WO2020102323A1

WO2020102323A1 - Sustained-release pharmaceutical compositions comprising a therapeutic agent for treating diseases due to reduced bone density or cartilage loss and uses thereof

Info

Publication number: WO2020102323A1
Application number: PCT/US2019/061143
Authority: WO
Inventors: Keelung Hong; Hao-Wen Kao; Yi-Yu Lin; Jonathan FANG; Weiwei FANG
Original assignee: Taiwan Liposome Co Ltd; TLC Biopharmaceuticals Inc
Current assignee: Taiwan Liposome Co Ltd; TLC Biopharmaceuticals Inc
Priority date: 2018-11-14
Filing date: 2019-11-13
Publication date: 2020-05-22
Anticipated expiration: 2021-05-14
Also published as: JP7431419B2; EP3880176A1; JP2023510658A; EP3880176A4; TW202031247A; CN113056259A; TWI729562B

Abstract

The present invention relates to a pharmaceutical composition comprising at least one liposome and a therapeutic agent for treating a disease due to reduced bone density or cartilage loss with a high drug to lipid ratio and encapsulation efficiency. The pharmaceutical composition improves the pharmacokinetic profile and sustains the release of the therapeutic agent. Also provided is the method for treating a disease due to reduced bone density or cartilage loss using the pharmaceutical composition disclosed herein.

Description

SUSTAINED-RELEASE PHARMACEUTICAL COMPOSITIONS COMPRISING A THERAPEUTIC AGENT FOR TREATING DISEASES DUE TO REDUCED BONE DENSITY OR CARTILAGE LOSS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Application No. 62/767,254, filed on November 14, 2018, the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention is directed to a sustained-release pharmaceutical composition for treating diseases due to reduced bone density or cartilage loss, with a high drug to lipid ratio and a high encapsulation efficiency using at least one trapping agent. The high drug to lipid ratio, high drug encapsulation efficiency and sustained release profile of the pharmaceutical composition reduce the frequency of drug administration, increase patient compliance and improve the therapeutic outcome.

BACKGROUND

[0003] Bone remodeling is a physiological process determined by the sequential and coordinated interaction involving osteoclasts and osteoblasts, as well as osteocytes, inflammatory cells and mediators. The balance between osteoblasts and osteoclasts activity maintains the bone homeostasis. Functional disorders of osteoclasts increase bone resorption and causes various bone and joint diseases, for instance, osteoporosis, osteopetrosis, rheumatoid arthritis, osteoarthritis, bone tumor and Paget’s bone disease.

[0004] Cathepsin K, a cysteine protease expressed at high levels in osteoclasts, plays crucial roles in degradation of bone matrix composed of hydroxyapatite and protein, especially type I collagen. Cathepsin K is also involved in the cleavage of type II collagen in human articular cartilage. Recent studies show that once or twice daily oral administration of cathepsin K inhibitors prevent both bone loss and cartilage degeneration. It is highly desirable to maintain the therapeutic concentration of a cathepsin K inhibitor and minimize the frequency of administration to treat diseases due to reduced bone density and cartilage loss.

[0005] Liposomes as a drug delivery system has been widely used for developing sustained- release formulations for various drugs. Drug loading into liposomes can be attained either passively (the drug is encapsulated during liposome formation) or remotely/actively (creating a transmembrane pH- or ion-gradient during liposome formation and then the drug is loaded by the driving force generated from the gradients after liposome formation) (US Patent No. 5,192,549 and 5,939,096). Although the general method of drug loading into liposomes is well documented in the literature, only a small number of therapeutic agents were loaded into liposomes with high encapsulation efficiency. This is because various factors can affect the drug to lipid ratio and encapsulation efficiency of a liposomal drug, including but not limited to, the physical and chemical properties of the therapeutic agent, for example, hydrophilic/hydrophobic characteristics, dissociation constant, solubility and partition coefficient, lipid composition, trapping agent, reaction solvent, and particle size (Proc Natl Acad Sci U S A. 2014; 111(6): 2283-2288 and Drug Metab Dispos. 2015; 43 (8): 1236-45).

[0006] There remains an unmet need for a sustained-release formulation with a high drug to lipid ratio and high drug encapsulation efficiency to reduce dosing frequency of cathepsin K inhibitors and improve therapeutic outcome. The present invention addresses this need and other needs.

SUMMARY OF THE INVENTION

[0007] In one embodiment, a sustained release pharmaceutical composition comprises (a) at least one liposome comprising a bilayer membrane, said bilayer membrane comprises at least one lipid; (b) a trapping agent; and (c) a therapeutic agent for treating diseases due to reduced bone density or cartilage loss, wherein the molar ratio of the therapeutic agent to the lipid is equal to or higher than about 0.1 is provided.

[0008] According to another embodiment of the present invention, methods are provided for treating a disease due to reduced bone density or cartilage loss, comprising the steps of administering the pharmaceutical composition described herein to a subject in need thereof.

[0009] Also provided are the uses of the pharmaceutical composition described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of diseases due to reduced bone density.

[0010] Further provided is a medicament for treating a disease due to reduced bone density or cartilage loss, comprising a therapeutically effective amount of the sustained pharmaceutical composition described herein.

[0011] The terms“invention,”“the invention,”“this invention” and“the present invention” used in this patent are intended to refer broadly to all of the subject matter of this patent and the patent claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Embodiments of the invention covered by this patent are defined by the claims below, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all drawings and each claim.

[0012] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a line graph showing the plasma L-006235 concentration in rats after intraarticular injection of free L-006235 and liposomal L-006235.

DETAILED DESCRIPTION OF THE INVENTION

[0014] As employed above and throughout the disclosure, the following terms, unless otherwise herein, the singular forms“a,”“an” and“the” include the plural reference unless the context clearly indicates otherwise.

[0015] All numbers herein may be understood as modified by“about.” As used herein, the term“about” refers to a range of ± 10% of a specified value.

[0016] An “effective amount” as used herein, refers to a dose of the pharmaceutical composition described herein to reduce the symptoms and signs of diseases due to bone resorption and reduced bone density, such as decreased/increased bone mass, loss of subchondral bone or cartilage, joint pain or joint swelling in arthritis. The term“effective amount” and“therapeutically effective amount” are used interchangeably.

[0017] The term“treating,”“treated,” or“treatment” as used herein includes preventative (e.g. prophylactic), palliative, and curative methods, uses or results. The terms“treatment” or “treatments” can also refer to compositions or medicaments. Throughout this application, by treating is meant a method of increasing bone density or reducing cartilage loss and hence, reducing or delaying one or more symptoms or signs of diseases due to reduced bone density or cartilage loss or the complete amelioration of diseases due to reduced bone density or cartilage loss as detected by art-known techniques. Art recognized methods are available to detect diseases due to reduced bone density or cartilage loss and its symptoms. These include, but are not limited to, clinical examination, imaging (for example, bone mineral densitometry, X rays, dual-energy X-ray absorptiometry, magnetic resonance imaging, computed tomography, ultrasound and nuclear imaging), measurement of biomarkers (for example, detection of C- reactive protein, anti-cyclic citrullinated peptide, serum alkaline phosphatase, creatine kinase BB isoenzyme, tartrate-resistant acid phosphatase, matrix metalloproteinase-3, C-terminal telopeptide of type I collagen, C-telopeptide of type II collagen, N-terminal telopeptide of type I collagen, N-terminal propeptide of collagen IIA and serum hyaluronan) in body fluid (for example, serum, urine or synovial fluid) or biopsy/histopathological evaluation (for example, cartilage and subchondral bone staining), to name a few. For example, a disclosed method is considered to be a treatment if there is at least 1% increase in bone density, as measured by bone mineral densitometry, or about a 1% reduction in one or more symptoms of the disease due to reduced bone density or cartilage loss in a subject when compared to the subject prior to treatment or control subjects. Thus, the reduction can be about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.

[0018] The term“disease due to reduced bone density or cartilage loss” as used herein, encompasses a variety of types and subtypes of bone and joint diseases due to bone resorption or cartilage degradation of various etiologies and causes, either known or unknown, including, but not limited to, osteoporosis, osteopetrosis, rheumatoid arthritis, osteoarthritis, bone tumor, bone fracture, and Paget’s bone disease.

[0019] The term“subject” can refer to a vertebrate having or at risk of developing a disease due to reduced bone density or cartilage loss or to a vertebrate deemed to be in need of treatment to increase bone density and/or repair cartilage. Subjects include all warm-blooded animals, such as mammals, such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

[0020] Liposome

[0021] The terms“liposome,”“liposomal” and related terms as used herein are characterized by an interior aqueous space sequestered from an outer medium by one or more bilayer membranes forming a vesicle. In certain embodiments, the interior aqueous space of the liposome is substantially free of a neutral lipid, such as triglyceride, non-aqueous phase (oil phase), water-oil emulsions or other mixtures containing non-aqueous phase. Non-limiting examples of liposomes include small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), and multi-lamellar vesicles (MLV) with an average diameter ranges from 50-500 nm, 50-450 nm, 50-400 nm, 50-350 nm, 50-300 nm, 50-250 nm, 50-200 nm, 100-500 nm, 100-450 nm, 100-400 nm, 100-350 nm, 100-300 nm, 100-250 nm or 100-200 nm.

[0022] Bilayer membranes of liposomes are typically formed by at least one lipid, i.e. amphiphilic molecules of synthetic or natural origin that comprise spatially separated hydrophobic and hydrophilic domains. Examples of lipid, including but not limited to, dialiphatic chain lipids, such as phospholipids, diglycerides, dialiphatic glycolipids, single lipids such as sphingomyelin and glycosphingolipid, and combinations thereof. Examples of phospholipid according to the present disclosure include, but not limited to, 1 ,2-dilauroyl-,v/7- glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-.s77-glycero-3-phosphocholine (DMPC), 1 ^-dipalmitoyl-sn-glycero-S -phosphocholine (DPPC) , 1 -palmitoyl-2-stearoyl-.s/7-glycero-3- phosphocholine (PSPC), 1 -palmitoyl-2-oleoyl-.s77-glycero-3-phosphatidylcholine (POPC), 1,2- distearoyl-.s77-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoy 1 -,s77-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1 ,2-dimyristoyl-.s77-glycero-3- phospho-(l’-rac-glycerol) (sodium salt) (DMPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(r- rac-glycerol) (sodium salt) (DPPG), 1 -palmitoyl-2-stearoyl-.s7/-glycero-3-phospho-( 1 -rac- glycerol) (sodium salt) (PSPG), 1 ,2-distearoyl-,s77-glycero-3-phospho-( 1 -rac-glycerol ) (sodium salt) (DSPG), 1 ,2-dioleoyl-,s77-glycero-3-phospho-( 1 -rac-glycerol ) (DOPG), 1,2- dimyristoyl-.s77-glycero-3-phospho-L-serine (sodium salt) (DMPS), 1 ,2-dipalmitoyl-,s77- glycero-3-phospho-L-serine (sodium salt) (DPPS), 1 ,2-distearoyl-,s77-glycero-3-phospho-L- serine (sodium salt) (DSPS), 1 ,2-dioleoyl-.s77-glycero-3-phospho-L-serine (DOPS), 1,2- dimyristoyl-.s77-glycero-3-phosphate (sodium salt) (DMPA), 1 ,2-dipalmitoyl-.s77-glycero-3- phosphate (sodium salt) (DPPA), 1 ,2-distearoyl-OT-glycero-3 -phosphate (sodium salt) (DSPA), 1 ,2-dioleoyl-,s77-glycero-3-phosphate (sodium salt) (DOPA), 1 ,2-dipalmitoyl-.s77-glycero-3- phosphoethanolamine (DPPE), /V-(carbonyl-methoxypolyethyleneglycol)- 1 ,2-dipalmitoyl-.s77- glycero-3-phosphoethanolamine (PEG-DPPE), 1 -palmitoyl-2-oleoyl-.s77-glycero-3- phosphoethanolamine (POPE), 1 ,2-distearoyl-.s77-glycero-3-phosphoethanolamine (DSPE), N- (carbonyl-methoxypolyethyleneglycol )- 1 ,2-distearoyl-,s77-glycero-3-phosphoethanolamine (PEG-DSPE), 1 ,2-dioleoy l-.s77-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dipalmitoyl-,s77- glycero-3-phospho-(r-myo-inositol) (ammonium salt) (DPPI), 1 ,2-distearoyl-.s77-glycero-3- phosphoinositol (ammonium salt) (DSPI), l,2-dioleoyl-5^,n-glycero-3-phospho-(r -myo inositol) (ammonium salt) (DOPI), cardiolipin, L-a-phosphatidylcholine (EPC), and L-a- phosphatidylethanolamine (EPE). In some embodiments, the lipid is a lipid mixture of one or more of the foregoing lipids, or mixtures of one or more of the foregoing lipids with one or more other lipids not listed above, membrane stabilizers or antioxidants. [0023] In some embodiments, the mole percent of the lipid in the bilayer membrane is equal or less than about 80, 79, 78, 77, 76, 75, 74, 73, 72, 71,70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45 or any value or range of values therebetween (e.g., about 45-80%, about 45-75%, about 45-70%, about 45-65%, about 50-80%, about 50-75%, about 50-70% or about 50-65%).

[0024] In some embodiments, the lipid of the lipid bilayer is a mixture of a first lipid and a second lipid. In some embodiments, the first lipid is selected from the group consisting essentially of phosphatidylcholine (PC), HSPC, DOPC, POPC, DSPC, DPPC, DMPC, PSPC and combination thereof and the second lipid is selected from the group consisting essentially of a phosphatidylethanolamine, phosphatidylglycerol, PEG-DSPE, DPPG, DOPG and combination thereof. In other embodiments, the mole percent of the first lipid in the bilayer membrane is equal or less than about 79.9, 79.5, 79.1, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44,

43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30 or any value or range of values therebetween (e.g., about 30-79.9%, about 30-79.5%, about 30-79.1%, about 30-70%, about 35-65% or about 40-60%) and the mole percent of the second lipid in the bilayer membrane is equal or higher than 0.1 or 0.5 to less than about 25, 24, 23 or any value or range of values therebetween (e.g., about 0.1-25%, about 0.1-24%, about 0.1-23%, about 0.5-25%, about 0.5-24%, about 0.5-23%, about 0.7-25%, about 0.7-24% or about 0.7-23%).

[0025] Bilayer membranes of liposomes further comprise less than about 55 mole percentage of steroids, preferably cholesterol. In certain embodiments, the mole % of cholesterol in the bilayer membrane is about 20-55%, about 20-50%, about 20-45%, about 25-55%, about 25- 50%, about 25-45%, about 30-55%, about 30-50% or about 30-45%.

[0026] In one exemplary embodiment, the mole % of the lipid and cholesterol in the bilayer membrane is about 45-80%: 20-55% or 50-75%: 25-50%. In another exemplary embodiment, the mole % of the first lipid, the second lipid and cholesterol in the bilayer membrane is about 30-79.9%: 0.1%-25%: 20-55%, 30-75%: 0.1-25%: 20-50% or 35-70%: 0.5-25%: 20-45% and the first phospholipid is HSPC and the second phospholipid is DSPE-PEG2000.

[0027] Remote Loading

[0028] The term“remote loading” as used herein is a drug loading method which involves a procedure to transfer drugs from the external medium across the bilayer membrane of the liposome to the interior aqueous space by a polyatomic ion-gradient. Such gradient is generated by encapsulating at least one polyatomic ion as a trapping agent in the interior aqueous space of the liposome and replacing the outer medium of the liposome with an external medium with a lower polyatomic ion concentration, for example, pure water, sucrose solution or saline by known techniques, such as column separation, dialysis or centrifugation. A polyatomic ion gradient is created between the interior aqueous space and the external medium of the liposomes to trap the therapeutic agent in the interior aqueous space of the liposomes. Exemplary polyatomic ion as trapping agents include, but are not limited to, sulfate, sulfite, phosphate, hydrogen phosphate, molybdate, carbonate and nitrate. Exemplary trapping agents include, but are not limited to, ammonium sulfate, ammonium phosphate, ammonium molybdate, ammonium sucrose octasulfate, triethylammonium sucrose octasulfate, dextran sulfate, or a combination thereof.

[0029] In an embodiment, the concentration of triethylammonium sucrose octasulfate is about 10 to about 200 mM, about 50 to about 150 mM or about 60 to about 100 mM. In another embodiment, the concentration of ammonium sulfate is about 100 to about 600 mM, about 150 to about 500 mM or about 200 to about 400 mM.

[0030] In accordance with the invention, the liposome encapsulating a trapping agent can be prepared by any of the techniques now known or subsequently developed. For example, the MLV liposomes can be directly formed by a hydrated lipid film, spray-dried powder or lyophilized cake of selected lipid compositions with trapping agent; the SUV liposomes and LUV liposomes can be sized from MLV liposomes by sonication, homogenization, microfluidization or extrusion.

[0031] Pharmaceutical Compositions

[0032] The present invention is directed to a sustained release pharmaceutical composition, comprising (a) at least one liposome comprising a bilayer membrane; (b) a trapping agent; and (c) a therapeutic agent for treating disease due to reduced bone density or cartilage loss, wherein the bilayer membrane comprises at least one lipid and the molar ratio of the therapeutic agent to the lipid is above or equal to about 0.1.

[0033] In one embodiment, the sustained release pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, diluent, medium for an active ingredient, a preservative, cryoprotectant or a combination thereof. In one exemplary embodiment, the weight percent of the bilayer membrane is about 0.1-15%; the weight percent of the trapping agent is about 0.1-12%; and the weight percent of the pharmaceutically acceptable excipient (such as sucrose, histidine, sodium chloride and ultrapure water), diluent, medium for the active ingredient, a preservative, cryoprotectant or a combination thereof is about 75.0-99.9%. [0034] In certain embodiments, the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is a cathepsin K inhibitor. Non-limiting examples of cathepsin K inhibitor include balicatib (C23H33N5O2), odanacatib (C25H27F4N3O3S), L-006235

(C24H30N6O2S), ONO-5334 (C21H34N4O4S), MIV-711 and relacatib (C27H32N4O6S). In another embodiment, the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is non-water soluble or hydrophobic. The sustained release profile of the pharmaceutical composition prolongs the half-life, the therapeutic concentration and the duration of action of the therapeutic agent for treating a disease due to reduced bone density or cartilage loss, and hence, sustains the therapeutic effect and reduces the administration frequency of the therapeutic agent.

[0035] In one aspect, the sustained release profile of the pharmaceutical composition is due to a high drug encapsulation efficiency. The encapsulation efficiency of the pharmaceutical composition is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0036] In another aspect, the sustained release profile of the pharmaceutical composition is due to the higher therapeutic agent to lipid molar ratio. In an exemplary embodiment, the molar ratio of the therapeutic agent for treating a disease due to reduced bone density or cartilage loss to the one or more lipids is above or equal to about 0.1, alternatively above or equal to about 0.1 to less than about 20, less than about 15, less about 10, less than about 5, less than about 4, less than about 3, less than about 2 or less than about 1.5.

[0037] In yet another aspect, the half-life of the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is extended by at least 2-fold, at least 5-fold, at least 7.5- fold, at least 10-fold, or at least 20-fold compared to that of a free therapeutic agent for treating a disease due to reduced bone density or cartilage loss.

[0038] The invention also provides methods of treating a disease due to reduced bone density or cartilage loss, comprise the administration of an effective amount of the pharmaceutical composition as described herein to a subject in need thereof, whereby the symptoms and/or signs of the disease due to reduced bone density or cartilage loss in the subject are reduced.

[0039] The pharmaceutical composition is formulated to be suitable for injection, such as intraarticular, subcutaneous, subdermal, intradermal, transdermal or intramuscular route. The pharmaceutical composition is also formulated to be administered as a transdermal patch.

[0040] The dosage of the pharmaceutical composition of the present invention can be determined by the skilled person in the art according to the embodiments. Unit doses or multiple dose forms are contemplated, each offering advantages in certain clinical settings. According to the present invention, the actual amount of the pharmaceutical composition to be administered can vary in accordance with the age, weight, condition of the subject to be treated, any existing medical conditions, and on the discretion of medical professionals.

[0041] In one embodiment, the pharmaceutical compositions disclosed herein display a significant extended-release profile of the therapeutic agent for treating a disease due to reduced bone density or cartilage loss. For example, the pharmaceutical composition of the present invention extended the half-life of L-006235 to 56.6 hours in rats, which is 16.6 times longer compared to the half-life of L-006235 in rats (3.4 hours) via oral administration (J Med Chem 2005 1:48(24) :7520-34). These pharmaceutical compositions are developed to reduce the dosing frequency from once to twice daily to once every two days, once every three days, once every four days, once every five days, once every six days, weekly, once every two weeks, once a month, once every two months, once every three months, once every four months, once every five months or once every six months.

Examples

[0042] Embodiments of the present invention are illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention. During the studies described in the following examples, conventional procedures were followed, unless otherwise stated.

Example 1. Preparation of Liposomal L-006235 Formulation

[0043] Empty liposomes were prepared by a lipid film hydration-extrusion method. Bilayer membrane components, HSPC, cholesterol, and DSPE-PEG2000 (mole percentage of 59.5/39.6/0.9), were dissolved in an organic solvent, for example, chloroform and dichloromethane. A thin lipid film was formed by removing the organic solvent under vacuum in a rotary evaporator. The dry lipid film was hydrated in an aqueous solution containing 300 mM ammonium sulfate at 60°C for 30 min and the empty liposomes were formed with ammonium sulfate encapsulated in the aqueous core. Other trapping agent, such as triethylammonium sucrose octasulfate, was also used. After five freeze-thaw cycles between liquid nitrogen and water at 60°C, the empty liposomes were subsequently extruded ten times through polycarbonate filter membranes with a pore size of 0.2 pm. Unencapsulated trapping agent was removed by dialysis method or diafiltration method against a 9.4% sucrose solution or 0.9% NaCl solution to create a polyatomic ion gradient between the inner aqueous phase and the outer aqueous phase of the empty liposomes.

[0044] A reaction mixture containing 3.0 mg/mL of L-006235 (commercially available from DC Chemicals, China), empty liposomes (with 6.0 mM of lipids), and 50 mM histidine buffer (pH 6.5) was incubated at 60°C for 15 min. The unencapsulated L-006235 of the reaction mixture was separated by a Sephadex™ G-50 Fine gel (GE Healthcare, USA) or dialysis bag (Spectrum Labs, USA) against a 9.4% sucrose solution to obtain a liposomal L-006235 formulation. To calculate the drug to lipid molar ratio (D/L) of the liposomal L-006235 formulation, the concentration of encapsulated L-006235 of the liposomal L-006235 formulation was measured using HPLC and the concentrations of lipids of the liposomal L- 006235 formulation were measured using an ultraviolet/visible (UV/Vis) spectrophotometer.

[0045] The encapsulation efficiency was calculated by dividing the drug to lipid molar ratio (D/L) of the liposomal L-006235 formulation by the nominal D/L of the reaction mixture, which was calculated by dividing the concentration of L-006235 by the lipid concentration of the empty liposomes. The particle size distribution was measured by a dynamic light scattering instrument (Zetasizer Nano-ZS90, Malvern, USA).

[0046] Using 300 mM ammonium sulfate as a trapping agent, the liposomal L-006235 formulation has a final D/L of 1.00, an encapsulation efficiency of 93.4%, and the mean diameter of the liposomes was 193.7 nm.

Example 2. Preparation of Various Liposomal Cathepsin K Inhibitor Formulations

[0047] Cathepsin K inhibitors used in this example included L-006235 and balicatib (MedChem Express, USA). The empty liposomes were prepared according to Example 1, with the following trapping agents: (1) 300 mM of ammonium sulfate and (2) 75 mM of triethylammonium sucrose octasulfate. The loading procedures of liposomal L-006235 formulations were based on Example 1. The liposomal balicatib formulation was prepared as follows: a reaction mixture containing 2 mg/mL of balicatib, empty liposomes and 50 mM histidine buffer (pH 6.5) was incubated at 60°C for 15 minutes. Unencapsulated drug was removed by Sephadex™ G-50 Fine gel (GE Healthcare, USA) to obtain a liposomal balicatib formulation. The D/L ratio of the liposomal formulations in this example were calculated according to the steps in Example 1. Table 1 shows the loading profiles of L-006235 and balicatib. [0048] Table 1 The loading profile of different cathepsin K inhibitors

EE, encapsulation efficiency; n.d., not determined.

Example 3. Pharmacokinetics (PK) Study of Liposomal L-006235 Lormulation

[0049] An in vivo PK evaluation of the liposomal L-006235 formulation was performed using 7-8 weeks old female Sprague-Dawley rats. The rats were housed in a holding room which operated on a 12-hr light/12-hr dark circadian cycle and the rats had free access to water and food.

[0050] The rats were divided into two groups (n - 4 in each group). The rats in the first group each received an intraarticular injection of 2.5 mg/kg of free L-006235, prepared by dissolving L-006235 in a 9.4% sucrose solution, containing 0.06 N HC1, with a final concentration of 10.0 mg/mL and the rats in the second group each received an intraarticular injection of 5.0 mg/kg of liposomal L-006235 formulation, prepared according to Example 1 with a final concentration of 18.1 mg/mL. Blood samples were collected at 5, 15, 30 mins, 1, 2, 4, 8, 24, 48, 72, 96 and 168 hours post- injection. Plasma samples were obtained by centrifugation and analyzed using liquid chromatography-tandem mass spectrometry. The plasma concentrations versus time curves were analyzed using a noncompartmental analysis model in PKSolver (Comput Methods Programs Biomed. 2010;99(3):306-314). The PK parameters of the two L- 006235 formulations are summarized in Table 2.

[0051] The results in Table 2 show the dose-normalized C_max (C_max/D) of liposomal L-006235 formulation was 40.5% of that of the free L-006235 formulation, and the half-life (t ) of liposomal L-006235 formulation (56.6 hours) was significantly longer compared to that of the free L-006235 formulation (4.5 hours). The dose-normalized area under the curve (AUCo-_t/D) of the liposomal L-006235 formulation indicates that 89.7% of L-006235 was released from the liposomal L-006235 formulation 168 hours post-injection compared to the AUCo-_t/D of the free L-006235 formulation, which indicates that 100% of L-006235 was released 24 hours post injection.

[0052] Table 2 PK parameters derived from rats after a single intraarticular injection of free L-006235 formulation or liposomal L-006235 formulation

Cmax/D, dose-normalized Cma_X; AUCo-t/D, dose-normalized AUCo-t; ALCo- D, dose-normalized AUCo-mf.

[0053] In addition, FIG. 1 shows plasma L-006235 was undetectable 24 hours post free L- 006235 injection whereas plasma L-006235 was detected up to 168 hours after the administration of the liposomal L-006235 formulation. The results support a conclusion that the claimed pharmaceutical composition sustained the release of cathepsin K inhibitors.

Claims

1. A pharmaceutical composition, comprising

(a) at least one liposome comprising a bilayer membrane, said bilayer membrane comprises at least one lipid;

(b) a trapping agent; and

(c) a therapeutic agent for treating a disease due to reduced bone density or

cartilage loss,

wherein the molar ratio of the therapeutic agent to the lipid is equal to or higher than about 0.1.

2. The pharmaceutical composition of claim 1, wherein the mean particle size of the liposomal is from about 50 nm to 500 nm.

3. The pharmaceutical composition of claim 1, wherein the bilayer membrane further comprises cholesterol.

4. The pharmaceutical composition of claim 3, wherein the mole percentage of the cholesterol in the bilayer membrane is about 20 to about 55%.

5. The pharmaceutical composition of claim 1, wherein the trapping agent is selected from the group consisting of triethylammonium sucrose octasulfate, ammonium sulfate and a combination thereof.

6. The pharmaceutical composition of claim 5, wherein the concentration of triethylammonium sucrose octasulfate is about 10 to 200 mM.

7. The pharmaceutical composition of claim 5, wherein the concentration of ammonium sulfate is about 100 to 600 mM.

8. The pharmaceutical composition of claim 1, wherein the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is a cathepsin K inhibitor.

9. The pharmaceutical composition of claim 1, wherein the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is selected from the group consisting essentially of balicatib, odanacatib, L-006235, ONO-5334, MIV-711, relacatib and a combination thereof.

10. The pharmaceutical composition of claim 1, wherein the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is encapsulated in the liposome with an encapsulation efficiency higher than about 50%.

11. A method of treating a disease due to reduced bone density or cartilage loss, comprising: administering a pharmaceutical composition to a subject in need thereof, said

pharmaceutical composition comprising:

(a) at least one liposome comprising a bilayer membrane, the bilayer membrane comprises at least one lipid;

(b) a trapping agent; and

(c) a therapeutic agent for treating a bone and joint disease,

wherein the molar ratio of the therapeutic agent to the lipid is equal to or higher than about

0.1.

12. The method of claim 11, wherein the half-life of the therapeutic agent for treating a disease due to reduced bone density or cartilage loss is extended by at least 2-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, or at least 20-fold compared to that of the free therapeutic agent for treating a disease due to reduced bone density or cartilage loss.

13. The method of claim 11, wherein the pharmaceutical composition is administered at least once every three days, at least once every week, at least once every two weeks or at least once a month.

14. The method of claim 11, wherein the pharmaceutical composition is administered by injection.

15. The method of claim 14, wherein the injection includes intraarticular, subcutaneous, subdermal, intradermal or intramuscular route.