of verapamil microspheres was injected in osteoarthritic model mice knee joints. Other test groups included verapamil and diltiazem, alone or in combination, not encapsulated in microspheres. The microsphere loaded with verapamil was referred to as “controlled release” verapamil or “CR.” As shown in FIG. 1, CR-A (controlled release verapamil 3 µg burst and 0.25 µg / d 20d) reduced tibial cartilage degradation by 13% as compared to one dose of 3ug verapamil (4%) or 6 doses (no reduction). Additionally, CR-A increased the amount of normal collagen whereas this was not seen with either 1x or 6x verapamil. The above results point to potential advantages of the controlled release formulation. No increased inflammation was seen when normal rat knees were injected with CR-A.

[0143] Introduction: A study was done to determine the efficacy of verapamil and Diltiazem, alone or in combination, in inhibiting lesions of medial meniscal tear-induced cartilage degeneration in rats. Additional trials with CR verapamil were performed to see if there was any benefit to loading the calcium channel regulator in a microparticle.

[0144] Unilateral medial meniscal tear in 275-300 gram rats will result in rapidly progressive cartilage degenerative changes characterized by chondrocyte and proteoglycan loss, fibrillation, osteophyte formation and chondrocyte cloning (1, 2, 7). Utilization of younger rats (without tidemark) results in lesions more typical of those seen in osteochondrosis and extremely large osteophytes. This model is performed by transection of the medial collateral ligament just below its attachment to the meniscus so that when the joint space opens, the meniscus is reflected toward the femur. The meniscus is cut at its narrowest point (away from the ossicles) taking care not to damage the tibial surface and making sure that the resulting transection produces separate, freely movable anterior and posterior meniscus halves.

[0145] Progressive cartilage degenerative changes occur and by 3-6 weeks post-surgery, tibial

[0036] 35  \\4154‐0697‐7112  v4      cartilage degeneration may be focally severe on the outer 1 / 3 of the tibia with degenerative changes of lesser severity in the middle and inner 1 / 3. Osteophytes are ultimately quite large (medial tibia) and progressively increase in size. The model is progressive and results in total cartilage loss (to eburnated bone) in 12 months in virtually all rats, with lesions that are reasonably consistent. Rats resume weight bearing immediately post-surgery and gait analysis suggests little if any change in load bearing of the operated knee. Due to the rapid progression of cartilage degeneration, protective effects are not always apparent in the outer 1 / 3 of the tibial cartilage, although zonal analysis may reveal effects of treatment in the middle and inner 1 / 3. Substantial subchondral and epiphyseal bone changes occur in the medial tibia subjacent to the areas of greatest lesion severity. These range in magnitude and type from increased basophilia of the calcified cartilage layer with small fractures into subchondral bone to overt collapse of articular cartilage into areas of bone resorption in the epiphysis with surrounding sclerosis of bone. Therefore, this model offers the opportunity to evaluate not only chondroprotective effects of agents but also bone preserving activities.

[0146] The model is of relatively short duration and animals are very consistent in their response to the surgery. Broad-spectrum matrix metalloproteinases (MMPs) inhibitors are consistently active in this model. This model has also been used to evaluate repair strategies. Evaluation of repair strategies in rodent models must always take into consideration the marked tendency for rodent marginal zones and pleuripotential marrow cells to proliferate to various irritant stimuli.

[0147] Test Article Identification and Preparation: Verapamil and Diltiazem were used for formulation for dosing.

[0148] Test System Identification: Male Lewis rats weighing 274-312 grams (mean 289) on day 0 were obtained from Charles River Labs. Animals were identified by a distinct mark at the base of the tail delineating group and animal number. After randomization, all cages were labeled with protocol number, group numbers, and animal numbers with appropriate color-coding.

[0149] Environment and Husbandry: Upon arrival, animals were housed 4 / cage in shoe-box polycarbonate cages with wire tops, wood chip bedding and suspended food and water bottles. The cages conformed to the guidelines cited in the Guide for the Care and Use of Laboratory Animals.

[0150] Animals were acclimated for 7 days prior to being randomized into groups. An attending

[0037] 36  \\4154‐0697‐7112  v4      veterinarian was on site or on call during the live phase of the study, and no concurrent medications were given. During the acclimation and study periods, animals were housed in a laboratory environment with temperatures ranging between 67-76°F and relative humidity between 30-70%. Automatic timers provided 12 hours of light and 12 hours of dark. Animals were allowed access ad libitum to Harlan Teklad Rodent Chow and fresh municipal tap water.

[0151] Animal care, including room, cage, and equipment sanitation conformed to the guidelines cited in the Guide for the Care and Use of Laboratory Animals.

[0152] Experimental Design: Animals were anesthetized with Isoflurane and the right knee area was prepared for surgery. A skin incision was made over the medial aspect of the knee and the medial collateral ligament was exposed by blunt dissection, and then transected. The medial meniscus was cut through the full thickness to simulate a complete tear. Skin and subcutis were closed with 4-0 Vicryl suture. Dosing by the intra-articular (IA) route began four days after surgery and continued as indicated below until day 24. Treatment groups were as follows: GroupN Treatment 1 15 S r r + S lin D 4 (#1-8) nd D 4812162024 (#9-15) Table 1

[0153] At necropsy, the right (operated) knee joint from all animals and the left from animals 1-5 of group 1 were trimmed of muscle and connective tissue and collected into 10% neutral buffered formalin. The patella was removed to allow proper fixation of the joints.

[0154] Observations, Measurements, and Specimens: Rats were observed daily for abnormal swelling or gait alterations. Serum was collected at necropsy. Right knees were lavaged with 100 µl of saline and patellae were collected and frozen. Left (unoperated) knees were also lavaged and left patellae were collected from animals 1-5 of group 1. Samples were frozen for shipment to sponsor. Weight bearing (left vs. right) was recorded on

[0038] 37  \\4154‐0697‐7112  v4      days 7 and 27 using an incapacitance meter and the following method:

[0155] On the days of evaluation for pain, animals were placed in the plexiglass housing of the incapacitance meter and allowed to acclimate for approximately 2-5 minutes. The position of the animal was such that each hind paw rested on a separate force plate. The force exerted on each plate was measured three times, and each measurement was the average force over the course of three seconds. The mean of these three readings constituted 1 data point. All measurements were used, regardless of whether the value was positive (leaning to the left) or negative (leaning toward the right). Right paw force was compared to left for each group to confirm that animals were showing pain. Difference in force (left minus right) was determined both as an absolute value and as a percentage of the total hind leg force and compared between groups, as was the percentage of the total body weight that was carried on the hind legs.

[0156] Morphologic Pathology Methods: Following three days in 10% formic acid decalcifier, the operated joints were cut into two approximately equal halves in the frontal plane and embedded in paraffin. Three sections were cut from each operated knee at approximately 200 µm steps and stained with toluidine blue. A single section was cut from each unoperated knee. A total of 335 toluidine blue sections (105 operated knees x 3 + 20 non-surgery knees) were prepared for this study.

[0157] All three sections of each knee were analyzed microscopically. In scoring the three sections, the worst-case scenario for the two halves on each slide was determined for general cartilage degeneration, proteoglycan loss, collagen damage, and osteophyte formation. The values for each parameter were then averaged across the 3 sections to determine overall subjective scores. In addition, for some parameters (noted below), regional differences across the tibial plateau were taken into consideration by dividing each section into three zones (1-outside, 2-middle, 3-inside). In the surgical OA model, the outside (z1) and middle (z2) thirds are most severely affected, and milder changes are present on the inside third (z3). When zones are scored individually, scores are assigned based on % area of the zone affected. Zone areas are delineated using an ocular micrometer.

[0158] The following parameters were measured and / or scored:

[0159] General cartilage degeneration includes the important parameters of chondrocyte death / loss, proteoglycan loss, and collagen loss or fibrillation. Cartilage degeneration in the tibia was scored none to severe (numerical values 0-5) for each zone (area defined by

[0039] 38  \\4154‐0697‐7112  v4      micrometer) using the following criteria: 0=no degeneration 1=minimal degeneration, within the zone 5-10% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present 2= mild degeneration, within the zone 11-25% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present 3=moderate degeneration, within the zone 26-50% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present 4=marked degeneration, within the zone 51-75% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present. 5=severe degeneration, within the zone 76-100% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present.

[0160] A 3-zone sum for cartilage degeneration was also calculated. The same process was applied to evaluation of the femoral cartilage with the exception that lesions were not analyzed based on zones since the lesions aren’t generally distributed over the surface in a zonal pattern. The total width of the load-bearing surface (approximately 2000 µm for the femur) was determined and the above criteria were applied to the most severely affected 1 / 3, 2 / 3 or 3 / 3. For example, if 1 / 3 of the total area (lesion may be in the center of the plateau covering about 667 µm) has minimal degeneration (5-10% of total area has loss of chondrocytes and / or matrix), a score of 1 is assigned. If that minimal degeneration extends over the entire surface (3 / 3) then the score is 3. If the entire femoral cartilage is absent as a result of severe diffuse degeneration, then the score is 15.

[0161] In addition to this overall cartilage degeneration score, collagen matrix damage was scored separately in order to identify more specific effects of agents.

[0162] Collagen damage across the medial tibial plateau (most severely affected section of the two halves) was quantified by measuring the total width of the following: -Any damage (fibrillation ranging from superficial to full thickness loss). -Severe damage (total or near total loss of collagen to tidemark, >90% thickness)

[0040] 39  \\4154‐0697‐7112  v4      -Marked damage (extends through 61-90% of the cartilage thickness) -Moderate damage (extends thru 31-60% of the cartilage thickness) -Mild damage (extends through 11-30% of the cartilage thickness) -Minimal damage (very superficial, affecting upper 10% only)

[0163] In addition to the above subjective general cartilage scoring, two cartilage degeneration width measurements were taken:

[0164] Total Tibial Cartilage Degeneration Width (µm) is a micrometer measurement of total extent of tibial plateau affected by any type of degeneration (cell loss, proteoglycan loss or collagen damage). This measurement extends from the origination of the osteophyte with adjacent cartilage degeneration (outside 1 / 3) across the surface to the point where tangential layer and underlying cartilage appear histologically normal.

[0165] Substantial Cartilage Degeneration Width (µm) reflects areas of tibial cartilage degeneration in which both chondrocyte and proteoglycan loss extend through greater than 50% of the cartilage thickness. In general, the collagen damage is mild (25% depth) or greater for this parameter, but chondrocyte and proteoglycan loss extend to at least 50% or greater of the cartilage depth.

[0166] A micrometer depth of any type of lesion (both chondrocyte and proteoglycan loss, but may have good retention of collagenous matrix and no fibrillation), expressed as a ratio of depth of changed area vs. depth to tidemark, was taken in the area of greatest lesion severity in each of the 3 zones across the tibial surface at the midpoint of the zone. This measurement is the most critical analysis of any type of microscopic change present. The denominator can serve as an average measure of cartilage thickness in each of the 3 zones for comparison of anabolics when measures are taken at the midpoint of the zone.

[0167] Scoring of the osteophytes and categorization into small, medium and large was done with an ocular micrometer. Marginal zone proliferative changes are 200 µm in order to be measured and designated as osteophytes. Scores are assigned to the largest osteophyte in each section (typically found in the tibia) according to the following criteria: 1=small up to 299 µm 2=moderate 300-399 µm 3=large 400-499 µm 4=very large 500-599 5=very large !600

[0168] The actual osteophyte measurement (tidemark to furthest distance point extending toward

[0041] 40  \\4154‐0697‐7112  v4      synovium) was also recorded.

[0169] The femoral cartilage degeneration score and the 3-zone sum of the tibial cartilage degeneration scores (mean of 3 levels) were summed to create a total cartilage degeneration score. The mean osteophyte score for each joint was added to this value to create a total joint score.

[0170] Image analysis: In order to quantitate and compare the cartilage matrix preservation, cartilage area measurements were taken from the most severely affected section of each animal. Photomicrographs were taken with a CoolSNAP-Pro microscope camera and loaded into ImagePro Plus software. The following measurements were taken from tracings of these photomicrographs: 1. Total area from the tidemark to the surface (or projected surface in degenerated areas) over 6 cm (2 zones) of the tibial plateau, measured from the inner edge of the osteophyte 2. Area of non-viable matrix (cartilage with less than 50% chondrocytes, proteoglycan, and intact collagen) and no matrix within the total area 3. Area of no matrix within the total area,

[0171] The area of non-viable matrix was subtracted from the total area to get the area of viable matrix, and the area of no matrix was subtracted from the total area to get the area of any matrix (collagen matrix with or without chondrocytes and proteoglycan). These two values were then compared back to the total area to derive the percent viable matrix area and the percent any matrix area, which were compared between groups.

[0172] Synovial reaction, if abnormal, was described (should be mainly fibrosis) and characterized with respect to inflammation type and degree but was not included in the score.

[0173] Damage to the calcified cartilage layer and subchondral bone (worst case scenario for all sections) was scored using the following criteria: 0=No changes 1=Increased basophilia at tidemark, no fragmentation of tidemark, no marrow changes or if present minimal and focal 2=Increased basophilia at tidemark, minimal to mild focal fragmentation of calcified cartilage of tidemark, mesenchymal change in marrow involves 1 / 4 of total area but generally is restricted to subchondral region under lesion 3=Increased basophilia at tidemark, mild to marked focal or multifocal fragmentation

[0042] 41  \\4154‐0697‐7112  v4      of calcified cartilage (multifocal), mesenchymal change in marrow is up to 3 / 4 of total area, areas of marrow chondrogenesis may be evident but no major collapse of articular cartilage into epiphyseal bone (definite depression in surface) 4=Increased basophilia at tidemark, marked to severe fragmentation of calcified cartilage, marrow mesenchymal change involves up to 3 / 4 of area and articular cartilage has collapsed into the epiphysis to a depth of 250 !m or less from tidemark (see definite depression in surface cartilage) 5=Increased basophilia at tidemark, marked to severe fragmentation of calcified cartilage, marrow mesenchymal change involves up to 3 / 4 of area and articular cartilage has collapsed into the epiphysis to a depth of greater than 250 µm from tidemark. A treatment group Mean±SE for each score and measurement was determined.

[0174] Animal Disposition: Animal carcasses were disposed of according to Bolder BioPATH procedures.

[0175] Statistical Analysis: Statistical analysis of histopathology parameters was done by comparing group values using the Student’s two-tailed t-test with significance set at p#0.05. Specimen and Raw Data Storage

[0176] Statement of Effect of Deviations on the Quality and Integrity of the Study: There were no deviations from the protocol.

[0177] Results: Live Phase Parameters. All animals resumed weight bearing immediately post- surgery upon recovery from anesthesia and there was no evidence of excessive post- operative swelling indicative of joint infection. Animals given verapamil + Diltiazem Q4D gained significantly less weight than the vehicle control. Left legs bore significantly more weight than right legs for all surgery groups at both time points.

[0178] Animals treated with verapamil Q4D bore significantly more weight on the right legs compared to the Q4D vehicle controls on day 27. This was true when data was evaluated both as a ratio of left to right weight bearing and as a percentage of hind leg force. There were no other significant differences between surgery groups and the vehicle controls.

[0179] Morphologic Pathology: Vehicle control animals dosed Q4D on days 4-24 had cartilage degeneration that was most severe in the outer 2 / 3 of the medial tibia and least severe on the inner 1 / 3. Osteophytes were present in all animals and were large to very large, with a mean measurement of 470 µm. Femoral cartilage degeneration was present in six of seven animals and had a mean score of 1.62. The total joint score was 9.81. Vehicle control animals dosed on day 4 only had generally more severe lesions, which were

[0043] 42  \\4154‐0697‐7112  v4      significantly different from the Q4D controls for tibial cartilage degeneration (zone 2 and total), substantial cartilage degeneration widths, and the total joint score without the femur.

[0180] Animals given verapamil+Diltiazem Q4D were similar to the Q4D controls for general pathology parameters, with a 1% decrease in the total joint score. The width of marked collagen degeneration was significantly decreased by 90%, although collagen degeneration was not significantly affected otherwise. Cartilage areas were similar to the controls.

[0181] Animals given verapamil on day 4 and saline Q4D on days 8-24 were similar to the Q4D controls for general pathology parameters, with a 3% decrease in the total joint score. Collagen degeneration widths and cartilage areas were similar to the controls.

[0182] Animals given verapamil Q4D were similar to the Q4D controls for general pathology parameters, with a 2% decrease in the total joint score. Collagen degeneration widths and cartilage areas were similar to the controls.

[0183] Animals given Diltiazem Q4D were similar to the Q4D controls for general pathology parameters, with a 6% decrease in the total joint score. Collagen degeneration widths and cartilage areas were similar to the controls.

[0184] Animals given controlled release verapamil formulation A (GJ121008) with an injection on day 4 were similar to the day 4 controls for general pathology parameters, with an 8% decrease in the total joint score. Collagen degeneration widths and cartilage areas were similar to the controls.

[0185] Animals given controlled release verapamil formulation B (GJ121808) with an injection on day 4 were similar to the day 4 controls for general pathology parameters, with a 5% decrease in the total joint score. The width of mild collagen degeneration was significantly decreased by 40%, although collagen degeneration was not significantly affected otherwise. Cartilage areas were similar to the controls.

[0186] Non-surgery knees injected with controlled release verapamil on day 4 (right) or 25 (left) had no cartilage lesions with the exception with one right knee from an animal given formulation A (GJ121008), which had minimal degeneration in zone 3 of the tibia. This is likely a spontaneous change. Minimal to mild subacute inflammation was seen in the majority of the knees injected on day 4 (right) from non-surgery animals and minimal to mild acute inflammation or synovitis was seen in the majority of the knees injected on day 2.

[0044] 43  \\4154‐0697‐7112  v4

[0187] Thus CR-A (controlled release verapamil 3ug burst and 0.25 µg / d for 20d) reduced tibial cartilage degradation by 13% verses one dose of 3ug verapamil (4%) or 6 doses (no reduction) (See FIG. 1). Reduction under 10% is likely within experimental error of the model. In addition, CR-A increased the amount of normal collagen whereas this was not seen with either 1x or 6x verapamil. The above results evidence the unexpected synergistic advantages of the controlled release microsphere encapsulated formulation.

[0188] Further experiments with calcium channel blockers injected into osteoarthritis patient’s joints are disclosed in U.S. Pat. No. 7,767,710, with examples 1 – 9 from that document incorporated herein by reference. Example 2: Spray dried dispersion (SDD) Formulations for Providing Extended Release of Verapamil in Synovial Fluid

[0189] Active loading, PLGA molecular weight, and PLGA hydrophobicity (lactide:glycolide (L:R) ratio,) were identified as the appropriate handles for tuning verapamil release kinetics. Three grades of PLGA were used to produce 10% and 20% active dispersions in the first round of SDD manufacture. The glass transition temperature (Tg) of each prototype batch was measured between 40-50°C. Phase separation and particle fusion / agglomeration should be considered in manufacture, storage, and aqueous suspensions of SDD. Verapamil free base is a liquid at room temperature and the HCl salt is unlikely to recrystallize from a dilute environment, such as an amorphous dispersion.

[0190] The SDDs exhibited pH dependent dissolution kinetics across a biorelevant range in aqueous media, which can be attributed to the pH dependent solubility of verapamil. Variable release should be expected in vivo and buffer composition and suspension hold times are likely to have a significant impact on free verapamil levels prior to administration. Two-week dissolution results indicated that active loading and PLGA molecular weight had the most profound impact on rate of verapamil release, with L:R ratio having a lesser influence. Most formulations exhibited biexponential kinetics, which suggests contributions from 2 separate populations of drug to the overall release profile. The most likely scenario is fast release from drug at the particle surface and slow release from embedded drug. Efforts were also directed to balancing initial burst and release rate of the slow population.

[0045] 44  \\4154‐0697‐7112  v4

[0191] Four additional SDDs were produced in a second round of manufacture. Challenges in collecting material from the dryer were encountered during manufacture of a 30% active dispersion, as well as a subsequent 20% active dispersion - targeting larger particle size. Each of the second round SDDs exhibited particle surface fusion. While each of the second round SDDs was secondary dried below the ICH limit for acetone, a 3-fold reduction in acetone content was achieved by tray drying at 40°C / 15%RH relative to vacuum drying at 30°C. Based on the experiments, 20%A, 75:25 L:G, 4- 15 kDa PLGA SDD is a preferred SDD formulation. In vitro dissolution results provide evidence for successful performance in vivo.

[0192] API Thermal Properties: A melt-quench experiment was performed to determine the melting point (Tm), and glass transition temperature (Tg) of verapamil HCl. Crystalline verapamil HCl was heated through the melt at 5°C / min before quenching the molten API in liquid nitrogen. The Tg was then measured using a heating rate of 2.5°C / min with ± 1.5°C / min modulation. Experimental results are listed in FIG.2.

[0193] Verapamil HCl exhibits a moderate Tm / Tg and the free base is a liquid at room temperature. Recrystallization of the HCl salt from a dilute environment, such as an amorphous dispersion, is anticipated to be low risk.

[0194] Given the low Tg of verapamil HCl, low Tm of the free base, and low reported values for Tg of PLGA (lit. 40-60°C), particle fusion / agglomeration is anticipated to be a risk during manufacture, on storage, and in aqueous suspensions of spray-dried dispersion (SDD), as even partial conversion to the free base could be problematic. Example 3: API and Polymer Organic Solubility

[0195] Solubility of verapamil HCl and selected grades of PLGA were evaluated in solvents commonly used for spray drying. Measurements were made by visual observation after mixing overnight at 25°C.

[0196] As shown in FIG. 3, all materials exhibited adequate solubility for manufacture of low loading SDDs from acetone. While the 75:25 L:G, 4-15 kDa grade of PLGA was not evaluated directly, it's solubility is expected to be greater than or equal to the higher molecular weight analog.

[0046] 45  \\4154‐0697‐7112  v4      Example 4: Aqueous Solubility

[0197] Aqueous solubility of verapamil was measured as a function of pH in phosphate- buffered saline (PBS). The experimental pH range was selected based on literature values for synovial fluid and previously used suspension vehicles. Solutions of verapamil HCl were prepared at 4 mgA / mL (free base basis) and allowed to mix overnight at 37°C. Dissolved concentrations were measured by HPLC after centrifugation at 386,000 RCF. Experimental results are shown in FIG.4 and FIG.5.

[0198] Solubility trends with pH were as expected for a basic compound with pKa of 8.46. A theoretical fit to the data suggests that the prepared sample concentration was too low for accurate solubility determination at pH 6.5.

[0199] While the release rate from spray dried particles was expected to be controlled primarily by PLGA, solubility of verapamil is likely to be a contributing factor. This could have implications for release in vivo with patient-to-patient variability in synovial fluid pH. Composition of suspension vehicles was also carefully considered as acceptable hold times suggested high dependence on pH. Example 5: SDD Formulation Selection

[0200] The primary goal in verapamil SDD formulation development is to provide extended drug release through 6 months in synovial fluid. Active loading, PLGA molecular weight, and PLGA hydrophobicity (lactide:glycolide or L:G ratio) were identified as the appropriate handles for initial screening. Six prototype SDD formulations were selected for manufacture. Their compositions and rationale for selection are listed in FIG.6. Example 6: SDD Manufacturing

[0201] The six initial formulations were sprayed on a Bend Lab Dryer with 35 kg / hr drying gas capacity (BLD-35). The chosen solvent system is 100% acetone, which is a good solvent for the dispersion polymer as well as the API. Solids concentration was kept constant at 4wt% total solids for all formulations. Spray dryer thermodynamics (solution flow rate, outlet temperature, drying gas flow rate) and atomization conditions (nozzle choice and nozzle pressure) were chosen based on a combination

[0047] 46  \\4154‐0697‐7112  v4      of historical data (not included) and the thermal properties of the ingoing API and polymer, with the goal of achieving sufficient droplet drying in the spray chamber and production of an amorphous solid material at the chamber outlet. After spray dying, the SDDs were secondary dried in a vacuum tray dryer to remove residual solvent. Good yields were achieved for all six formulations.

[0202] Appearance: All of the SDD formulations were white in color. Varying levels of aggregation or clumping was also observed.

[0203] SEM: All prototype SDD batches exhibit mostly spherical particle morphology (FIG. 7). Larger, collapsed spheres were observed in formulations containing the highest molecular weight polymer. Formulations containing the lowest molecular weight polymer appear to have an appreciable number of particles < 10 μm in diameter. Evidence of strings or crystalline material was only observed in the 10%A SDD containing 50:50 L:G PLGA (FIG.8).

[0204] Particle Size Distribution: A standard dry Malvern method was not viable for measuring the particle size distribution of the SDDs. A standard pressure titration process for determining method dispersive pressure was not achievable due to the aggregation behavior of the SDD material. Large pieces of material did not properly convey in the sample feed and were observed sticking along the feed component surface. Measured distributions were not repeatable and heavily skewed due to the large, non-dispersed agglomerates. A wet Malvern method approach was suggested. Suspending the SDD material in an anti-solvent may lead to more favorable particle dispersion and more measurable particle size. An initial wet Malvern method was developed during analysis of the second round of SDD formulation, as described below.

[0205] DSC: Modulated differential scanning calorimetry (mDSC) was used to measure glass transition temperatures of the verapamil SDDs. When SDDs are stored at a condition that is well below the Tg (ideally 20°C or more), mobility in the glass is very low and phase separation and crystallization do not typically occur over relevant time scales. Recrystallization of verapamil HCl is unlikely to occur from amorphous dispersions.

[0048] 47  \\4154‐0697‐7112  v4

[0206] As shown in FIG. 9, the dry Tg of each SDD and ingoing polymer was measured between 40-50°C. Any exposure to moisture could result in further depression of Tg. Long-term storage of the formulations should preferably include temperature control and protection from moisture to help mitigate risk to physical stability. Representative thermograms for each SDD are shown in FIG.10. Example 7: Powder X-Ray Diffraction

[0207] The SDDs were evaluated for crystallinity by powder x-ray diffraction (PXRD). Representative diffractograms can be found in FIG.11. While each SDD is likely be amorphous, some evidence of crystalline material was detected in the 10%A dispersion with 50:50 L:G, 30-60 kDa PLGA. Small peaks observed at 6 and 19 two- theta are not consistent with verapamil HCl and may explain the unexpected crystalline material observed in this sample by SEM. Example 8: Assay and Related Substances

[0208] The SDDs were evaluated for assay and related substances. Assay was measured to be on target for each of the prototype formulations. Impurity profiles of the 20% active SDDs were largely in-line with the in-going API. The 10% active SDDs contain unknown impurities at relative retention time (RRT) 0.11 and RRT 0.76 with levels that trend with order of manufacture. This observation, along with the absence of these unknowns in the second round of SDDs, suggest that RRT 0.11 and RRT 0.76 are not active pharmaceutical ingredient (API) related. It's possible that these impurities are tied to the unexpected crystalline material detected in the specific batch of 10%A, 50:50 L:G, 30-60 kDa PLGA formulation by SEM and PXRD. Example 9: Drug Release as a Function of pH

[0209] The impact of pH on initial rate of release from verapamil SDDs was studied to better understand mechanism of release, expected variance in vivo, and acceptable suspension compositions / hold times. Sample solutions were prepared at 200 μgA / mL in PBS between pH 6.5 and 8.0 and placed on a nutating mixer at 37°C. Based on previous solubility measurements, all samples were evaluated under sink

[0049] 48  \\4154‐0697‐7112  v4      conditions. At selected timepoints, the concentration of dissolved verapamil was determined after centrifugation at 386,000 RCF.

[0210] As shown in FIGs 12A, 12B, and 13, the initial rate of release is dependent on verapamil solubility (pH), active loading, and grade of PLGA in the SDDs. The pH dependency suggests variable release should be expected in vivo and that buffer composition and suspension hold times are likely to have a significant impact on free verapamil levels prior to administration, and one or both will preferably be adjusted as required by the needs of the situation.

[0211] It appears that verapamil release is faster than desired for most of the prototype formulations. The 10%A, 75:25 L:G, 66-107 kDa PLGA formulation would be most likely to achieve the target release profile if zero order kinetics were maintained through six months. This formulation’s release is also relatively stable in terms of pH change when compared to the other formulations. Example 10: Extended Sink Dissolution

[0212] Dissolution of verapamil SDDs was evaluated through 2 weeks in PBS at pH 7.4 with and without 0.2% sodium hyaluronate. The pH of the dissolution media was selected based on simulated synovial fluid. The selected concentration of hyaluronic acid falls between values of levels measured in patients with and without osteoarthritis. Experimental details were otherwise consistent with those described in the section above. It should be noted that the dissolved concentration was measured after centrifugation at 15,800 RCF for the 3, 7, and 14 day timepoints due to equipment limitations.

[0213] Results of the 2-week dissolution test are displayed in FIGs.14A and 14B. In general, release profiles of the prototype SDDs were in line with expectations. Active loading and PLGA molecular weight appear to have the most profound impact on rate of verapamil release with lactide:glycolide ratio having a lesser influence. Most formulations exhibit biexponential kinetics, which suggests contributions from 2 separate populations of drug to the overall release profile. The most likely scenario is fast release from drug at the particle surface and slow release from embedded drug.

[0050] 49  \\4154‐0697‐7112  v4      Note that the 20%A, 50:50 L:G, 30-60 kDa PLGA formulation shows some discontinuity in release between 7 and 14 days.

[0214] Release profiles were projected through 6 months by deriving rate constants for each drug population (fast and slow) from biexponential fits to experimental data. Projections are shown in FIGs. 15A and 15B and the fits used to generate each projection can be found in FIGs. 16A – 16C. Samples with poor fits were excluded from the projections. Balance between initial burst and release rate of the slow population should preferably be considered during formulation optimization. Example 11: SDD Formulation Selection

[0215] Four additional verapamil SDDs were manufactured in an effort to optimize formulation performance and select a lead for progression to scale-up activities. The composition of each dispersion and rationale for selection is listed in FIG.17.

[0216] SDD Manufacturing Summary: The four additional formulations were sprayed at Lonza on a Bend Lab Dryer with 35 kg / hr drying gas capacity (BLD-35). The chosen solvent system was maintained as 100% Acetone. Solids concentration was maintained at 4wt% total solids for three of the additional formulations. Solids concentration was increased to 6wt% for the re-manufacture of the potential lead formulation to increase dispersion particle size.

[0217] Spray dryer thermodynamics (solution flow rate, outlet temperature, drying gas flow rate) and atomization conditions (nozzle choice and nozzle pressure) were chosen for the first three additional formulations based on the initial round of feasibility spray drying. The selected thermodynamics were modified (outlet temperature, nozzle choice, and nozzle pressure) for the re-manufacture of the potential lead formulation to achieve lower residual solvent and an increase in dispersion particle size.

[0218] After spray dying, the first three additional formulation SDDs were secondary dried in a vacuum tray dryer to remove residual solvent. Humidified tray drying was chosen for the remanufacture SDD to achieve lower residual solvent.

[0219] Good yields were achieved for the first two formulations. Yields were not optimal for the second two formulations. Material was observed accumulating within a coupler that joins the material collection container to the bottom of the cyclone within the

[0051] 50  \\4154‐0697‐7112  v4      process train for the second two formulations. This material remained in the coupler for the third formulation and was manually extracted for the fourth formulation.

[0220] Appearance: The round 2 SDDs were evaluated for appearance and representative images can be found in FIG. 18 While each of the round 2 SDD samples contained white powder, significant clumping was observed in the 30%A, 75:25 L:G, 66-107 kDa PLGA SDD as well as the new batch of 20%A, 75:25 L:G, 4-15 kDa PLGA SDD.

[0221] SEM: Particle morphology of the round 2 SDDs was evaluated by SEM and representative images can be found in FIG.19. Both the 15%A, 75:25 L:G, 4-15 kDa PLGA and 25%A, 75:25 L:G, 66-107 k Da PLGA SDDs displayed mostly spherical morphology. Several instances of particle surface fusion were observed in the other two dispersions, which may be related to the previously described clumping. The images indicate that efforts to increase particle size of the 20%A, 75:25 L:G, 4-15 kDa PLGA SDD were successful.

[0222] Particle Size Distribution: A wet Malvern method was developed for measuring particle size of the second round SDDs. Particle size measurements of the second round SDDs using this method are shown in FIG. 20. Aggregates or agglomerates of material larger than 100 μm were observed despite the switch to the wet Malvern method, likely an artifact of the material collection issues observed during second round manufacture. True individual particle size distributions are likely smaller than 100 μm, but not directly measurable using this method due to the strong aggregation or agglomeration behavior. Further assessment of a particle size measurement approach is recommended.

[0223] DSC: Thermal properties of the round 2 SDDs were determined by mDSC. As shown in FIG. 21, measured Tgs were in the same range as values obtained for the first round of formulations. Variance in Tg between batches of 20%A, 75:25 L:G, 4-15 kDa PLGA SDD manufactured in rounds 1 and 2 is likely the result of differences in residual solvent content.

[0224] PXRD: The round 2 SDDs were evaluated for crystallinity by PXRD. No evidence of crystalline material was detected.

[0052] 51  \\4154‐0697‐7112  v4      Example 12: Round 2 Assay and Related Substances

[0225] The round 2 SDDs were evaluated for assay and related substances. Assay values were on target for each formulation and impurity profiles were consistent with the in- going API. Example 13: Round 2 Extended Sink Dissolution

[0226] Test conditions used for sink dissolution of the round 2 SDDs were similar to those used for the initial round of formulations. In round 2, sample solutions were vortexed, briefly, before placing on a nutating mixer and the dissolved concentration was measured after centrifugation at 386,000 RCF for each timepoint. The 20%A, 75:25 L:G, 4-15 kDa PLGA SDD from round 1 was included for control in the dissolution test. A notable increase in initial burst was observed in the control with vortexing (FIG. 22). Expectations for improved wetting in suspension and the impact on initial burst should be considered in selecting lead SDD formulations.

[0227] Dissolution results for the round 2 SDDs are shown in FIG.23. Projections for release through 6 months and fits to the experimental data can be found in FIGs. 24 and 25. Increasing particle size does not appear to have a substantial impact on release rate, as minimal differences in dissolution were observed between the two batches of 20%A, 75:25 L:G, 4-15 kDa PLGA SDD. Likewise, the projected release was not significantly altered by reducing the active loading from 20%A to 15%A. Undesirable release profiles were obtained for 25%A and 30%A dispersions in the high MW polymer.

[0228] Lead Formulation Selection: The 20%A, 75:25 L:G, 4-15 kDa PLGA SDD is recommended as the lead SDD formulation. In vitro dissolution results show evidence for adequate performance in vivo. While SDD stability has yet to be evaluated, long- term storage is anticipated to require temperature control and protection from moisture to mitigate risk to physical stability.

[0053] 52  \\4154‐0697‐7112  v4

Claims

CLAIMS 1. A pharmaceutical composition comprising active pharmaceutical ingredient (API) encapsulated poly(lactic-co-glycolic acid) (PLGA) microspheres, wherein the composition comprises at least about 10% active active pharmaceutical ingredient (API) (wt%), a lactide:glycolide (L:G) ratio of 75:25, wherein the weight of the microspheres is between about 4 to about 15 kDa, and wherein the pharmaceutical composition is a spray dried dispersion (SDD).

2. The pharmaceutical composition of claim 1, wherein the composition comprises at least about 20% active API (wt%).

3. The pharmaceutical composition of any one of the previous claims, wherein the active API is a calcium channel regulator.

4. The pharmaceutical composition of claim 3, wherein the calcium channel regulator is amlodipine, bepridil, diltiazem, hypochloride, felodipine, gallopamil, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, devapamil, emopamil, or a combination thereof.

5. The pharmaceutical composition of claim 3 or 4, comprising a hydrochloride salt of the calcium channel regulator.

6. The pharmaceutical composition of any one of claims 3-5, wherein the calcium channel regulator is verapamil.

7. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises about 20% active API (wt%), and wherein the active API is verapamil.

8. A method for treating arthritis in a patient in need thereof, comprising administering to the patient an effective dose of a pharmaceutical composition of any one of claims 1-7.

9. The method of claim 8, wherein the arthritis is osteoarthritis.53  \\4154‐0697‐7112  v4     10. The method of any one of claims 8-9, wherein the administering is by injection into an intraarticular space of a joint of the patient.

11. The method of claim 10, wherein the joint is a hip joint, a joint of a foot, a joint of an ankle, a knee joint, a joint of a hand, a joint of a spine, a shoulder joint, or a combination thereof.

12. The method of any one of claims 8-11, wherein the patient is administered a single injection.

13. The method of any one of claim 8-11, wherein the injection comprises the API in solution and the API in microspheres.

14. The method of claim 13, wherein the injection comprises between about 0.5 mg to about 2.0 mg of API in solution.

15. The method of claim 13 or 14, wherein the injection comprises at least about 32 mg of API in microspheres.

16. The method of any one of claims 8-15, wherein the composition further comprises a vehicle appropriate for injection into the human body.

17. The method of claim 16, wherein the vehicle include physiological saline.

18. The method of any one of claims 8-17, wherein the API is released from the microspheres for a duration of at least three months.

19. The method of claim 18, wherein the duration is between about three to about six months.

20. The method of claim 18 or 19, wherein the duration is about six months.54  \\4154‐0697‐7112  v4     21. The method of any one of claims 8-20, wherein the pharmaceutical composition delivers at least about 32 mg of API over about six months.

22. The method of any one of claims 8-20, comprising a single injection of about 0.5-2.0 mg of verapamil in solution and the pharmaceutical composition comprising about 32 mg of verapamil encapsulated PLGA microspheres, into an intraarticular space of a joint of the patient, wherein the pharmaceutical composition is delivered over about six months.55  \\4154‐0697‐7112  v4