Patent Publication Number: US-2022211621-A1

Title: Liposomal sustained-release compositions containing a therapeutic drug and use thereof

Description:
RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 62/838,571, filed Apr. 25, 2019, the contents of which are incorporated by reference in their entirety. 
    
    
     BACKGROUND 
     Technical Field 
     The present disclosure relates to a drug delivery system for delivery of a therapeutic drug. The present disclosure also relates to a pharmaceutical composition that has an improved release profile. 
     Description of Related Art 
     Liposomes are self-assembled, lipid based vesicles composed of phospholipid bilayers with an aqueous interior. These vesicles have been utilized as drug carriers for sustained drug delivery for decades. Liposomes as drug delivery vehicles alter the pharmacokinetic profile of the drug, which is usually reflected in the properties of slow drug release systemically or at the disease site, high administered doses with less frequent drug administration, and reduced side effects and toxicity with respect to a portion of drugs. 
     A liposomal drug formulation can be tailored to achieve slow drug release in vivo, which would prolong the therapeutic effect of the drug. However, multiple factors might affect the properties of the liposomes, such as the type of phospholipids used, phospholipid to cholesterol ratio, trapping agent used for drug loading, and possibly the lamellarity of the liposome. 
     A trend of prolonged release is projected in a direction such that the higher the drug per unit of used lipid in the liposomal composition is, the longer the drug remains in the lipid based vehicles of the liposomal composition (Johnston et al., Biochim Biophys Acta. 2006 January;1758(1):55-64, Chountoulesi et al., J Liposome Res. 2018 September;28(3):249-258, and Johnston et al. Journal of Liposome Research, 18:145-157, 2008). Thus, a desired sustained-release liposomal composition should possess a high drug to lipid ratio. Nevertheless, the increased amount of a therapeutic agent, especially a highly potent drug, will raise the potential of enhanced side effects or undesired toxicity. 
     There is therefore an unmet need in the art for a beneficial and effective lipid based delivery system for a highly potent drug that has an improved release profile and lower undesired side effects or toxicity. The compositions and methods of the present disclosure satisfy these and other needs. 
     SUMMARY 
     The present disclosure provides a liposomal sustained-release composition, comprising a pharmaceutically effective amount of a therapeutic agent, and one or more lipids, wherein the therapeutic agent and total lipids are present at a predetermined ratio, whereby the liposomal sustained-release composition has a prolonged release profile of the therapeutic agent compared to a composition having the therapeutic agent and one or more lipids at a ratio higher than the predetermined ratio. 
     To improve upon existing treatment paradigms for diseases and to take advantage of the benefits of slow, sustained drug release, we developed a liposomal sustained-release composition comprising liposome-entrapped therapeutic agent and a predetermined amount of free therapeutic agent in an aqueous suspension for enhanced treatment of disease with desired pharmacokinetics. 
     The present disclosure provides a liposomal sustained-release composition for use in the treatment of disease having the advantages of: achieving a therapeutic effect with a much lower drug dose, quicker onset of action, reducing adverse drug reactions and systemic effects, increasing the residence time of drug in the target site via sustained release from the liposomal drug formulation, decreasing the frequency of drug administration, and potentially improving patient outcomes and compliance. The drug dose of the liposomal sustained-release composition for treating disease can significantly increase the therapeutic effects when compared to the unformulated therapeutic agent. 
     In some embodiments, the predetermined ratio of the therapeutic agent to the total lipids ranges from 0.0001 mol/mol to 0.15 mol/mol, optionally 0.0001 mol/mol to 0.14 mol/mol, optionally 0.0001 mol/mol to 0.13 mol/mol, optionally 0.0001 mol/mol to 0.12 mol/mol, optionally 0.0001 mol/mol to 0.11 mol/mol, and optionally 0.0001 mol/mol to 0.1 mol/mol, optionally 0.0001 mol/mol to 0.09 mol/mol, optionally 0.0001 mol/mol to 0.08 mol/mol, optionally 0.0001 mol/mol to 0.07 mol/mol, optionally 0.0001 mol/mol to 0.06 mol/mol, optionally 0.0001 mol/mol to 0.05 mol/mol, optionally 0.0001 mol/mol to 0.04 mol/mol, optionally 0.0001 mol/mol to 0.03 mol/mol, optionally 0.0001 mol/mol to 0.02 mol/mol, and optionally 0.0001 mol/mol to 0.01 mol/mol. 
     In some embodiments, the pharmaceutically effective amount of the therapeutic agent ranges from 0.0001 mg/mL to 10 mg/mL, optionally 0.0001 mg/mL to 7.5 mg/mL, optionally 0.0001 mg/mL to 5 mg/mL, optionally 0.0001 mg/mL to 2.5 mg/mL, optionally 0.0001 mg/mL to 2 mg/mL, optionally 0.0001 mg/mL to 1 mg/mL, and optionally 0.0001 mg/mL to 0.5 mg/mL. 
     In some embodiments, the liposomal sustained-release composition comprises a phospholipid concentration ranging from about 0.01 mM to about 100 mM, optionally about 0.1 mM to about 100 mM, and optionally about 0.1 mM to about 60 mM. 
     In certain embodiments, the therapeutic agent could be, but is not limited to: an antineoplastic agent, an analgesic, an anesthetic, or an immune modulator. 
     In some embodiments, the antineoplastic agent is selected from the group consisting of antihormonals, antifolates, antimicrotubule agents, alkylating agents, antimetabolites, antibiotics, topoisomerase inhibitors, antivirals, and cytotoxic agents. 
     In some embodiments, the antineoplastic agent is selected from the group consisting of vinca alkaloids, auristatins, cryptophycins, maytansines, anthracyclines, calicheamicins, duocarymycins, pyrrolobenzodiazepine (PBD) dimers, and α-amanitin. 
     In some embodiments, the analgesic is an α-adrenergic receptor (α-AR) agonist, an opioid, a nonsteroidal anti-inflammatory drug (NSAID), or a N-methyl-D-aspartate (NMDA) receptor antagonist. 
     In some embodiments, the α-AR agonist is an α 2 -AR agonist. 
     In some embodiments, the analgesic is selected from the group consisting of dexmedetomidine, clonidine, morphine, fentanyl, hydromorphone, codeine, oxycodone, hydrocodone, tramadol, ondansetron, dexamethasone, methadone, alfentanil, remifentanil, aspirin, ibuprofen, naproxen, indomethacin, ibuprofen, naproxen, fenoprofen, tolmetin, sulindac, meclofenamate, keoprofen, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, flurbiprofen, diclofenac, acetylsalicylic acid, sodium acetylsalicylic acid, calcium acetylsalicylic acid, salicylic acid, sodium salicylate, dizocilpine, and ketamine. 
     In some embodiments, the anesthetic is an ester-based local anesthetic, an amide-based local anesthetic, an α-AR agonist, an opioid, a barbiturate compound, a benzodiazepine compound, or a NMDA receptor antagonist. 
     In some embodiments, the α-AR agonist is an α 2 -AR agonist. 
     In some embodiments, the anesthetic is selected from the group consisting of benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, piperocaine, propoxycaine, procaine, proparacaine, tetracaine, articaine, bupivacaine, cinchocaine/dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, trimecaine, saxitoxin, tetrodotoxin, menthol, eugenol, dexmedetomidine, clonidine, morphine, fentanyl, hydromorphone, oxycodone, sufentanil, buprenorphine, butorphanol, diamorphine, levorphanol, pethidine, dexamethasone, methadone, alfentanil, remifentanil, nalbuphine, oxymorphone, pentazocine, amobarbital, methohexital, thiamylal, thiopental, diazepam, lorazepam, midazolam, dizocilpine, and ketamine. 
     In some embodiments, the immune modulator is a toll-like receptor (TLR) agonist or a sphingosine 1-phosphate receptor agonist. 
     In some embodiments, the TLR agonist is a TLR7 agonist or a TLR8 agonist. 
     In certain embodiments, the immune modulator is an imidazoquinoline compound. 
     In some embodiments, the immune modulator is selected from the group consisting of resiquimod, imiquimod, gardiquimod, CL075, SM-011, isatoribine, SM-360320, fingolimod, ponesimod, siponimod, ozanimod, and ceralifimod. 
     In some embodiments, the therapeutic agent is a highly potent agent. 
     In some embodiments, the liposomal sustained-release composition according to the present disclosure comprises liposomes, wherein the liposomes entrap the therapeutic agent. 
     In some embodiments, the liposomes of the liposomal sustained-release composition have a mean particle diameter of from about 50 nm to about 400 nm. Suitable liposomes of the composition according to the present disclosure may comprise: 
     a lipid bilayer comprising one or more phospholipids, a sterol, and an optional polyethylene glycol (PEG)-modified lipid; and an aqueous interior encompassed by the lipid bilayer and entrapping the therapeutic agent. 
     In accordance with the present disclosure, the one or more phospholipids is selected from the group consisting of hydrogenated soy phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), phosphatidylethanolamine lipid, and a combination thereof. 
     In some embodiments, the liposomal sustained-release composition comprises PEG-modified lipid at a molar percentage ranging from 0.001 mol % to 10 mol % on the basis of the total phospholipids and sterol. 
     In some embodiments, the PEG-modified lipid comprises a PEG moiety with an average molecular weight ranging from about 500 g/mol to about 5,000 g/mol. 
     In some embodiments, the PEG-modified lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)] (DSPE-PEG). 
     In some embodiments, the one or more phospholipids of the liposomal sustained-release composition is a neutral phospholipid and the DSPE-PEG of the liposome is present in the composition at a molar percentage ranging from about 0.001 mol % to about 6 mol % on the basis of the total phospholipid and sterol. 
     In some embodiments, the molar ratio of the total phospholipids to sterol ranges from about 1:1 to about 3:1. 
     In some embodiments, the therapeutic agent is entrapped inside the liposome in the aqueous interior space of the liposome via a transmembrane pH gradient-driven remote loading method using a trapping agent. 
     In some embodiments, the trapping agent is ammonium sulfate, ammonium mesylate, ammonium tosylate, triethylammonium sucrose octasulfate, dextran sulfate, or a combination thereof. 
     In some aspects, the present disclosure provides a pharmaceutical composition for use in a treatment of a disease, which comprises a pharmaceutically effective amount of the liposomal sustained-release composition in accordance with the present disclosure, wherein the disease is selected from the group consisting of cancer, infectious disease, and autoimmune disease. 
     In some embodiments, the liposomal sustained-release composition is administered at a daily therapeutic dose of the therapeutic agent of not more than 10 mg/day. In some embodiments, the composition is administered by single, double, or multiple (e.g., three or more) administrations. In some embodiments, the composition is administered weekly, biweekly, or monthly. 
     In another aspect, the present disclosure provides a method for treating cancer, infectious disease, and/or autoimmune disease, the method comprising: administering a pharmaceutically effective amount of the liposomal sustained-release composition of the present disclosure. 
     In some embodiments, the liposomal sustained-release composition is administered at a daily therapeutic dose of the therapeutic agent of not more than 10 mg by single, double, or multiple (e.g., three or more) administrations weekly, biweekly, or monthly. 
     In yet another aspect, the present disclosure provides a method for controlling release of a therapeutic agent, comprising steps of:
         introducing a liposomal composition into an environment, wherein the composition comprises:
           a pharmaceutically effective amount of a therapeutic agent, and   one or more lipids,   
           wherein the therapeutic agent to the total lipids is at a predetermined ratio,   the liposomal composition has a prolonged release profile of the therapeutic agent compared to a composition having the therapeutic agent and one or more lipids at a ratio higher than the predetermined ratio.       

     Other objectives, advantages, and novel features of the disclosure will become apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A and 1B  are graphs showing drug retention of liposomal compositions comprising resiquimod as the therapeutic agent with a formulation concentration of 0.05 mg/mL in 90% human plasma (the therapeutic agent concentration in 90% human plasma is 0.005 mg/mL) ( FIG. 1A ) or in 10% human plasma (the therapeutic agent concentration in 10% human plasma is 0.005 mg/mL) ( FIG. 1B ). 
         FIG. 2  is a graph showing drug retention of liposomal compositions comprising resiquimod as the therapeutic agent with a formulation concentration of 1 mg/mL in 90% human plasma, whereby the final therapeutic agent concentration in 90% human plasma is 0.1 mg/mL. 
         FIG. 3  is a graph depicting drug retention of liposomal compositions comprising resiquimod as the therapeutic agent with a fixed drug to lipid ratio of 0.19 mol/mol (D/L =0.19 mol/mol) in 90% human plasma (the formulation concentration of the therapeutic agent is 0.05 mg/mL or 2.00 mg/mL and the therapeutic agent concentration in 90% human plasma is 0.005 mg/mL or 0.2 mg/mL, respectively). 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. 
     As used herein, the singular forms “a”, “an,” and “the” include the plural reference unless the context clearly indicates otherwise. 
     All numbers herein may be understood as modified by “about,” which, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to obtain a desired amount of liposomal drug, unless otherwise specified. 
     The term “treating,” “treated,” or “treatment” as used herein includes preventive (e.g., prophylactic), palliative, and curative uses or results. 
     The term “pharmaceutically effective amount” as used herein denotes an amount which, compared with a corresponding subject who has not received this amount, has the following consequence: improved treatment, healing, prevention or elimination of a disease, syndrome, condition, complaint, disorder or side-effects or also the reduction in the advance of a disease, complaint or disorder. The term “pharmaceutically effective amount” also encompasses the amounts which are effective for increasing normal physiological function. In a particular embodiment, the pharmaceutically effective amount can be 0.005 mg/m 2 /day to 3 mg/m 2 /day, optionally 0.08 mg/m 2 /day to 1 mg/m 2 /day, and optionally 0.3 mg/m 2 /day to 1 mg/m 2 /day. 
     The term “subject” includes a vertebrate having cancer and/or a disease affecting pulmonary function. In some embodiments, the subject is a warm-blooded animal, such as a mammal, including a human. 
     As used herein, the term “drug to lipid ratio” (“D/L”) refers to the ratio of therapeutic agent to total lipids. The content of therapeutic agent in a free form or liposomal form was determined by UV-Vis absorbance measurements. The phospholipid content or concentration of liposome and liposomal drug was determined by assaying the phosphorus content of liposome and liposomal drug samples using a phosphorus assay (adapted from G. Rouser et al., Lipids 1970, 5, 494-496). 
     As used herein, the term “mol %” means the percentage of moles of a given component of a mixture relative to the total moles of that mixture. 
     Liposome 
     The term “liposome” as used herein refers to a particle characterized by having an aqueous interior space sequestered from an outer medium by a membrane of one or more bilayer membranes forming a vesicle. Bilayer membranes of liposomes are typically formed by lipids, i.e., amphiphilic molecules of synthetic or natural origin that comprise spatially separated hydrophobic and hydrophilic domains. In certain embodiments of the present disclosure, the term “liposomes” refers to small unilamellar vesicle (SUV) liposomes in which one lipid bilayer forms the membrane. 
     In general, liposomes comprise a lipid mixture typically including one or more lipids selected from the group consisting of: dialiphatic chain lipids, such as phospholipids; diglycerides; dialiphatic glycolipids; single lipids such as sphingomyelin and glycosphingolipid; steroids such as cholesterol and derivatives thereof and a combination thereof. 
     Examples of phospholipids according to the present disclosure include, but are not limited to, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1-palmitoyl-2-stearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (PSPG), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-distearoyl-sn-gly cero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-myo-inositol) (ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) (ammonium salt) (DOPI), cardiolipin, L-α-phosphatidylcholine (EPC), and L-α-phosphatidylethanolamine (EPE). 
     Polyethylene Glycol (PEG)-Modified Lipid 
     A polyethylene glycol-modified lipid comprises a polyethylene glycol moiety conjugated to a lipid. In some embodiments, the PEG moiety has a molecular weight from about 500 to about 20,000 daltons. In some embodiments, the PEG-modified lipid is mixed with the phospholipids to form liposomes with one or more bilayer membranes. In some embodiments, the amount of PEG-modified lipid ranges from 0.0001 mol % to 40 mol %, optionally from 0.001 mol % to 30 mol %, and optionally from 0.01 mol % to 20 mol %, on the basis of the total phospholipids and sterol. In some embodiments, the amount of PEG-modified lipid is no more than 6 mol %, no more than 5 mol %, no more than 3 mol %, or no more than 2 mol %, on the basis of the total phospholipids and sterol. In some embodiments, the PEG-modified lipid has a PEG moiety with an average molecular weight ranging from 500 g/mol to 5,000 g/mol. In some embodiments, the PEG-modified lipid is phosphatidylethanolamine lipid linked to a polyethylene glycol group (PEG-PE). In some embodiments, phosphatidylethanolamine lipid includes is but not limited to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine. In some embodiments, PEG-modified phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG). 
     Liposomal Sustained-Release Compositions 
     The terms “liposomal drug formulation,” “liposomal composition,” and “liposomal sustained-release composition” are used interchangeably in the present disclosure. The liposomal sustained-release composition in accordance with the present disclosure includes, but is not limited to, liposomes with entrapped therapeutic agent prepared by entrapping the therapeutic agent in the aqueous interior of the liposome via pass loading or a transmembrane pH gradient-driven remote loading method. In some embodiments, the transmembrane pH gradient is created by using a trapping agent for remote loading of the therapeutic agent into liposomes. In various embodiments, the trapping agent is selected from the group consisting of ammonium sulfate, ammonium mesylate, ammonium tosylate, triethylammonium sucrose octasulfate, dextran sulfate, and a combination thereof. 
     In certain embodiments, the liposome with the entrapped therapeutic agent comprises (a) a lipid bilayer comprising one or more phospholipids, a sterol, and a polyethylene glycol (PEG)-modified lipid, including but not limited to a PEG-modified phosphatidylethanolamine; and (b) an aqueous interior encompassed by the lipid bilayer entrapping a therapeutic agent. 
     In some embodiments, the one or more phospholipids is a neutral phospholipid. In some embodiments, the PEG-modified lipid is DSPE-PEG and the amount of DSPE-PEG in the liposome ranges from 0.001 mol % to 6 mol % on the basis of the total phospholipid and sterol. 
     In some embodiments, the liposomes with the entrapped therapeutic agent have a mean particle diameter between 50 nm and 400 nm. 
     Therapeutic Agents 
     The term “therapeutic agent” as used herein includes a substance that alleviates a symptom of or prevents development of a disease or disorder. 
     In one aspect, therapeutic agents according to the present disclosure refer to substances that are prescribed at the lowest effective dosage, with a dosing regimen given an adequate trial, and with the patient carefully followed with periodic determinations made about the need for continued therapy. Suitable therapeutic agents may be, but are not limited to: a growth factor, an immune modulator, an anti-inflammatory compound, an antibiotic, a compound that promotes angiogenesis, a compound that inhibits angiogenesis, a chemotactic agent, an agent that promotes apoptosis, an agent that inhibits apoptosis, a mitogenic agent, an analgesic, an anesthetic, an anti-coagulation agent, a procoagulation agent, and/or a contrast agent for imaging studies. 
     In some embodiments, the therapeutic agents may also include those active agents that are to be administered for long-term maintenance to patients such as cardiovascular drugs, including blood pressure, pacing, anti-arrhythmia, beta-blocking drugs, and calcium channel based drugs. 
     In some embodiments, the therapeutic agents may also include active agents for epilepsy or other movement disorders. 
     In some embodiments, these therapeutic agents may also include long-term medications such as contraceptives and fertility drugs. They could comprise neurologic agents such as dopamine and related drugs as well as psychological or other behavioral drugs. The therapeutic agents of the present disclosure may also include chemical scavengers such as chelators, antioxidants and nutritional agents. 
     In some embodiments, the therapeutic agent is directed to antineoplastic agents. A partial listing of the antineoplastic agents by classification is as follows. 
     Structure-based classes: fluoropyrimidines—5-fluorouracil, fluorodeoxyuridine, ftorafur, 5′-deoxyfluorouridine, UFT, S-1capecitabine; pyrimidine; nucleosides—deoxycytidine, cytosine arabinoside, 5-azacytosine, gemcitabine, 5-azacytosine-arabinoside; purines-6-mercaptopurine, thioguanine, azathioprine, allopurinol, cladribine, fludarabine, pentostatin, 2-chloro adenosine; platinum analogues—cisplatin, carboplatin, oxaliplatin, tetraplatin, platinum-DACH, ormaplatin, CI-973, JM-216; anthracyclines/anthracenediones—doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone; epipodophyllotoxins—etoposide, teniposide; camptothecins—irinotecan, topotecan, lurtotecan, silatecan, 9-amino camptothecin, 10,11-methylenedioxy camptothecin, 9-nitro camptothecin, TAS103, 7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(s)-camptothecin, 7-(2-n-isopropylamino)ethyl)-20(s)-camptothecin; hormones and hormonal analogues—diethylstilbestrol, tamoxifen, toremefine, tolmudex, thymitaq, flutamide, bicalutamide, finasteride, estradiol, trioxifene, droloxifene, medroxyprogesterone acetate, megesterol acetate, aminoglutethimide, testolactone and others; vinca alkaloids—vincristine, vinblastine, vindesine, vinorelbine; taxanes—paclitaxel, docetaxel, abraxane, taxotere. 
     Mechanism-based classes: antihormonals—see classification for hormones and hormonal analogues, anastrozole; antifolates—methotrexate, aminopterin, trimetrexate, trimethoprim, pyritrexim, pyrimethamine, edatrexate, MDAM; antimicrotubule agents—taxanes and vinca alkaloids; alkylating agents (classical and non-classical)—nitrogen mustards (mechlorethamine, chlorambucil, melphalan, uracil mustard), oxazaphosphorines (ifosfamide, cyclophosphamide, perfosfamide, trophosphamide), alkylsulfonates (busulfan), nitrosoureas (carmustine, lomustine, streptozocin), thiotepa, dacarbazine and others; antimetabolites—purines, pyrimidines and nucleosides, listed above; antibiotics—anthracyclines/anthracenediones, bleomycin, dactinomycin, mitomycin, plicamycin, pentostatin, streptozocin; topoisomerase inhibitors—camptothecins (topo I), epipodophyllotoxins, m-AMSA, ellipticines (topo II); antivirals—AZT, zalcitabine, gemcitabine, didanosine, and others; miscellaneous cytotoxic agents—hydroxyurea, mitotane, fusion toxins, pyrazinamide, bryostatin, retinoids, butyric acid and derivatives, pentosan, fumagillin, and others. 
     In some embodiments, the therapeutic agent is an analgesic. 
     In certain embodiments, the analgesic can be, but not limited to, an α-adrenergic receptor (α-AR) agonist, an opioid, a nonsteroidal anti-inflammatory drug (NSAID) or a N-methyl-D-aspartate (NMDA) receptor antagonist. 
     In some embodiments, the α-AR agonist is an α 2 -AR agonist, such as dexmedetomidine. 
     In some embodiments, the analgesic is selected from the group consisting of dexmedetomidine, clonidine, morphine, fentanyl, hydromorphone, codeine, oxycodone, hydrocodone, tramadol, ondansetron, dexamethasone, methadone, alfentanil, remifentanil, aspirin, ibuprofen, naproxen, indomethacin, ibuprofen, naproxen, fenoprofen, tolmetin, sulindac, meclofenamate, keoprofen, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, flurbiprofen, diclofenac, acetylsalicylic acid, sodium acetylsalicylic acid, calcium acetylsalicylic acid, salicylic acid, sodium salicylate, dizocilpine, and ketamine. 
     In some embodiments, the therapeutic agent is an anesthetic. 
     In certain embodiments, the anesthetic can be, but is not limited to, an ester-based local anesthetic, an amide-based local anesthetic, an α-AR agonist, an opioid, a barbiturate compound, a benzodiazepine compound, or a NMDA receptor antagonist. 
     In some embodiments, the α-AR agonist is an a 2 -AR agonist, such as dexmedetomidine. 
     In some embodiments, the anesthetic is selected from the group consisting of benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, piperocaine, propoxycaine, procaine, proparacaine, tetracaine, articaine, bupivacaine, cinchocaine/dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, trimecaine, saxitoxin, tetrodotoxin, menthol, eugenol, dexmedetomidine, clonidine, morphine, fentanyl, hydromorphone, oxycodone, sufentanil, buprenorphine, butorphanol, diamorphine, levorphanol, pethidine, dexamethasone, methadone, alfentanil, remifentanil, nalbuphine, oxymorphone, pentazocine, amobarbital, methohexital, thiamylal, thiopental, diazepam, lorazepam, midazolam, dizocilpine, and ketamine. 
     In some embodiments, the therapeutic agent is an immune modulator. 
     In certain embodiments, the immune modulator is a toll-like receptor (TLR) agonist that induces innate immune activation. 
     In some embodiments, the therapeutic agent according to the present disclosure is a TLR7 agonist or a TLR8 agonist. Non-limiting examples of TLR7 agonists comprise resiquimod, imiquimod, gardiquimod, CL075, SM-011, the N9-pyridinylmethyl analogues recited in Jones et al., Bioorg Med Chem Lett. 2011 Oct. 1;21(19):5939-43 (as illustrated below), isatoribine, and SM-360320. 
     
       
         
         
             
             
         
       
     
     In certain embodiments, the immune modulator is a sphingosine 1-phosphate receptors (S1PR) agonist, which sequesters lymphocytes to peripheral lymphoid organs and away from the sites of chronic inflammation. 
     Non-limiting examples of S1PR agonists include fingolimod, ponesimod, siponimod, ozanimod, and ceralifimod. 
     In certain embodiments, the immune modulator is an imidazoquinoline compound. 
     Suitable imidazoquinoline compounds include, but are not limited to, resiquimod, imiquimod, gardiquimod, CL075, SM-011, and dactolisib. 
     The term “highly potent agent” refers to powerfully active pharmaceutical ingredients needing only very small quantities to exhibit a therapeutic effect. The highly potent agent in accordance to the present disclosure comprises active pharmaceutical ingredients with a daily therapeutic dose of not more than 10 mg/day. 
     In the field of pharmacology, potency is a measure of drug activity expressed in terms of the amount required to produce an effect of given intensity. A highly potent agent (e.g., fentanyl, alprazolam, risperidone) evokes a given response at low concentrations, while an active agent of lower potency evokes the same response only at higher concentrations. 
     In some embodiments, the highly potent agent according to the present disclosure includes, but is not limited to, microtubule disrupting agents, such as auristatins, cryptophycins and maytansines; and DNA modifying agents, such as anthracyclines, calicheamicins, duocarymycins, PBD dimers, and amanitins. 
     Non-limiting examples of the highly potent agent according to the present disclosure comprises vincristine, resiquimod, imiquimod, gardiquimod, fingolimod, dexmedetomidine, auristatins, cryptophycins, maytansines, anthracyclines, calicheamicins, duocarymycins, PBD dimers, and amanitins. 
     In various embodiments, the liposomal sustained-release composition according to the present disclosure has a predetermined ratio of the therapeutic agent to the total lipids ranging from 0.0001 mol/mol to 0.15 mol/mol, optionally 0.0001 mol/mol to 0.14 mol/mol, optionally 0.0001 mol/mol to 0.13 mol/mol, optionally 0.0001 mol/mol to 0.12 mol/mol, optionally 0.0001 mol/mol to 0.11 mol/mol, and optionally 0.0001 mol/mol to 0.1 mol/mol, optionally 0.0001 mol/mol to 0.09 mol/mol, optionally 0.0001 mol/mol to 0.08 mol/mol, optionally 0.0001 mol/mol to 0.07 mol/mol, optionally 0.0001 mol/mol to 0.06 mol/mol, optionally 0.0001 mol/mol to 0.05 mol/mol, optionally 0.0001 mol/mol to 0.04 mol/mol, optionally 0.0001 mol/mol to 0.03 mol/mol, optionally 0.0001 mol/mol to 0.02 mol/mol, and optionally 0.0001 mol/mol to 0.01 mol/mol. 
     Treatment of Diseases or Disorders 
     The liposomal sustained-release composition according to the present disclosure may be used to treat a disease or disorder according to a dosing regimens comprising administering to a mammal a pharmaceutically effective amount of the therapeutic agent as a unit dosage according to a continuous schedule, e.g., having a dosing interval selected from the group consisting of once-weekly dosing, twice-weekly dosing, thrice-weekly dosing, biweekly dosing, and bimonthly dosing. 
     In some embodiments, the liposomal sustained-release composition according to the present disclosure is for use in treatment of cancer or tumor including, but not limited to, lung cancer or tumor, breast cancer or tumor, liver cancer or tumor, hepatocellular carcinoma, pancreatic cancer or tumor, bowel and colon cancer or tumor, colorectal cancer or tumor, bladder cancer or tumor, ovarian cancer or tumor, prostate cancer or tumor, skin cancer or tumor, brain cancer or tumor, or malignancies affecting the bone marrow (including leukemias) and lymphoproliferative systems, such as Hodgkin&#39;s and non-Hodgkin&#39;s lymphoma. 
     In certain embodiments, the liposomal sustained-release composition according to the present disclosure comprises a TLR agonist that modulates toll-like receptor activity and is used in the treatment of neoplasia including, but not limited to, melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, sarcomas, Kaposi&#39;s sarcoma, renal cell carcinoma, leukemias, myelogeous leukemia, chronic lymphocytic leukemia, and/or multiple myeloma. 
     In some embodiments, the liposomal sustained-release composition in accordance with the present disclosure is used in the treatment of infectious diseases including, but not limited to, viral infection such as genital warts, common warts, plantar warts, respiratory syncytial virus, hepatitis B, hepatitis C, Dengue virus, molluscum contagiosum, vaccinia, variola, lentivirus, human immunodeficiency virus (HIV), human papilloma virus (HPV), cytomegalovirus (CMV), varicella zoster virus (VZV), rhinovirus, enterovirus, adenovirus, coronavirus (e.g., SARS), influenza, parainfluenza, mumps virus, measles virus, papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus, and/or filovirus. 
     In some embodiments, the liposomal sustained-release composition in accordance with the present disclosure is used in the treatment of bacterial, fungal, and/or protozoal infections including, but not limited to, tuberculosis and mycobacterium avium, leprosy, pneumocystis carinii, cryptosporidiosis, histoplasmosis, toxoplasmosis, trypanosome infection, leishmaniasis, infections caused by bacteria of the genus  Escherichia, Enterobacter, Salmonella, Staphylococcus, Klebsiella, Proteus, Pseudomonas, Streptococcus , or  Chlamydia , or fungal infections such as candidiasis, aspergillosis, histoplasmosis, or cryptococcal meningitis. 
     In some embodiments, the liposomal sustained-release composition according to the present disclosure is used in the treatment of an autoimmune disease including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, irritable bowel syndrome, Crohn&#39;s disease, inflammatory bowel disease (IBD), Hashimoto&#39;s thyroiditis, Graves&#39; disease, Addison&#39;s disease, diabetes mellitus, idiopathic thrombocytopenic purpura, eosinophilic fasciitis, or antiphospholipid syndrome. 
     In some embodiments, the liposomal sustained-release composition according to the present disclosure is for use in pain relief or prevention of pain. 
     In some embodiments, the liposomal sustained-release composition according to the present disclosure is for use in anesthesia practice including, but not limited to, sedation, anxiety reduction, pain reduction, or delirium. 
     The disclosure will be further described with reference to the following specific, non-limiting examples. 
     EXAMPLES 
     The following examples illustrate the preparation and properties of certain embodiments of the present disclosure. 
     Example 1 Preparation of Liposomal Compositions 
     A. Preparation of Empty Liposomes 
     Liposomes were prepared via either a solvent injection method or a thin-film hydration method. The process for preparing empty liposomes using a solvent injection method is described as follows: HSPC, cholesterol and DSPE-mPEGr000 were mixed at a molar ratio of 3:2:0.045-0.3 and then were dissolved in absolute ethanol at 50 to 65° C. in a glass vial. The lipid solution was placed in a sonication bath to complete the lipid dissolution. Afterwards, the lipid solution was injected into a trapping agent solution under stirring (500 rpm) at 55 to 65° C., where the volume of the trapping agent solution is 4 to 10 times of the volume of lipid solution and the trapping agent solution can be 250 mM to 300 mM of ammonium sulfate, 300 mM ammonium sulfate in combination with 0.3 mM dextran sulfate, or 75 mM to 124 mM of triethylamine sucrose octasulfate to form a pro-liposome suspension. The obtained pro-liposome suspension was stirred (500 rpm) at 55 to 65° C. for 10 min followed by extruding for 5 to 10 times through a 0.2 μm polycarbonate membrane and then 3 to 5 times through a 0.1 μm polycarbonate membrane at 65° C. until the liposomes were in the size range of 90 to 160 nm. The liposome suspension was then dialyzed against a 9.4% (w/v) sucrose solution or saline using Float-A-Lyzer® dialysis devices (Spectra/Por®, MWCO: 50 kDa) at 4° C. under stirring (100 rpm) to remove the external trapping agents and ethanol. The volume of the dialysis solution was 100 times of the liposome suspension in each dialysis batch and three dialysis batches were applied at intervals of 12 hours. The resulting liposome suspension was filtered through a 0.2 μm polytetrafluoroethylene (PTFE) membrane to obtain the sterilized empty liposomes. The various formulations of empty liposomes prepared for drug loading are listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Liposome formulations 
               
            
           
           
               
               
               
            
               
                   
                 Lipids (molar ratio) 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 DSPE- 
                 Trapping agent 
               
               
                 Formulation 
                 HSPC 
                 Cholesterol 
                 mPEG 2000   
                 solution 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 I 
                 3 
                 2 
                 0.045 
                 300 mM ammonium 
               
               
                   
                   
                   
                   
                 sulfate 
               
               
                 II 
                 3 
                 2 
                 0.3 
                 250 mM ammonium 
               
               
                   
                   
                   
                   
                 sulfate 
               
               
                 III 
                 3 
                 2 
                 0.045 
                 300 mM ammonium 
               
               
                   
                   
                   
                   
                 sulfate/0.3 mM 
               
               
                   
                   
                   
                   
                 dextran sulfate 
               
               
                 IV 
                 3 
                 2 
                 0.045 
                 75 mM triethylamine 
               
               
                   
                   
                   
                   
                 sucrose octasulfate 
               
               
                 V 
                 3 
                 2 
                 0.045 
                 123.5 mM 
               
               
                   
                   
                   
                   
                 triethylamine sucrose 
               
               
                   
                   
                   
                   
                 octasulfate 
               
               
                   
               
            
           
         
       
     
     B. Drug Loading into Liposomes to Obtain Liposomal Compositions 
     Therapeutic agent as identified below was dissolved in 9.4% (w/v) sucrose or saline at the concentration of 20 mg/mL as a stock solution. Hydrochloric acid (HCl) was added into the therapeutic agent stock solution (to a final concentration up to 0.5 N) to accelerate therapeutic agent dissolution if necessary. L-histidine (L-His) buffer stock solution was prepared at the concentration of 31 mg/mL in 9.4% (w/v) sucrose or saline and was adjusted to pH 6.0 to 7.8 using 6 N HCl. Drug loading was performed by mixing the empty liposomes (prepared in Example 1-A), therapeutic agent stock solution, L-His stock solution, and 9.4% (w/v) sucrose or saline in a predetermined amount to result in a certain drug to lipid ratio (D/L) and followed by incubation at 20 to 60° C. for 30 min to obtain the liposomal composition. Liposomal compositions with D/L higher than 0.15 mol/mol were prepared as comparison examples. The drug loading profiles for some embodiments of the liposomal compositions are listed in Table 2 to Table 5. 
     C. Encapsulation Efficiency (EE) Analysis of Liposomal Compositions 
     The free form and liposomal form of therapeutic agent in the obtained liposomal compositions were separated by size exclusion chromatography through Sepharose® CL-4B (GE Healthcare) or Sephadex® G-50 Fine (GE Healthcare) resins. 2 mL of gel was packed in one column and the gel was washed by ddH 2 O and mobile phase solution (e.g., 9.4% ((w/v)) sucrose, 9.4% ((w/v)) sucrose containing L-His or saline, etc.) for at least 5 column volumes, respectively. 0.1 or 0.2 mL of the liposomal composition was added to the column drop-wisely and evenly distributed on the surface of the gel. After the sample immersing to the gel, 0.45 mL of the mobile phase solution was added to wash the column, followed by adding 1 mL or 0.7 mL of mobile phase solution to elute the liposomal form of drugs. The encapsulation efficiency was determined by 
     
       
         
           
             
               
                 
                   amount 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   of 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   therapeutic 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   agent 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   in 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   liposomal 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   form 
                 
                 
                   amount 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   of 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   therapeutic 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   agent 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   in 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   total 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   form 
                 
               
               × 
               100 
               ⁢ 
               % 
             
             , 
           
         
       
     
     in which the total form (containing free form and liposomal form of therapeutic agent) was prepared by 10 times or 3.5 times dilution of the liposomal composition to the mobile phase solution. For concentration determination of the therapeutic agents, liposomal- and total form of therapeutic agents were mixed with 100% methanol (MeOH) where the volume of MeOH was at least 4 times of the samples. The mixtures were then sonicated for 10 min to break the liposomes and subsequently diluted with diluents if further dilution was needed. The assays of the therapeutic agents were performed either in HPLC or UV plate reader. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Drug loading profiles of liposomal compositions 
               
               
                 with resiquimod as the therapeutic agent 
               
            
           
           
               
               
               
               
               
            
               
                   
                 D/L 
                 Drug 
                   
                 L-His 
               
               
                 Sample 
                 (mol/ 
                 conc. 
                 Empty 
                 (pH 6.5) 
               
               
                 name 
                 mol) 
                 (mg/mL) 
                 liposome 
                 conc. (mM) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 JCCR848-05 
                 0.439 
                 6 
                 Formulation I 
                 50 
               
               
                 JCCR848-06 
                 0.146 
                 2 
               
               
                 CHYR848-011 
                 0.192 
                 2 
                   
                 20 
               
               
                 CHYR848-014 
                 0.594 
                 0.05 
                 Formulation II 
                 20 
               
               
                 CHYR848-015 
                 0.198 
               
               
                 CHYR848-016 
                 0.059 
               
               
                 CHYR848-017 
                 0.020 
               
               
                 CHYR848-018 
                 0.010 
               
               
                 CHYR848-019 
                 0.005 
               
               
                 CHYR848-020 
                 0.594 
               
               
                 CHYR848-021 
                 0.198 
                 0.5 
               
               
                 CHYR848-022 
                 0.059 
               
               
                 CHYR848-023 
                 0.594 
                 6 
               
               
                 CHYR848-024 
                 0.198 
                 2 
               
               
                 CHYR848-025 
                 0.059 
                 0.6 
               
               
                 CHYR848-026 
                 0.020 
                 0.2 
               
               
                 CHYR848-027 
                 0.010 
                 0.1 
               
               
                 CHYR848-029 
                 0.576 
                 1 
                 Formulation III 
                 20 
               
               
                 CHYR848-030 
                 0.192 
               
               
                 CHYR848-031 
                 0.096 
               
               
                 CHYR848-032 
                 0.058 
               
               
                 CHYR848-033 
                 0.576 
               
               
                 CHYR848-034 
                 0.192 
               
               
                 CHYR848-035 
                 0.096 
               
               
                 CHYR848-036 
                 0.058 
                 0.1 
               
               
                 CHYR848-037 
                 0.019 
               
               
                 CHYR848-038 
                 0.010 
               
               
                 CHYR848-039 
                 0.005 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Drug loading profiles of liposomal sustained-release 
               
               
                 compositions with imiquimod as the therapeutic agent 
               
            
           
           
               
               
               
               
               
            
               
                   
                 D/L 
                 Drug 
                   
                 L-His 
               
               
                 Sample 
                 (mol/ 
                 conc. 
                 Empty 
                 (pH 6.5) 
               
               
                 name 
                 mol) 
                 (mg/mL) 
                 liposome 
                 conc. (mM) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 CHYIMQ-009 
                 0.502 
                 4 
                 Formulation I 
                 10 
               
               
                 CHYIMQ-010 
                 0.754 
                 1 
               
               
                 CHYIMQ-011 
                 0.251 
                 1 
               
               
                 CHYIMQ-012 
                 0.126 
                 1 
               
               
                 CHYIMQ-013 
                 0.075 
                 1 
               
               
                 CHYIMQ-014 
                 0.025 
                 0.1 
               
               
                 CHYIMQ-015 
                 0.013 
                 0.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Drug loading profiles of liposomal sustained-release 
               
               
                 compositions with fingolimod as the therapeutic agent 
               
            
           
           
               
               
               
               
               
            
               
                   
                 D/L 
                 Drug 
                   
                 L-His 
               
               
                 Sample 
                 (mol/ 
                 conc. 
                 Empty 
                 (pH 7.0) 
               
               
                 name 
                 mol) 
                 (mg/mL) 
                 liposome 
                 conc. (mM) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 JCCFIN18 
                 0.196 
                 2 
                 Formulation I 
                 50 
               
               
                 JCCFIN19 
                 0.393 
                 4 
               
               
                 JCCFIN20 
                 0.589 
                 6 
               
               
                 JCCFIN04 
                 0.785 
                 8 
               
               
                 CHYFIN01 
                 0.589 
                 1 
               
               
                 CHYFIN02 
                 0.196 
                 1 
               
               
                 CHYFIN03 
                 0.098 
                 1 
               
               
                 CHYFIN04 
                 0.059 
                 1 
               
               
                 CHYFIN05 
                 0.020 
                 0.1 
               
               
                 CHYFIN06 
                 0.010 
                 0.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Drug loading profiles of liposomal sustained-release compositions 
               
               
                 with dexmedetomidine as the therapeutic agent 
               
            
           
           
               
               
               
               
               
            
               
                   
                 D/L 
                 Drug 
                   
                 L-His 
               
               
                 Sample 
                 (mol/ 
                 conc. 
                 Empty 
                 (pH 6.5) 
               
               
                 name 
                 mol) 
                 (mg/mL) 
                 liposome 
                 conc. (mM) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 YW4_NL 
                 0.241 
                 2 
                 Formulation I 
                 10 
               
               
                 CHYDEX01 
                 0.904 
                 1 
               
               
                 CHYDEX02 
                 0.301 
                 1 
               
               
                 CHYDEX03 
                 0.15 
                 1 
               
               
                 CHYDEX04 
                 0.090 
                 1 
               
               
                 CHYDEX05 
                 0.030 
                 0.1 
               
               
                 CHYDEX06 
                 0.015 
                 0.1 
               
               
                   
               
            
           
         
       
     
     Example 2 In Vitro Release Profiles of Liposomal Compositions 
     Diafiltration using Amicon® Ultra-0.5 Centrifugal Filter Devices (50 kDa, Millipore®) can be performed prior to in vitro release experiments if the encapsulation efficiencies of the liposomal compositions were less than 70%. 
     9.4% sucrose containing 20 mM of L-His buffer was used as a diluent for dilution of liposomal compositions if different formulation concentrations were applied with the same D/L liposomal composition. The in vitro release profiles of the liposomal composition were carried out by mixing liposomal compositions and release medium. The dilution factor of the liposomal compositions in release medium was ten in all in vitro release experiments. The release conditions were either in 90% human plasma or 10% human plasma at room temperature for 5 min. The encapsulation efficiencies of the liposomal compositions in 90% or 10% human plasma were analyzed as described in 
     Example 1-C. Drug retention was calculated by 
     
       
         
           
             
               
                 EE 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 of 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 liposomal 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 composition 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 after 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 in 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 vitro 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 release 
               
               
                 EE 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 of 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 liposomal 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 composition 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 before 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 in 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 vitro 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 release 
               
             
             × 
             100 
             ⁢ 
             
               % 
               . 
             
           
         
       
     
     The liposomal compositions used in the in vitro release experiments, the release conditions and results are listed in Table 6. The release results are also shown in  FIG. 1A  to  FIG. 3 . 
     As illustrated in  FIGS. 1A-1B , the results indicated that the lower the D/L, the better the drug retention ability of liposomal composition having resiquimod as the therapeutic agent. The results shown in  FIG. 1A  were acquired from the data of IVR01 (D/L=0.19 mol/mol) and IVR02 (D/L=0.01 mol/mol), and the results shown in  FIG. 1B  were acquired from the data of IVR04 (D/L=0.19 mol/mol) and IVR05 (D/L=0.01 mol/mol) listed in Table 6. 
       FIG. 2  demonstrates the same trend of drug retention ability as  FIGS. 1A-1B : the lower the D/L (when D/L&lt;0.19 mol/mol), the better the drug retention ability of liposomal composition having resiquimod as the therapeutic agent. The results shown here were acquired from the data of IVR13 (D/L=0.58 mol/mol), IVR14 (D/L=0.19 mol/mol), IVR15 (D/L=0.10 mol/mol), and IVR16 (D/L=0.06 mol/mol) listed in Table 6. 
     Referring to  FIG. 3 , the results indicate that a liposomal composition having high D/L (0.19 mol/mol) exhibits poor drug retention in human plasma when applied in low concentration (0.05 mg/mL). The results shown in  FIG. 3  were acquired from the data of IVR01 (drug conc.=0.05 mg/mL) and IVR12 (drug conc.=2.00 mg/mL) listed in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Conditions and results of in vitro release experiments 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Resiquimod 
                   
                   
               
               
                   
                   
                   
                 Resiquimod 
                 conc. in 
               
               
                 IVR 
                   
                   
                 conc. in 
                 release 
                   
                 Drug 
               
               
                 trial 
                   
                 D/L 
                 formulation 
                 medium 
                 Release 
                 retention 
               
               
                 no. 
                 Sample 
                 (mol/mol) 
                 (mg/mL) 
                 (mg/mL) 
                 medium 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 IVR01 
                 CHYR848- 
                 0.192 
                 0.05 
                 0.005 
                 90% 
                 0.0 
               
               
                   
                 011 
                   
                   
                   
                 human 
               
               
                 IVR02 
                 CHYR848- 
                 0.01 
                 0.05 
                 0.005 
                 plasma 
                 14.9 
               
               
                   
                 018 
               
               
                 IVR03 
                 CHYR848- 
                 0.192 
                 1 
                 0.1 
                   
                 44.3 
               
               
                   
                 011 
               
               
                 IVR04 
                 CHYR848- 
                 0.192 
                 0.05 
                 0.005 
                 10% 
                 50.9 
               
               
                   
                 011 
                   
                   
                   
                 human 
               
               
                 IVR05 
                 CHYR848- 
                 0.01 
                 0.05 
                 0.005 
                 plasma + 
                 79.2 
               
               
                   
                 018 
                   
                   
                   
                 80% PBS 
               
               
                 IVR06 
                 CHYR848- 
                 0.192 
                 1 
                 0.1 
                   
                 89.6 
               
               
                   
                 011 
               
               
                 IVR10 
                 CHYR848- 
                 0.594 
                 1 
                 0.1 
                 90% 
                 21.2 
               
               
                   
                 020 
                   
                   
                   
                 human 
               
               
                 IVR11 
                 CHYR848- 
                 0.198 
                 1 
                 0.1 
                 plasma 
                 20.5 
               
               
                   
                 024 
               
               
                 IVR12 
                 CHYR848- 
                 0.198 
                 2 
                 0.2 
                   
                 33.8 
               
               
                   
                 024 
               
               
                 IVR13 
                 CHYR848- 
                 0.576 
                 1 
                 0.1 
                   
                 33.2 
               
               
                   
                 029 
               
               
                 IVR14 
                 CHYR848- 
                 0.192 
                 1 
                 0.1 
                   
                 30.6 
               
               
                   
                 030 
               
               
                 IVR15 
                 CHYR848- 
                 0.096 
                 1 
                 0.1 
                   
                 41.7 
               
               
                   
                 031 
               
               
                 IVR16 
                 CHYR848- 
                 0.058 
                 1 
                 0.1 
                   
                 48.8 
               
               
                   
                 032