Injectable in situ cross-linked depots for ultra-long-term delivery of hydrophilic drugs

Abstract

A therapeutic or diagnostic delivery system, and methods of making and using the same, are disclosed. The system is a solvent free, injectable, biodegradable, and in situ crosslinking depot (ISCD) platform for ultra-long-term release of hydrophilic drugs.

Description

[0001] INJECTABLE IN SITU CROSS-LINKED DEPOTS FOR ULTRA-LONG-TERM DELIVERY OF

[0002] HYDROPHILIC DRUGS

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] This invention was made with government support under award number 1 R21 DA057701 -01 (agreement 2022A003259) from the National Institutes of Health. The government has certain rights in the invention.

[0005] Background of the Invention

[0006] Patient adherence to medications is a major obstacle towards achieving effective treatment of numerous diseases, especially in the treatment of chronic conditions where patients are required to take therapeutics throughout their entire life1-3. Sustained release technologies, such as long-acting injectables and implants simplify dosing schedules and minimize side effects by maintaining steady blood levels of the drug4-11. These benefits reduce the need for frequent medical visits and enhance treatment regimen adherence, which is particularly advantageous in developing countries with limited healthcare infrastructure and resources.

[0007] Multiple long-acting implants and injectables are in clinical use for the treatment and prevention of different diseases.12 13Although implantable devices have shown success for the longterm delivery of both hydrophobic14-15and hydrophilic drugs9 16, they require invasive, timeconsuming medical procedures for insertion, which may pose significant challenges, particularly in low resource settings and low-middle income countries. Furthermore, implants in general tend to be more susceptible to local inflammation when compared to injectable alternatives.17-18Conventional injectable platforms such as microparticles, and in s / tu-forming implants (ISFI) have been documented to achieve prolonged release of hydrophobic drugs but have poor ability to achieve similar long-term release of hydrophilic drugs19-22. In the case of ISFI, which typically consist of a hydrophobic polymer, poly (lactic-co-glycolic acid) (PLGA) dissolved in N-methyl-2-pyrrolidone (NMP), efflux of the solvent during phase conversion tends to release a significant amount of drug as initial burst, which increases with the hydrophilicity of the drug. Microparticles and ISFI based approaches also promote significant influx and efflux of water due to their large pores, leading to fast diffusion of hydrophilic drugs.21 23-24Moreover, certain long-acting injectable approaches employ manufacturing processes such as wetmilling,25-26which require the drug to be hydrophobic and are fundamentally incompatible with hydrophilic drugs.6’8’12’20’23’27

[0008] Hydrophilic drugs constitute a major fraction of all the drugs used for the treatment and management of chronic conditions. Examples include antipsychotics (e.g., olanzapine, risperidone, and quetiapine), antidepressants (e.g., fluoxetine, sertraline, and citalopram), anticonvulsants (e.g., carbamazepine, phenytoin, and valproic acid), anti-inflammatory drugs (e.g., ibuprofen, naproxen, and celecoxib), antibiotics (e.g., amoxicillin, vancomycin, and gentamicin), and treatments for substance abuse (e.g., naltrexone, buprenorphine, and methadone), which often consist of hydrophilic drugs. Therefore, there is an unmet need to develop an injectable and biodegradable platform that enables ultra-long-term delivery of hydrophilic drugs. Additionally, the platform should be designed to allow for retrieval in the event of local or systemic drug toxicity.

[0009] Summary of Invention

[0010] Provided herein is an extended-release injectable drug delivery platform, in situ cross-linked depots (ISCD), capable of providing continuous delivery of both hydrophilic and hydrophobic drugs for over 6 months. The main component of ISCD is a vinyl end functionalized, low-molecular-weight liquid hydrophobic pre-polymer - polycaprolactone (PCL). This pre-polymer effectively suspends both hydrophilic and hydrophobic drugs, transitioning into a solid monolithic depot upon injection with a radical initiator and an accelerator. ISCD has two key features, which enable ultra-long-term release of hydrophilic drugs. These include a solvent-free design and a dense mesh network, both attributed to the use of ultra-low-molecular weight PCL. The liquid state of the pre-polymer eliminates the need for a solvent, minimizing the risk of high burst release, which is commonly associated with solvent exchange processes in ISFL Additionally, the ultra-low molecular weight of methacrylated PCL forms a dense mesh upon cross-linking, which limits water influx / efflux, thereby controlling the drug release. In vitro, ISCD demonstrated ultra-long-term release of multiple drugs of varying hydrophilicity for at least six months. We also identified design parameters that can tailor the polymer network to tune the drug release kinetics and degradation of ISCD. Notably, modulating intrinsic factors, such as decreasing the concentrations of cross-linkers or using a higher molecular weight of the pre-polymer, increases drug release. Additionally, integrating external hydrophilic polymeric additives including alongside the hydrophobic pre-polymer can enhance drug release and depot degradation rate, which can be further fine-tuned by varying the degree of cross-linking of the polymer additive. In vivo, ISCD formulations loaded with tenofovir alafenamide (TAF) or naltrexone (NAL) with water solubilities of 5.63 and 100 mg / mL, respectively, showed sustained release for at least six months with plasma concentrations within the therapeutic window. Impressively, ISCD also demonstrated ultra-long-term release of the hydrophobic drug tacrolimus (TAC), achieving sustained release for at least 200 days in vivo. We have thoroughly evaluated the biocompatibility, retrievability and drug delivery capabilities of our ISCD platform to ensure its safety and adaptability. ISCD has also demonstrated ultra-long-term release of various combinations of antiretroviral drugs used clinically to prevent and treat HIV. Overall, we believe that ISCD holds promise for further development as an ultralong-acting platform for a variety of other diseases where patient adherence is critical.

[0011] The invention comprises, consists of, or consists essentially of the following features or steps, in any combination.

[0012] In one aspect, the present invention is an injectable composition comprising:

[0013] (i) at least one crosslinkable low molecular weight hydrophobic pre-polymer with a molecular weight of less than 10000 Da (e.g., less than 5000 Da, less than 2000 Da, less than 1000 Da, less than 600 Da);

[0014] (ii) at least one crosslinking initiator and accelerator pair (e.g., a redox pair); and

[0015] (iii) at least one active agent (e.g., any active agent described herein).

[0016] In another aspect, the present invention is an injectable composition comprising: (i) at least one hydrophobic methacrylated or acrylated pre-polymer with a molecular weight of less than 10000 Da (e.g., less than 5000 Da, less than 2000 Da, less than 1000 Da, less than 600 Da);

[0017] (ii) at least one organic peroxide (initiator, e.g., any initiator described herein) and a secondary or tertiary amine (accelerator, e.g., any accelerator described herein) redox pair for cross linking; and

[0018] (iii) at least one active agent (e.g., any active agent described herein).

[0019] In some embodiments of the aforementioned aspects, the injectable composition is substantially solvent-free.

[0020] In another aspect, the present invention is an injectable composition comprising:

[0021] (i) at least one hydrophobic methacrylated or acrylated pre-polymer with a molecular weight of less than 10000 Da (e.g., less than 5000 Da, less than 2000 Da, less than 1000 Da, less than 600 Da);

[0022] (ii) at least one organic peroxide (initiator, e.g., any initiator described herein) and a secondary or tertiary amine (accelerator, e.g., any accelerator described herein) redox pair for cross linking;

[0023] (iii) at least one active agent (e.g., any active agent described herein); and

[0024] (iv) a solvent additive to dissolve the pre-polymer and / or to prolong the curing time of the pre- polymer / initiator / accelerator mixture.

[0025] In some embodiments, the solvent additive is selected from vinyl acetate, ethyl acetate, propyl acetate, butyl acetate or a combination thereof. In specific embodiments, the solvent additive is present at a concentration between 5-50% w / v. In specific embodiments, the solvent additive is present at a concentration between 15-30% w / v.

[0026] In some embodiments of any of the forementioned aspects, the active agent is a therapeutic agent.

[0027] In some embodiments of any of the forementioned aspects, the active agent is a prophylactic agent.

[0028] In some embodiments of any of the forementioned aspects, the active agent is a diagnostic agent.

[0029] In some embodiments of any of the forementioned aspects, the composition comprises of one active agent.

[0030] In some embodiments of any of the forementioned aspects, the composition comprises at least two active agents.

[0031] In some embodiments of any of the forementioned aspects, the active agent is suspended in the pre-polymer using ultra-sonication.

[0032] In some embodiments of any of the forementioned aspects, the active agent is suspended in the pre-polymer using micronization.

[0033] In some embodiments of any of the forementioned aspects, the active agent is dissolved in the pre-polymer using a co-solvent. In another aspect, the present invention is a depot formed from the injectable composition crosslinks to form a depot, e.g., transitioning from a liquid to a solid phase, with a curing time ranging between 1-10 min.

[0034] In some embodiments, the depot is a solid monolithic depot.

[0035] In some embodiments, the active agent is incorporated into the depot by physical entrapment. In some embodiments, the active agent is incorporated into the depot by covalent conjugation with the polymer.

[0036] In some embodiments, the active agent is distributed throughout the depot and released from the depot.

[0037] In some embodiments, the depot releases at least 80% of the active agent in a time 2.5 times greater than tso in the post-injection environment.

[0038] In some embodiments, the time during which 10% of the active agent is released from the depot is equal to or greater than of tso in the post-injection environment.

[0039] In some embodiments, tso is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or at least 15 months.

[0040] In some embodiments of the aforementioned composition or the depot, wherein the injectable composition comprises at least one polymeric additive to tune the release kinetics of the encapsulated active agent.

[0041] In specific embodiments, the polymeric additive is either a low molecular weight noncrosslinking hydrophobic polymer or a low molecular weight cross-linking or non-cross-linking hydrophilic polymer, with molecular weight less than 10000 Da (e.g., less than 5000 Da, less than 2000 Da, less than 600 Da). In specific embodiments, the polymeric additive is selected from polyethylene glycol (PEG), Poly(ethylene glycol) methacrylate (PEGMMA), Polyethylene glycol) dimethacrylate (PEGDMA), vinyl acetate, polydimethylsiloxane (PDMS), or polycaprolactone (PCL). In specific embodiments, the polymeric additive is present at a concentration between 5-90% w / v. In further specific embodiments, the polymeric additive is present at a concentration between 25-75% w / v.

[0042] In some embodiments of the composition or the depot of any of the forementioned embodiments, the hydrophobic pre-polymer comprises a pre-polymer with a molecular weight less than 2000 Da (e.g., less than 1000 Da, less than 600 Da, less than 300 Da).

[0043] In some embodiments of the composition or the depot of any of the forementioned embodiments, the hydrophobic pre-polymer comprises a polymer with a molecular weight less than 1000 Da (e.g., less than 600 Da, less than 300 Da).

[0044] In some embodiments of the composition or the depot of any of the forementioned embodiments, the hydrophobic pre-polymer comprises a pre-polymer with a molecular weight less than 600 Da (e.g., less than 300 Da).

[0045] In some embodiments of the composition or the depot of any of the forementioned embodiments, the hydrophobic pre-polymer is a liquid below 45 °C. In some embodiments of the composition or the depot of any of the forementioned embodiments, the hydrophobic pre-polymer comprises polycaprolactone dimethylacrylate (PCLDMA) or polycaprolactone trimethacrylate (PCLTMA) or both.

[0046] In some embodiments of the composition or the depot of any of the forementioned embodiments, the initiator is benzoyl peroxide.

[0047] In some embodiments of the composition or the depot of any of the forementioned embodiments, the accelerator is a secondary or tertiary amine or a combination of amines selected from a group consisting of: N,N-dimethyl-p-toluidine, N,N-dimethylaniline, N-p-hydroxyethyl-aniline, N,N-di(p-hydroxyethyl)-aniline, N,N-di(p-hydroxyethyl)-p-toluidine, N-methyl-aniline, and N-methyl-p- toluidine. In specific embodiments, the accelerator is N,N-dimethyl-p-toluidine.

[0048] In some embodiments of the composition or the depot of any of the forementioned embodiments, the concentration of crosslinking initiator and accelerator is between 0.025-1 % w / v each.

[0049] In some embodiments of the composition or the depot of any of the forementioned embodiments, the concentration of crosslinking initiator and accelerator is between 0.05%-0.5% w / v each.

[0050] In some embodiments of the composition or the depot of any of the forementioned embodiments, the concentration of crosslinking initiator and accelerator is between 0.1 %-0.3% w / v each.

[0051] In some embodiments of the composition or the depot of any of the forementioned embodiments, the injectable composition has a viscosity between 0.01-1 Pa.sa (e.g., 0.01 Pa.sa, 0.05 Pa.sa, 0.10 Pa.sa, 0.15 Pa.sa, 0.20 Pa.sa, 0.25 Pa.sa, 0.30 Pa.sa, 0.35 Pa.sa, 0.40 Pa.sa, 0.45 Pa.sa, 0.50 Pa.sa, 0.55 Pa.sa, 0.60 Pa.sa, 0.65 Pa.sa, 0.70 Pa.sa, 0.75 Pa.sa, 0.80 Pa.sa, 0.85 Pa.sa, 0.90 Pa.sa, 0.95 Pa.sa, or 1 .00 Pa.sa).

[0052] In some embodiments of the composition or the depot of any of the forementioned embodiments, the composition comprises between 1 % and 50% w / w of the active agent (e.g., 1 % w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w).

[0053] In some embodiments of the composition or the depot of any of the forementioned embodiments, the composition comprises between 2% and 12% w / w of the active agent (e.g., 2% w / w, 3% w / w, 4% w / w, 5% w / w, 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11 % w / w, 12% w / w).

[0054] In some embodiments of the composition or the depot of any of the forementioned embodiments, the composition comprises at least 10 mg / mL of active agent (e.g., 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, more than 30 mg / mL, more than 50 mg / mL, more than 100 mg / mL).

[0055] In some embodiments of the composition or the depot of any of the forementioned embodiments, the composition comprises 30 to 150 mg / mL of active agent (e.g., 30 mg / mL, 30 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL). In some embodiments of the composition or the depot of any of the forementioned embodiments, the active agent is hydrophobic (e.g., as described herein).

[0056] In some embodiments of the composition or the depot of any of the forementioned embodiments, the active agent is hydrophilic (e.g., as described herein).

[0057] In some embodiments of the composition or the depot of any of the forementioned embodiments, the active agent is selected from the group consisting of: Analgesics, Antianxiety Drugs, Antiarrhythmics, Antibacterials, Antibiotics, Anticoagulants and Thrombolytics, Anticonvulsants, Antidepressants, Antiemetics, Antifungals, Antihistamines, Antihypertensives, Anti-Inflammatories, Antineoplastics, Antipsychotics, Antipyretics, Antiretrovirals, Antivirals, Barbiturates, Beta-Blockers, Bronchodilators, Corticosteroids, Cytotoxics, Diuretics, Hormones, Hypoglycemics Immunosuppressives, Muscle Relaxants, Sedatives, Tranquilizer, and Vitamins or a combination thereof.

[0058] In some embodiments of the composition or the depot of any of the forementioned embodiments, the active agent comprises of a small molecule, or a biologic selected from a group consisting of: protein, peptide, enzyme and nucleic acid.

[0059] In some embodiments of the composition or the depot of any of the forementioned embodiments, the active agent is selected from the group consisting of: tenofovir alafenamide, emtricitabine, abacavir sulfate, lamivudine, zidovudine, tacrolimus, naltrexone, and a combination thereof.

[0060] In some embodiments of the composition or the depot of any of the forementioned embodiments, the injectable composition can be injected subcutaneously or intramuscularly into a subject and cures to form an implant in situ.

[0061] In some embodiments of the composition or the depot of any of the forementioned embodiments, the composition can be cured outside the subject to form an implant, which is then implanted subcutaneously or intramuscularly into a subject.

[0062] In another aspect, the present invention is an injectable composition comprising of:

[0063] (i) at least one hydrophobic methacrylated or acrylated polymer with a molecular weight of less than 10000 Da;

[0064] (ii) at least one organic peroxide (initiator, e.g., an initiator as described herein) and a secondary or tertiary amine (accelerator, e.g., an accelerator as described herein) redox pair for cross linking;

[0065] (iii) at least one therapeutic, prophylactic, or diagnostic agent; and

[0066] (iv) at least one polymeric additive to tune the release kinetics of the encapsulated agent.

[0067] In another aspect, the present invention is an injectable composition comprising of:

[0068] (i) at least one hydrophobic methacrylated or acrylated polymer with a molecular weight of less than 10000 Da;

[0069] (ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking;

[0070] (iii) at least one therapeutic, prophylactic, or diagnostic agent; and

[0071] (iv) at least one polymeric additive to tune the release kinetics of the encapsulated agent. (v) a solvent additive to dissolve the pre-polymer and / or to prolong the curing time of the pre- polymer / initiator / accelerator mixture.

[0072] In another aspect, the present invention is a kit comprising the components needed to make an injectable composition or depot according to any of the forementioned aspects or embodiments thereof.

[0073] In some embodiments, the kit comprises of:

[0074] (i) a pre-polymer or a pre-polymer and active agent mixture; and

[0075] (ii) a separate mixture comprising BPO and DMT.

[0076] In some embodiments, the kit further comprising instructions for preparing an injectable composition with different active agents.

[0077] In another aspect, the invention is a method of treating a subject in need thereof, the method comprising the steps:

[0078] (i) prepare an injectable composition according to any of the above-mentioned aspects or embodiments thereof;

[0079] (ii) administer the injectable composition to a subject;

[0080] (iii) the composition crosslinks to form a depot in vivo; and

[0081] (iv) the depot formed releases the active agent to the subject over time.

[0082] In some embodiments, step (i) of the above method of treatment comprises using a kit of the present invention.

[0083] In some embodiments, the subject is a mammal. In specific embodiments, the subject is a human, dog, cat, or farm animal.

[0084] In some embodiments, step (ii) of the above method of treatment comprises administration of the injectable composition subcutaneously or intramuscularly to the subject.

[0085] In some embodiments of the above method of treatment, the depot is biodegradable.

[0086] In some embodiments, step (iv) of the above method of treatment comprises the release of at least 80% of the active agent in a time 2.5 times greater than tso.

[0087] In some embodiments of the above method of treatment, the time during which 10% of the active agent is released is equal to or greater than of tso.

[0088] In some embodiments of the above method of treatment, tso is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months (e.g., long acting), at least 7 months (e.g., ultra-long-acting), at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or at least 15 months (e.g., ultra-long-acting).

[0089] In another aspect, the present invention is a method of treating a subject in need thereof, the method comprising the steps:

[0090] (i) prepare an injectable composition according to any of the above-mentioned aspects or embodiments thereof;

[0091] (ii) the composition crosslinks to form an implant;

[0092] (iii) the implant is then implanted subcutaneously or intramuscularly into a subject; and

[0093] (iv) the implant releases the active agent to the subject over time. In some embodiments, step (i) of the above method of treatment comprises using a kit of the present invention.

[0094] In some embodiments, the subject is a mammal. In specific embodiments, the subject is a human, dog, cat, or farm animal.

[0095] In some embodiments, step (ii) of the above method of treatment comprises administration of the implant subcutaneously or intramuscularly to the subject.

[0096] In some embodiments of the above method of treatment, the implant is biodegradable.

[0097] In some embodiments, step (iv) of the above method of treatment comprises the release of at least 80% of the active agent in a time 2.5 times greater than tso.

[0098] In some embodiments of the above method of treatment, the time during which 10% of the active agent is released is equal to or greater than of tso.

[0099] In some embodiments of the above method of treatment, tso is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months (e.g., long-acting), at least 7 months (e.g., ultra-long-acting), at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or at least 15 months (e.g., ultra-long-acting).

[0100] In another aspect, the invention is a method of diagnosing a disease in a subject, the method comprising the steps:

[0101] (i) prepare an injectable composition according to any of the above-mentioned aspects or embodiments thereof, wherein the injectable composition comprises of a diagnostic agent;

[0102] (ii) administer the injectable composition to a subject;

[0103] (iii) the composition crosslinks to form a depot in vivo; and

[0104] (iv) the depot formed releases the diagnostic agent to the subject over time.

[0105] In some embodiments, step (i) of the above method of treatment comprises using a kit of the present invention.

[0106] In some embodiments, the diagnostic agent is a small molecule or biologic.

[0107] In some embodiments, the diagnostic agent is a fluorescent marker.

[0108] Achieving ultra long-term delivery of hydrophilic drugs via injectable system remains a major challenge. Ultra-long acting implants have demonstrated potential for both hydrophobic and hydrophilic drugs; however, they require an invasive insertion procedure conducted in specialized environments, which restricts their suitability for resource-limited settings.9 16 23 28Furthermore, implants tend to be more susceptible to local inflammation when compared to injectable alternatives.17-18The present invention is directed to a solvent free, injectable, biodegradable, and in situ crosslinking depot (ISCD) platform for ultra-long term release of hydrophilic drugs. ISCD consists of a low molecular weight pre-polymer methacrylated polycaprolactone (PCL). Both hydrophilic and hydrophobic drugs can be suspended / dissolved in the pre-polymer, and when injected along with a radical initiator and an accelerator, the polymer crosslinks in situ, resulting in a solid monolithic and degradable depot, integrating the unique advantages of injectability and retrievability. Low molecular weight PCL forms a dense mesh, which limits water influx / efflux and hence reduces the drug release. The liquid state of the polymer obviates the need for solvent, minimizing initial burst release due to solvent exchange process, as observed with in situ forming implants. Drug release and ISCD degradation can be tailored by modifying their polymer network via altering the concentration of accelerator and initiator, molecular weight of methacrylated PCL, or by incorporation of a hydrophilic polymer or a non-crosslinking polymer. We demonstrated sustained release of seven hydrophilic drugs with varying solubility or drug combinations for over seven months in vitro. Ultra-long term drug release and depot degradation was also demonstrated in rats for at least six months without any evidence of local inflammation or fibrosis. Excitingly, the platform also enabled ultra-long-term release of a model hydrophobic drug - tacrolimus for at least six months. This platform holds promise for developing ultra-long-acting therapies across a wide range of diseases.

[0109] Definitions

[0110] Abbreviations and terms that are commonly used in the fields of organic chemistry, medicinal chemistry, pharmacology, and medicine and are well known to practitioners in these fields are used herein. Representative definitions are provided below:

[0111] The term, “accelerator” as used herein refers to a compound or compounds employed for accelerating polymerization. The accelerator can be used in catalytic quantities or stoichiometric quantities.

[0112] The term, “accelerator pair” as used herein refers to a redox pair, which includes an organic peroxide, e.g., benzoyl peroxide (BPO), and an accelerator, e.g., N,N-dimethyl-p-toluidine (DMT).

[0113] The terms “active agent” as used herein refers to the active pharmaceutical ingredient, e.g., a therapeutic agent. The active agent can be distributed into the present invention, e.g., via physical entrapment or via covalent conjugation with the polymer. In some embodiments, the active agent is either suspended into the pre-polymer using ultra-sonication or micronization. In some embodiments, the active agent is dissolved into the pre-polymer or dissolved in a small amount of a co-solvent.

[0114] The terms “covalent conjugation with the polymer” as used herein refers to the active agent being bound to a polymer within the crosslinked composition of the invention. The active agent can be conjugated by reaction of a functional group on the active agent (e.g., a hydroxyl, amine, carboxyl, or sulfhydryl moiety) and a functional group on the polymer (e.g., a hydroxyl, amine, carboxyl, or sulfhydryl moiety). The covalent link can optionally include a linker, such as a diacid (e.g., succinic acid), a diamine, or a diol to form a covalent link between the active agent and the polymer. The covalent conjugation can be made to the pre-polymer prior to crosslinking. Alternatively, the active agent can be modified to include a crosslinkable functionality and undergoes crosslinking with the prepolymer at the time the crosslinked composition is prepared.

[0115] The term “amine,” as used herein, represents a moiety of formula N(R1)(R2)(R3), wherein each of R1, R2, and R3is, independently, selected from H, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl. The amine of the present invention can function as an accelerator in the formation of the crosslinked compositions of the invention.

[0116] The term, “crosslink” as used herein refers to one or more bonds that connect one prepolymer chain to another. A pre-polymer that is “crosslinkable” is a pre-polymer with functionality (or functionalities) capable of undergoing a reaction to form the crosslink bond(s) with another pre- polymer. Examples of a crosslinkable functionality of the present invention includes, but is not limited to, methacrylate, acrylate, amine, carboxyl, and hydroxyl. The crosslinking of two or more prepolymers can be aided by employing a “crosslinking initiator” in the reaction. Examples of crosslinking initiators of the present invention include thermal initiators, which may include, but are not limited to, tert-amyl peroxybenzoate; 4,4-azobis (4-cyanovaleric acid); 1 ,1 ’-azobis (cyclohexanecarbonitrile); 2,2’-azobisisobutyronitrile (AIBN); benzoyl peroxide; 2,2-bis (tertbutylperoxy) butane; 1 , 1 -bis(tert- butylperoxy)cyclohexane; 2,5-bis (tert-butylperoxy)-2,5-dimethylhexane; 2,5-bis(tert-butylperoxy)-2,5- dimethyl-3-hexyne; bis(1-(tert-butylperoxy)-1-methylethyl)benzene; 1 , 1 -bis (tert-butylperoxy)-3,3,5- trimethylcyclohexane; tert-butyl hydroperoxide; tert-butyl peracetate; tert-butyl peroxide; tert-butyl peroxybenzoate; tert-butylperoxy isopropyl carbonate; cumene hydroperoxide; cyclohexanone peroxide; dicumyl peroxide; lauroyl peroxide; 2,4- pentanedione peroxide; peracetic acid; tert-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, p- menthane hydroperoxide; 1 ,1 ,3,3-tetramethylbutyl hydroperoxide, acetyl peroxide, p-chlorobenzoyl peroxide, 2,4-dichlorobenzoyl peroxide, ditoluoyl peroxide, decanoyl peroxide, lauroyl peroxide, isobutyryl peroxide, diisononanoyl peroxide, pelargonyl peroxide, tert-butyl peroxyacetate, tert-butyl peroxymaleic acid, tert-butyl peroxy-isobutyrate, tert-butyl peroxypivalate, tert-butyl peroxycrotonate, tert-butyl-peroxy-(2-ethylhexanoate), 2,5-dimethyl-2,5-bis-(2-ethylhexanoylperoxy) hexane, 2,6- dimethyl-2,5-bis-(benzoylperoxy) hexane, 2,5-dimethyl-2,5-bis-(tert-butyl-peroxy) hexane, 2,5- dimethyl-2,5-bis-(tert-butylperoxy)-hexyne-3, di-tert-butyldiperoxyphthalate, 1 ,1 ,3,3- tetramethylbutylperoxy-2-ethyl-hexanoate, di-tert-butyl peroxide, di-tertamyl peroxide, tert-amyl-tert- butyl peroxide, 1 ,1-di-tert-butylperoxy-3,3,5-trimethyl cyclohexane, bis-(tert-butylperoxy)- diisopropylbenzene, n-butyl-4,4-bis-(tert-butylperoxy)valerate, dicumyl peroxide, acetyl acetone peroxide, methyl ethyl ketone peroxide, cyclohexanone peroxide, tert-butylperoxy isopropyl carbonate, 2,2-bis-(tert-butylperoxy) butane, di-(2-ethylhexyl) peroxydicarbonate, and bis-(4-tert- butylcyclohexyl) peroxydicarbonate.

[0117] The term, “depot” as used herein refers to a crosslinked formulation formed after mixing the pre-polymer with an initiator and accelerator, in the presence or absence of an active agent, and that provides a controlled release of an active agent into the body. A depot of the present invention may be, for example, a solid monolithic depot, e.g., a solid drug delivery system.

[0118] The term “hydrophilic” as used in the context of the present invention refers to (i) hydrophilic active agents; and / or (ii) the use of hydrophilic polymers in the crosslinked compositions of the invention to increase the water permeability of implants. In some embodiments, the hydrophilic active agents and hydrophilic polymers are dissolved in aqueous mixtures to prepare the crosslinked compositions. In the present invention, a hydrophilic active agent or hydrophilic polymer has a water solubility at pH 7.0 of greater than or equal to 0.1 mg / mL.

[0119] The term “hydrophobic” as used in the context of the present invention refers to (i) hydrophobic active agents; and / or (ii) the use of hydrophobic pre-polymers in the crosslinked compositions of the invention. In the present invention, a hydrophobic active agent or hydrophobic pre-polymer has a water solubility at pH 7.0 of less than 0.1 mg / mL. The term “injectable composition” as used herein refers to an injectable mixture (prior to solidification and crosslinking) which has low enough viscosity for injection by conventional 18-25- gauge needles (see, e.g., FIG 1A). If not otherwise specified, the viscosity value of the pre-polymeric solution (e.g., prior to crosslinking) or the injectable composition is given in Pa sa (Pascal second) unit. Preferably, the viscosity of the injectable composition is less than 0.8 Pa.sa. Preferably, the injectable composition of the present invention can be injected subcutaneously or intramuscularly.

[0120] “Low molecular weight pre-polymer” refers to a pre-polymer with a molecular weight less than 10000 Da. Preferably, the low molecular weight pre-polymer has a molecular weight < 2000 Da.

[0121] The terms “long-acting”, “long acting injectable”, “controlled release”, “sustained release”, and “extended release” as used interchangeably herein refer to a formulation that prolongs the release rate of an active agent from the post-injection environment. In preferred embodiments of the present invention, long-acting refers to when the crosslinked composition releases at least 80% of the active agent in a time 2.5 times greater than tso (see, e.g., Example 7). In some embodiments of the present invention, the long-acting formulation is a depot.

[0122] As used herein, “tso” is the time at which 50% of the administered active agent has been released from a crosslinked composition of the invention. Time tio is, correspondingly, the time at which 10% of the administered active has been released from a crosslinked composition of the invention. When the release curve is perfectly linear, tio = of tso. When there is an initial burst of released drug, tio is much less than of tso. In the compositions and methods of the invention tio can preferably be equal to or greater than of tso. Agent release from a crosslinked composition of the invention can be measured as described in Examples herein, e.g., Example 5 and FIG 17.

[0123] “Organic peroxides” are a class of organic compounds that contain oxygen in a bivalent (-O- O-) structure and are derivatives of hydrogen peroxide. Exemplary organic peroxides of the present invention include benzoyl peroxide (BPO), tert-butyl hydroperoxide, tert-butyl peroxide, cumene hydroperoxide, cyclohexanone peroxide, dicumyl peroxide, lauroyl peroxide, 2,4-pentanedione peroxide, tert-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5- dihydroperoxide, p-menthane hydroperoxide, 1 ,1 ,3,3-tetramethylbutyl hydroperoxide, acetyl peroxide, p-chlorobenzoyl peroxide, 2,4-dichlorobenzoyl peroxide, ditoluoyl peroxide, decanoyl peroxide, lauroyl peroxide, isobutyryl peroxide, diisononanoyl peroxide, pelargonyl peroxide, di-tert-butyl peroxide, ditertamyl peroxide, tert-amyl-tert-butyl peroxide, dicumyl peroxide, acetyl acetone peroxide, methyl ethyl ketone peroxide, and cyclohexanone peroxide.

[0124] The “post-injection environment” refers to the system where the pharmaceutical composition of the present invention takes effect. For example, the post-injection environment can be the subject.

[0125] “Pre-polymer” as used herein, refers to a monomer, group of monomers, or a low molecular weight polymer that serves as a reagent in the formation of a crosslinked polymer of the present invention.

[0126] The term “subject,” as used herein, represents a human or non-human animal (e.g., a mammal) that is suffering from, or is at risk of, disease or condition, as determined by a qualified professional (e.g., a doctor or a nurse practitioner) with or without known in the art laboratory test(s) of sample(s) from the subject. In some embodiments of the present invention, the subject is a mammal. In specific embodiments, the subject is a human, dog, cat, or farm animal. A farm animal includes animals raised for food or labor. Nonlimiting examples of farm animals include cow, horse, donkey, mule, pig, chicken, turkey, duck, goose, sheep, goat, rabbit, llama, camel, buffalo, ox, and the like.

[0127] “Substantially solvent-free” as used herein, refers to a composition that includes less than 5% w / w of solvent (e.g., less than 4% w / w, less than 3% w / w, less than 2% w / w, less than 1% w / w, or completely free of solvent). A solvent of the present invention is a low molecular weight molecule (e.g., ethanol, ethyl acetate, diethyl ether, acetone, dimethylformamide) for solubilizing (e.g., fully solubilizing, partially solubilizing, or improving solubility) other components in the composition. In particular embodiments of the present invention, the compositions are completely solvent free.

[0128] “Treatment” and "treating," as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, or cure a disease or condition. This term includes active treatment (treatment directed to improve the disease or condition); causal treatment (treatment directed to the cause of the associated disease or condition); palliative treatment (treatment designed for the relief of symptoms of the disease or condition); and supportive treatment (treatment employed to supplement another therapy). Exemplary treatments of the present invention include administration of active agents such as a therapeutic agent (agent used to improve, ameliorate, stabilize, prevent or cure a disease or condition), a prophylactic agent (agent used to prevent infections and disease), or a diagnostic agent (agent used to diagnose a subject). Exemplary active agents include Analgesics, Antianxiety Drugs, Antiarrhythmics, Antibacterials, Antibiotics, Anticoagulants and Thrombolytics, Anticonvulsants, Antidepressants, Antiemetics, Antifungals, Antihistamines, Antihypertensives, Anti- Inflammatories, Antineoplastics, Antipsychotics, Antipyretics, Antiretrovirals, Antivirals, Barbiturates, Beta-Blockers, Bronchodilators, Corticosteroids, Cytotoxic agents, Diuretics, Hormones, Hypoglycemics Immunosuppressives, Muscle Relaxants, Sedatives, Tranquilizer, and Vitamins.

[0129] Detailed Description of the Drawings

[0130] Some embodiments of the disclosure are described herein with reference to the accompanying figures (FIGs). The description, together with the figures make apparent to a person having ordinary skill in the art how some embodiments of the disclosure may be practiced. The figures are for the purpose of illustrative discussion and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the teachings of the disclosure. The figures herein are not shown to scale. Where dimensions are given in the text or figures, these dimensions are merely exemplary and do not limit the scope or spirit of the disclosed invention.

[0131] FIG 1 are schematics illustrating: A the injection of the composition and crosslinking into a depot (ISCD); B the liquid state of the injectable becomes a solid; C design parameters to tune the release kinetics.

[0132] FIG 2 shows the synthesis and characterization of PCLDMA. A. Schematic showing methacrylation of hydroxyl-functionalized PCL to yield PCLDMA. B.1H-NMR spectrum of PCLDMA. The chemically equivalent protons are labeled with the same color and their NMR signals are marked with the same colored box for ease of understanding. The NMR integration of each signal corresponds to the expected ratio of each type of hydrogen in the PCLDMA molecule.

[0133] FIG 3 is the schematic illustrating hydrolysis of polycaprolactone domains and subsequent collapse of ISCD. The ISCD consists of a cross-linked network of polymethacrylate chains (red) connected by ester bonds (dark blue). Hydrolysis of these ester bonds (light blue) disconnects the polymethacrylate chains, enabling the depot to degrade.

[0134] FIG 4 shows physiochemical characterization of the ISCD platform. A. The cross-linking time for PCLDMA at different BPO / DMT concentrations is shown. Cross-linking time was measured as the point at which the viscosity of the pre-polymer mixture, monitored with a rheometer, begins to increase rapidly, as shown in the inset. B. Exothermic heat released during the cross-linking of ISCD with varying concentrations of BPO / DMT, measured using DSC. C. Injection parameters for the Hagen-Poiseuille equation used to calculate the injection force for PCLDMA, with or without TAF, using a 23-gauge needle. D. Viscosities of PCLDMA, with or without TAF, measured using a rheometer. E. Injection force calculated for PCLDMA, with or without TAF. The maximum acceptable injection force is 80 N. F. In vitro release profile of TAF in PBS (37°C) from ISCD comprising either PCLDMA or PCLTMA. G. In vitro release profile of TAF in PBS (37°C) from ISCD loaded with concentrations of TAF. H. Compressive stress-strain curves for ISCD with or without TAF, measured using a mechanical tester. I. Elastic moduli and yield stress for ISCD with or without TAF. J. ISCD can be polymerized ex vivo into various shapes such as cylinders, pipes, or disks that can be used as ultra-long-acting implants. Data in A, B, E-G, and I are presented as mean ± standard deviation (n=3, replicates performed at least twice). Data in D and H are representative of a single experiment (repeated three times).

[0135] FIG 5 shows infrared thermal imaging confirming that there is no noticeable heat generated during ISCD polymerization.

[0136] FIG 6 shows in vitro cumulative release of TAF from ISCD loaded with different concentrations of TAF and incubated in PBS (37°C). Data are presented as mean ± standard deviation (n=3, experiments performed at least twice).

[0137] FIG 7 shows in vitro cumulative release of TAF from ISCD depots formed by injecting prepolymer mixture into PBS (37°C) compared with TAF release from pre-formed implants with cylindrical shape. Data are presented as mean ± standard deviation (n=3, experiments performed at least twice).

[0138] FIG 8 shows tailoring of the drug release kinetics and degradation of ISCDs in vitro and in vivo. A. In vitro release profiles of TAF in PBS (37°C) from ISCDs prepared with PCLDMA of different molecular weights (630 Da and 2100 Da). B. In vitro release profiles of TAF in PBS (37°C) from ISCDs prepared with varying BPO / DMT concentrations. C. Crosslinking density of unmodified ISCD prepared with different concentrations of BMP / DMT and ISCD containing different external polymer additives (25 wt%) (*P<0.05 and **P<0.01). D. In vitro release profiles of TAF in PBS (37°C) from unmodified ISCD (prepared using PCLDMA only) or ISCD containing 25 wt% of an external polymer additive (PEG, PCL, or PDMS) alongside PCLDMA. E. In vitro release profiles of TAF and F. and percentage depot degradation in PBS (37°C) for unmodified ISCD or ISCD containing 25 wt% of PEG with varying degrees of methacrylation. G. SEM images of unmodified ISCD or ISCD containing different external polymer additives (25 wt%) showing the cross-section of depot structure at week 1 post-incubation in PBS (37°C). H. Plasma level of TFV in rats injected with 500 pl of TAF-loaded ISFI (control) or TAF-loaded unmodified ISCDs of ISCD containing 25 wt% PEGMMA. All depots were loaded with 90 mg / mL of TAF. The inset shows plasma levels up to day 30. (*P<0.05 for the overall comparison of plasma levels of the two ISCDs over the entire study duration). I. In vivo daily release rate and J. cumulative release of TAF from unmodified ISCD or ISCD containing 25 wt% of PEGMMA, as determined by PK modeling. K. Camera images of TAF-loaded ISFI or TAF-loaded unmodified ISCD or ISCD containing 25 wt% of PEGMMA, retrieved from rats at month 7 post-injection, and L. Remaining TAF amount in the depots (*P<0.05 and ***P<0.001) and M. Remaining mass of the depot (*P<0.05). Due to the disintegration of ISFI within the animal, the remaining mass of the ISFI depots could not be measured. N. MRI images of subcutaneously injected unmodified ISCD or ISCD containing 25 wt% PEGMMA at different time points. Data in A, B, C, E, F, and G are presented as mean ± standard deviation (n=3, experiments performed at least twice). Data in H, L, and M are presented as mean ± standard deviation of technical repeats (n=3, experiment performed twice). Data in I and J present predictions from PK modeling of the average plasma levels of TFV obtained experimentally. P-value in H was determined using two-way ANOVA with Bonferroni correction, with time and different ISCD formulations as the two variables. The P-value in C and L was determined using one-way ANOVA with Tukey’s post hoc analysis. The P-value in M was determined using Student’s t-test.

[0139] FIG 9 is a bar graph showing swelling rate of different ISCD formulations studied in benzyl alcohol after a week. Data are presented as mean ± standard deviation (n=3, experiments performed at least twice).

[0140] FIG 10 shows cumulative release of TAF at day 42 post-incubation of different ISCD formulations in PBS (37 °C). (**P<0.01 , ****P<0.0001). Data are presented as mean ± standard deviation (n=3). The P-value was determined using one-way ANOVA with Tukey’s post hoc analysis.

[0141] FIG 11 shows the impact of incorporating external polymer additives with varying degrees of methacrylation into ISCD on TAF release and depot degradation. A. Month 7 cumulative release, and B. month 7 degradation rate of unmodified ISCD and ISCD containing 25 wt% of PEGs with different degree of methacrylation, when incubated in PBS (37 °C). (*P<0.05, **P<0.01 , ***P<0.001 , ****p<0.0001). Data are presented as mean ± standard deviation (n=3). The P-value was determined using one-way ANOVA with Tukey’s post hoc analysis.

[0142] FIG 12 shows the time profiles of the blood / plasma concentration in rats after a single IV dose of A. TAF (1 & 4 mg / kg) B. TAC (1 & 2 mg / kg), and C. NAL (1 & 2 mg / kg). Time profiles of the blood / plasma concentration in rats after subcutaneous injection of ISCD containing D. TAF, E. TAC, and F. NAL. Data presented as symbols reflect the mean ± standard deviation of three technical repeats (n=3). Lines represent the model-predicted drug concentrations.

[0143] FIG 13 shows the scheme of the pharmacokinetic (PK) model for subcutaneous injection of ISCD. The disposition kinetics (referred to as Systems) of analytes is characterized by two compartments (Csiood / piasma and CTissue), with first-order rate constants for elimination (kei), distribution (ki 2) , and redistribution (k2i), and Vc for the central volume of distribution. At the SC implant site (referred to as SC Depot), the release / absorption model assumes three sequential release phases, delineated by first-order release rate constants (ki, km, ks). Initially, a fraction (fi) of the ISCD implant is released (ki), leading to the maximum concentration in the central compartment. Subsequently, drug release continues with an intermediate phase (km) for a fraction of the total released drug mass (fm), followed by a sustained release phase (ks) for the remaining drug amount (1- fi- fm). The time delays associated with the intermediate (Tdm) and sustained-release (Tds) phase are characterized by a gamma distribution function with shape (N) and rate parameter (Td). F represents the bioavailability of ISCD implants.

[0144] FIG 14 A. shows the percentage of the initial amount TAF amount remaining in the explanted ISFI following the 2-month in vivo study in rats. B. ISFI explanted after the 2-month in vivo study is a fragmented solid. Data are presented as individual values for each animal.

[0145] FIG 15 shows the HPLC chromatograph of TAF-loaded ISCD explanted after a 7-month in vivo study.

[0146] FIG 16 illustrates the in vivo biocompatibility and safety of ISCD. A. Representative image (10X magnification) of an H&E stained section of local tissue, explanted with the ISCD depot one week after subcutaneous injection of 500 pl PCLDMA-based ISCD in rats. B. Left side shows high magnification (20X) representative images of H&E-stained sections of local tissue, explanted with the ISCD depot at different time points. Arrows show inflammatory cells. The right side shows representative immunofluorescence images of local tissue sections explanted at different time points and stained against CD3 and CD68 markers to visualize T cells and macrophages, respectively. Arrows show cells positive for CD3 or CD68. C. Fluorescence intensity quantified for CD3 and CD68 immunofluorescence. (*P<0.05, **P<0.01). D. Camera image taken during the procedure of retrieving ISCD from a rat, showing safe retrievability via a small incision. E. Plasma levels of TFV following ISCD removal. Data in C and E are presented as mean ± standard deviation of technical repeats (n=3). The P-value in C was determined using one-way ANOVA with Tukey’s post hoc analysis.

[0147] FIG 17 shows the versatility of the ISCD platform and human PK prediction. A. In vitro release profile of different drugs with varying water solubilities encapsulated into the ISCD platform. The release was studied in PBS (37 °C). B. Correlation of cumulative release at day 1 with different drugs with varying water. C. In vitro release profile of TAF and FTC, when loaded into the ISCD platform individually versus in combination. The release was studied in PBS (37 °C). D. In vitro release profile of ABC and LAM, when loaded into the ISCD platform individually versus in combination. The release was studied in PBS (37 °C). E. Plasma concentration of NAL in rats subcutaneously injected with 500 pl of NAL-loaded ISCD (45 mg / ml NAL). F. Whole blood concentration of TAC in rats subcutaneously injected with 500 pl of TAC-loaded ISCD (28 mg / ml TAC). In vivo daily release rate and cumulative release profile of G. NAL and H. TAC, as predicted by PK modeling of systemic drug levels in rats, following subcutaneous injection of 500 pl of NAL- or TAC-loaded ISCD. I. Convolution analysisbased prediction of human PK of a single subcutaneous dose of NAL-loaded ISCD in comparison to clinically established PK profile of once-daily oral dose of NAL, and once monthly intra-muscular injection - Vivitrol®. J. Convolution analysis-based prediction of human PK of a single subcutaneous dose of TAC-loaded ISCD (at different dosages) in comparison to clinically established PK profile of twice-daily oral doses of TAC. Data in A-D are presented as mean ± standard deviation (n=3, experiments performed at least twice). Data in E and F are presented as mean ± standard deviation of technical repeats (n=3). Data in G and H present predictions from PK modeling of the average plasma level of TAC and NAL obtained experimentally. Data in I and J present human PK prediction based on convolution analysis of experimentally obtained PK data of TAC and NAL in rats.

[0148] FIG 18 shows convolution analysis-based prediction of human PK of a single subcutaneous dose of TAC-loaded ISCD (at different dosages) up to 6 months.

[0149] FIG 19 is a bar graph showing that the addition of vinyl acetate in a 75 / 25% PCLDMA / vinyl acetate blend allows slower-curing ISCD formulations to avoid additional burst release, compared to 100% PCLDMA.

[0150] FIG 20 is a plot showing that the addition of vinyl acetate in a 75 / 25% PCLDMA / vinyl acetate blend lowers ISCD viscosity by 65%. Lines mark the maximum viscosity levels that keep injection force below 80 N, for injection through the corresponding gauge needle of 2 ml of formulation at 0.1 ml / s with a BD 3-ml syringe.

[0151] FIG 21 is a plot showing that the addition of biocompatible non-crosslinking solvents such as ethyl / propyl / butyl acetate into ISCD increase the curing time along with slight increase in TAF release rate as compared to vinyl acetate.

[0152] FIG 22 is a plot showing plasma concentration of semaglutide in rats after a single subcutaneous injection of ISCD composed of PCLDMA:PEGMMA 70:30 weight (wt) ratio and loaded with 10 mg / mL semaglutide.

[0153] FIG 23 shows plasma concentration of semaglutide in rats after a single subcutaneous injection of ISCD composed of PCLDMA:PEGMMA 70:30 or 50:50 wt ratio and loaded with 10 mg / mL semaglutide.

[0154] Detailed Description

[0155] The development of long-acting drug delivery systems is of immense importance in the field of healthcare, particularly in the context of chronic diseases where consistent medication adherence is critical for therapeutic efficacy.6-7 9 12 27 29-30The ISCD presented in this study represents a significant advancement in addressing critical challenges associated with existing drug delivery platforms.

[0156] One of the notable achievements of the ISCD platform is its ability to provide sustained release of both hydrophilic drugs, such as TAF and NAL, and hydrophobic drugs, such as TAC, for 180-200 days in vivo. This prolonged delivery time for hydrophilic drugs is previously underdeveloped.

[0157] To achieve this remarkable feat, we utilized a solvent-free formulation that consisted of a hydrophobic, low molecular weight polymer called PCLDMA (MW 630 Da). By employing BPO / DMT polymerizing agents, we were able to create a dense polymer mesh network with PCLDMA, which proved highly effective in efficiently entrapping drugs. This strategic approach minimized burst release and, in turn, enabled the sustained delivery of a wide variety of therapeutic agents. We investigated the intricate factors governing the controlled release kinetics of ISCDs, as evidenced by both in vitro and in vivo experiments. These factors encompassed alterations in the polymer mesh network, including variations in the length and hydrophilic nature of the polymer chain, concentrations of crosslinkers, and the density of crosslinking groups. We elucidated strategies to increase release rates by employing higher molecular weight PCLDMA, lower concentrations of BPO and DMT, the incorporation of noncrosslinkable polymers as spacers, and the utilization of more hydrophilic polymers. Under these conditions, we observed the formation of looser or more hydrophilic polymer networks, making them susceptible to water permeation and depot degradation by erosion or hydrolysis, resulting in higher drug release. Consequently, we harnessed these insights to develop ISCDs incorporating non- or singly methacrylated PEG into PCLDMA-based formulations, thereby introducing an alternative ISCD platform capable of achieving higher systemic levels of therapeutics compared to pure PCLDMA ISCDs.

[0158] We conducted a comprehensive evaluation of crucial aspects of long-acting injectables, including biocompatibility and retrievability, to ensure the adaptability and safety of the ISCD platform. We have demonstrated successful in vitro delivery of eight different small molecule drugs, each with a different hydrophilicity, for extended periods of time ranging from 150 to 360 days using ISCD, establishing ISCD as a versatile ultra-long-acting delivery platform. Furthermore, our results highlight the importance of considering the physicochemical properties of drugs when formulating ISCDs, with more hydrophilic drugs exhibiting higher release rates. Furthermore, we successfully achieved combined drug delivery using a single ISCD, aligning with the delivery patterns observed in commercial HIV targeting therapeutics. This adaptability holds significant relevance in the context of combination therapy, a treatment approach that is increasingly prevalent in addressing a variety of diseases.

[0159] Taken together, the ISCD represents a groundbreaking paradigm shift in long-acting drug delivery, offering the remarkable capability of sustained release of both hydrophilic and hydrophobic drugs over unprecedented durations. This study provides critical insights into the optimization of drug release kinetics and highlights the ISCD's biocompatibility, retrievability, and ability to deliver multiple drugs simultaneously. This platform promises to revolutionize healthcare by addressing the challenges of chronic diseases where consistent medication adherence is critical to therapeutic efficacy.

[0160] Methods

[0161] Synthesis of methacrylated polycaprolactone (PCL)

[0162] 20 ml of 530 Da PCL-diol (Sigma-Aldrich) was mixed with 200 ml dichloromethane in a sealed nitrogen-filled flask chilled to 0 °C. 33.8 mL of TEA (Sigma-Aldrich) was added to the flask via syringe under stirring at 400 rpm, and 36 mL MAA (Sigma-Aldrich) was subsequently added dropwise via syringe to have three molar equivalent of TEA and MAA per mol of hydroxy group from PCL-diol. The reaction proceeded for 15 hours under stirring at 0 °C. The reaction solvent was removed under reduced pressure and reconstituted in 200 mL of ethyl acetate. This product was washed three times each with saturated aqueous sodium bicarbonate, 0.1 N hydrochloric acid (HCI), and aqueous brine. Following the washing steps, the product was dried with anhydrous sodium sulfate, filtered under vacuum, and removed under reduced pressure. The crude product was run through a premanufactured silica column (Teledyne Isco) using five column volumes of a mixture of 90% heptane / 10% ethyl acetate, followed by five column volumes of pure ethyl acetate. Solvent was removed from the latter five fractions under reduced pressure to yield the purified product, PCLDMA, as a viscous yellow liquid. Substitution and purity were confirmed by1H NMR.

[0163] Mechanical characterization

[0164] ISCD depots were prepared in cylindrical PDMS molds (6.8 mm diameter, 2.8mm height) for compression test. The dimensions of the depots were then measured using a caliper. The ISCD depots were mounted between plates of a mechanical tester (ADMET) and compressive force was applied to the samples at a rate of 1 mm / min. The compressive strain and stress on the samples were measured and the compressive moduli were obtained from the linear region (0.15-0.25 mm / mm strain) in the stress strain curve.

[0165] Rheological characterization

[0166] A rheometer (Discovery HR-3, TA Instruments) equipped with a parallel plate with a gap size of 1 mm and a diameter of 20 mm was used to characterize rheological properties of ISCD prepolymer solutions. The solutions were prepared as outlined before but without adding polymerization agents and pipetted onto the rheometer. Any excess solution was trimmed with a spatula before these measurements. The viscosity of the solution was measured while shear rate was swept from 2 to 3700 s-1.

[0167] The force required to inject 1 mL of ISCD solution in 30 seconds was measured indirectly from the viscosities using the equation below. Briefly, dynamic viscosities, p, of ISCD solutions were measured and put into the equation below, where L is length of needle (15 mm); Q is volumetric flow rate (2 mL / min); D is inner diameter of syringe barrel (8.66 mm); and d is inner diameter of needle (0.337 mm).

[0168] F= 32gLQD2 d4

[0169] Preparation of ISCD for in vitro and in vivo samples

[0170] Pre-polymer solution, composed of methacrylated PCL with or without additives (i.e. PEG, PEGMMA, PEGDMA, PCL, and PDMS), was mixed with 0.1 -0.5 wt % of polymerization initiator, BPO. Drugs were incorporated into the polymer blend, followed by 15 seconds of ultrasonication to facilitate a homogeneous suspension. The polymerization accelerator, DMT, was added in at a concentration of 0.1-0.5 wt % and thoroughly mixed, only after all other components had been added. The formulation is drawn into a syringe and injected either into a sink for in vitro studies or subcutaneously into a rat for in vivo studies. The formulation underwent polymerization into a solid depot within 3-10 minutes after the addition of the accelerator.

[0171] Exothermic heat analysis

[0172] To quantify the exothermic heat generated during ISCD polymerization, 15 mg of PCLDMA was prepared with 0.1 , 0.3, and 0.5 wt% BPO. The mixture was pipetted onto an aluminum pan and combined with 0.1 , 0.3, and 0.5 wt% DMT, respectively. Immediately after mixing, the heat flow within the sample was measured for 20 minutes at room temperature. The total exothermic heat for the reaction was calculated by integrating the heat flow over time. For qualitative analysis, an infrared thermal imaging camera (Fluke Ti95 9Hz) was used to detect the temperature change in the depot during its polymerization.

[0173] In vitro drug analysis

[0174] 100 pl of drug-loaded pre-polymer mixtures were injected into a sink medium. While TAC- and AMX-loaded ISCDs were incubated with 20% methanol in PBS solution due to low water solubility, other ISCD formulations encapsulating different therapeutics were incubated with PBS in a shaker incubator at 37°C. To maintain sink conditions and prevent bacterial growth, the release medium was completely removed and replaced with fresh release medium every week. The release medium was collected at predetermined time points to be analyzed directly or after lyophilization and reconstitution with an appropriate solvent for analysis.

[0175] The in vitro cumulative release of TAF was analyzed using NMR. TAF release samples were stored in a -80 °C freezer and lyophilized after completely frozen. The remaining powder was reconstituted with 500 pL 0.1 mg / mL maleic acid in deuterium oxide (D2O), as an internal standard, and loaded into NMR tubes (NORELL). NMR experiments were performed on an Agilent MR 400 MHz automated NMR system equipped with a 5mm AutoX One probe at room temperature. 64 scans were conducted for each1H NMR spectrum recording. MestReNova was used for spectrum analysis. Baseline correction of the recorded spectra was performed manually in the software. The maleic acid alkene peak at approximately 6 ppm was integrated, whereas the ppm range of 8.1 -8.3 (adenine protons) was integrated and normalized to the maleic acid peak integration area. A 3-point calibration curve was made in a range of 75-300 pg / mL of TAF in D2O with 0.1 mg / mL maleic acid. The concentration was plotted against the normalized area of integration. The linear regression was generated in Excel and used for all TAF sample quantification.

[0176] The in vitro cumulative release of therapeutics, except TAF, in this study was determined by HPLC (Agilent 1260 Infinity II) and the cumulative drug release was calculated. Sample analyses were performed on a ZORBAX 300SB-C18 column (Agilent, 3.0 x 150 mm, 3.5 pm) at 30 °C, and all experiments were performed in triplicate. Samples were injected into the HPLC and chromatographic separation was achieved by gradient elution using different mobile phases and flow rates depending on the therapeutics, as described in Tables 1-3. Table 1 HPLC analysis details for different therapeutics

[0177] Table 2 Gradient program of the mobile phase for HPLC analysis of TAC

[0178] Table 3 Gradient program of the mobile phase for HPLC analysis of LAM, ABA and NAL

[0179] Scanning electron microscopy (SEM) analysis

[0180] Surface and microstructures of ISCD were evaluated by SEM. First, polymer solutions were prepared and polymerized in a PBS sink as described before. At predetermined time points, the implants were removed from the sink and dried under low pressure overnight. The lyophilized samples were subsequently mounted on an aluminum stub using carbon tape, and sputter coated with 6 nm of platinum (Leica EM ACE600). The coated samples were then imaged with Hitachi S- 4700 FE-SEM. In vitro degradation analysis

[0181] To evaluate the degradation rate of different ISCDs, a weight loss assay was conducted at designated time intervals (Day 1 / 2 / 4 / 7 / 14 / 28 / 30 / 42 / 60 / 80) at 37 °C. Depots were formed by injecting pre-polymer mixture into PBS (sink), followed by incubation in a shaker incubator at 37°C. Different samples were prepared for each time point. Sink was decanted at each pre-determined time point, and depots were subjected to freeze-drying for 24 hours to remove any residual sink solution. The weight loss (degradation) was quantified as a percentage according to the equation provided below.

[0182] Wois the original weight; W s the weight of the samples after degradation. Crosslinking density analysis

[0183] The degree of cross-linking of the depots was measured using an equilibrium swelling method and the Flory-Rehner equation,31described as follows, where l^.is the volume fraction of depot in swollen state; EJs molar volume of the solvent (benzyl alcohol), 103.9 mL / mol; is the Flory-Huggins polymer-solvent interaction parameter.

[0184] Crosslink density (

[0185] ISCD depots composed of PCLDMA with or without external polymer additives (PEG, PEGMMA, PEGDMA, PCL, or PDMS) with 0.3 wt% BPO / DMT and PCLDMA with 0.15 wt% BPO / DMT were prepared. The depots were incubated in benzyl alcohol for a week. The weight of each depot was measured before and after the incubation. The volume fraction of the depots in the swollen state was calculated from the increase in weight using the density of the solvent and polymer mixtures. The Flory-Huggins polymer-solvent interaction parameter was obtained from literature.32The parameters were plugged into the Flory-Rehner equation to determine the cross-linking density of each depot.

[0186] In vivo pharmacokinetic studies

[0187] Male Sprague-Dawley rats (6-8 weeks, Charles River Laboratories) were housed and handled in pathogen-free animal facilities at Brigham and Women's Hospital (BWH) in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at BWH. In vivo pharmacokinetic (PK) studies were conducted with different ISCD formulations, either pure PCLDMA or PCLDMA with 25% PEGMMA, with 0.3% BPO / DMT encapsulating tenfovir alafenamide (TAF), naltrexone (NAL), or tacrolimus (TAC). After mixing all ingredients, the ISCD drug formulations were administered subcutaneously with an 18 G needle over the shoulder of anesthetized rats. At predetermined time points, blood was collected from the tail vein of the rats into EDTA-coated tubes (EDTA = ethylenediaminetetraacetic acid) for up to 6-7 months. For analysis of systemic therapeutic levels in rats, whole blood was collected from the tail vein of the rat using a 25-gauge needle and collected in EDTA-coated tubes. For TAF and NAL samples, the blood sample was centrifuged at 1200g for 10 minutes at 4 °C to isolate plasma from the supernatant after centrifugation. The Naltrexone / Nalbuphine Forensic ELISA Kit (Neogen) was used to quantify NAL plasma concentrations according to the manufacturer's instructions. TAF concentrations in plasma samples were analyzed at the PPD Analytical Laboratory (PPD, Inc., Richmond, VA) using reversed-phase high-performance liquid chromatography with UV detection.

[0188] To extract TAC from the blood samples, 100 pL of whole blood were combined with 100 pL of MeOH and 50 pL of 0.1 M ZnSO4 in an Eppendorf tube, followed by thorough mixing through vortexing. Subsequently, 1 mL of ethyl acetate was added to the mixture and vortexed again. The resulting mixture underwent centrifugation at 14000 rpm for 5 minutes at room temperature. The supernatant was collected and dried, and the measurement of tacrolimus levels was carried out using the Tacrolimus (FK506) ELISA kit (Abbexa, abx515779). The dried TAC sample was reconstituted with the sample diluent buffer provided in the ELISA kit, and further sample analysis was performed following the manufacturer's instructions.

[0189] PK analysis and human PK prediction

[0190] The PK analysis of ISCD formulations utilized both non-compartmental and compartmental methods. The in vivo release rate (mg / day) from ISCD formulations was determined employing the area function method33, a deconvolution technique that relies on the relationship between observed drug concentration following subcutaneous injection of ISCD and the area intervals under both subcutaneous and IV drug concentration-time curves. For compartment modeling of ISCD PK profiles in rats, the disposition kinetics of each drug were first established using rat IV PK data obtained experimentally (TFV, TAC) and from literature (NAL)34 36with a two-compartment PK model. Subsequently, an appropriate absorption kinetic model was integrated to characterize the ISCD PK profiles. These absorption models describe the in vivo release rate profiles of ISCD, characterized by multiple first-order rates occurring in a sequential manner.

[0191] For human PK prediction for NAL and TAC following subcutaneous injection of ISCD, convolution analysis was employed, integrating the human disposition kinetic function and the release function of the ISCD formulation. The human disposition function was derived from human IV PK data for TAC37and NAL38obtained from the literature. The in vivo release functions estimated from the rat PCLDMA ISCD were used after applying simple allometry to release kinetic parameters based on average body size between rats and humans, with a typical allometry exponent of -0.25 for rate constants39 40.

[0192] Elemental CHN analysis

[0193] Residual TAF content within the depots was quantified using elemental analysis (CHN) performed at Midwest Microlab (Indianapolis, IN). Given that the polymer matrix used in the depots lacks nitrogen, the measured nitrogen content can be directly correlated with the remaining amount of TAF. The mass percentages of carbon, hydrogen, and nitrogen in the sample were analyzed, and the residual drug mass within the depots was back-calculated from these values. In vivo degradation

[0194] To assess in vivo degradation, weight loss of explanted ISCDs was measured. Rats received subcutaneous injections of 500 pL unmodified ISCDs or ISCDs containing 25% PEGMMA (both with 0.3% BPO / DMT but no drugs) (n = 3). After 7 months, the rats were euthanized, and the explanted depots were cleared of surrounding tissue. The explants were rinsed with DI water and freeze-dried for 24 hours to remove residual solvents. Weight loss was then calculated as a percentage of degradation using the equation provided below.

[0195] In vivo biocompatibility study

[0196] To investigate the inflammatory response caused by the implanted material, rats received injections of either unmodified ISCD or ISCD containing 25% PEGMMA, both formulated with 0.3% BPO / DMT but without any drugs. The depots were explanted at 1 week, 4 weeks, and 7 months postinjection. To assess tissue response over time, histological and immunohistochemical analyses were performed on cryosections of the explanted samples. After explantation, samples were fixed in 4% paraformaldehyde for 4 hours, followed by overnight incubation in 30% sucrose at 4 °C. Samples were then embedded in optimal cutting temperature compound (OCT) and flash frozen in liquid nitrogen. Frozen samples were then sectioned using a Leica Biosystems CM1950 Cryostat. 15-pm cryosections were obtained and mounted in positively charged slides. The slides were then processed for hematoxylin and eosin staining (Sigma) according to instructions from the manufacturer. The stained samples were preserved with DPX mountant medium (Sigma). Immunohistofluorescent staining was performed on mounted cryosections. Anti-CD3 (ab16669) and anti-CD68 (ab125212) (Abeam) were used as primary antibodies, and an Alexa Fluor 594-conjugated secondary antibody (Invitrogen) was used for detection. All sections were counterstained with DAPI (Invitrogen), and visualized on an Leica DMi8 widefield microscope.

[0197] Depot retrievability

[0198] \Ne subcutaneously administered 500 pL ISCD composed of PCLDMA, 90 mg / mL TAF and 0.3 % BPO / DMT with an 18 G needle to rats and retrieved the depots two weeks later. For the retrieval, the rats were anesthetized and their back was shaved to visualize the location of the depot. Under sterile conditions, a small cutaneous incision was made adjacent to the depot for atraumatic removal using forceps, followed by closure with sterile sutures. Blood (300-500 pL) was collected one hour before retrieval and at day 1 , 3, 7, 10, 14, and 21 post-retrieval to monitor plasma tenofovir (TFV) levels.

[0199] Histological and immunohistochemical analysis

[0200] Histological and immunohistochemical analysis were performed on cryosections of the explanted implant samples in order to characterize the inflammatory response elicited by the implanted material. After explanation, samples were fixed in 4% paraformaldehyde for 4 hours, followed by overnight incubation in 30% sucrose at 4 °C. Samples were then embedded in optimal cutting temperature compound (OCT) and flash frozen in liquid nitrogen. Frozen samples were then sectioned using a Leica Biosystems CM1950 Cryostat. 15-pm cryosections were obtained and mounted in positively charged slides. The slides were then processed for hematoxylin and eosin staining (Sigma) according to instructions from the manufacturer. The stained samples were preserved with DPX mountant medium (Sigma). Immunohistofluorescent staining was performed on mounted cryosections. Anti-CD3 (ab16669) and anti-CD68 (ab125212) (Abeam) were used as primary antibodies, and an Alexa Fluor 594-conjugated secondary antibody (Invitrogen) was used for detection. All sections were counterstained with DAPI (Invitrogen), and visualized on an Leica DMi8 widefield microscope.

[0201] Statistical information

[0202] All values are presented as mean ± standard deviation. Two-tailed Student's t-test was used to compare two experimental groups, and one-way ANOVA with Tukey's post hoc analysis was used to compare more than two groups using GraphPad (Software Inc., CA, USA), with P values defined as *< 0.05, **< 0.01 , “*< 0.001 , and ****< 0.0001 . One-way ANOVA with Tukey's post hoc analysis was used to determine the statistical significance of differences in swelling and cross-linking density of ISCD, with the mean of each group compared to the mean of the PCLDMAwith 0.3% w / w BPO / DMT (control) group. To compare the in vivo plasma TFV level of unmodified ISCD and ISCD containing 25% PEGMMA, we used a two-way ANOVA analysis with time and ISCD formulation as the two variables. One-way ANOVA with Tukey's post hoc analysis was used to determine the statistical significance of differences in residual TAF from explanted depots, with the mean of each group compared to the mean of the unmodified ISCD depot.

[0203] Compounds

[0204] Exemplary compounds of the present invention are listed in the following sections. Representative compounds of the present invention and their structures are illustrated in Tables 4-8.

[0205] Table 4: Representative Polymerization Reagents and Products for ISCD Table 5: Representative Organic Peroxides Table 6: Representative Secondary and Tertiary Amines

[0206] Table 7: Representative Additives

[0207]

[0208] Table 8 Representative active agents

[0209]

[0210] Non-limiting list of polymers that can be used for ISCD

[0211] Polymers of the present invention may include, but are not limited to, Poly(lactic-co-glycolide) diacrylate (PLGA DA), Poly(caprolactone) diacrylate (PCL DA), Polypropylene fumarate) diacrylate (PPF DA), Poly(methyl methacrylate) triacrylate (PMMATA), Poly(2-hydroxyethyl acrylate) (PHEA), Poly(2-hydroxyethyl methacrylate) (PHEMA), Poly(vinyl alcohol) (PVA), PEG diacrylate (PEG DA), PEG dimethacrylate (PEG DMA), Gelatin methacrylate (GelMA), Chitosan methacrylate (ChiMA), Alginate methacrylate (AMA), Hyaluronic acid methacrylate (HAMA), poly(ethyleneimine), poly(4- vinylpyridine), poly(vinylbenzyl chloride), poly(diallylammonium chloride), poly (glycidyl methacrylate), poly(allylamine), copolymers of vinylpyridine and dimethyldiallylammonium chloride, copolymers of vinylpyridine, dimethyladiallylammonium chloride, (3-acrylamidopropyl) trimethylammonium chloride with glycidyl acrylate or methacrylate.

[0212] Non-limiting list of initiators

[0213] The thermal initiators of the present invention may include peroxides, peracids, peracetates, persulfates, or the like. Exemplary thermal initiators include tert-amyl peroxybenzoate; 4,4-azobis (4- cyanovaleric acid); 1 ,1-azobis(cyclohexanecarbonitrile); 2,2-azobisisobutyronitrile (AIBN); benzoyl peroxide; 2,2-bis (tertbutylperoxy)butane; 1 ,1-bis(tert-butylperoxy)cyclohexane; 2,5-bis (tert- butylperoxy)-2,5-dimethylhexane; 2,5-bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne; bis (1 -(tert- butylperoxy)-1-methylethyl)benzene; 1 , 1 -bis (tert-butylperoxy)-3,3,5-trimethylcyclohexane; tert-butyl hydroperoxide;tert-butyl peracetate; tert-butyl peroxide; tert-butyl peroxybenzoate; tert-butylperoxy isopropylcarbonate; cumene hydroperoxide; cyclohexanone peroxide; dicumyl peroxide; lauroyl peroxide; 2,4- pentanedione peroxide; peracetic acid; tert-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, p-menthane hydroperoxide; 1 , 1 ,3,3- tetramethylbutylhydroperoxide, acetyl peroxide, p-chlorobenzoyl peroxide, 2,4-dichlorobenzoyl peroxide, ditoluoylperoxide, decanoyl peroxide, lauroyl peroxide, isobutyryl peroxide, diisononanoyl peroxide, pelargonylperoxide, tert-butyl peroxyacetate, tert-butyl peroxymaleic acid, tert-butyl peroxyisobutyrate, tert-butylperoxypivalate, tert-butyl peroxycrotonate, tert-butyl-Peroxy-(2-ethylhexanoate),

[0214] 2.5-dimethyl-2,5-bis-(2-ethylhexanoylperoxy) hexane, 2,6-dimethyl-2,5-bis-(benzoylperoxy) hexane,

[0215] 2.5-dimethyl-2,5-bis-(tert-butyl-peroxy) hexane, 2,5-dimethyl-2,5-bis-(tert-butylperoxy)-hexyne-3, di- tert-butyldiperoxyphthalate, 1 ,1 ,3,3-tetramethylbutylperoxy-2-ethyl-hexanoate, di-tert-buty I peroxide, di-tertamylperoxide, tert-amyl-tert-butyl peroxide, 1 ,1-di-tert-butylperoxy-3,3,5-trimethyl cyclohexane, bis-(tert-butylperoxy)-diisopropylbenzene, n-butyl-4,4-bis-(tert-butylperoxy)valerate, dicumyl peroxide, acetyl acetone peroxide, methyl ethyl ketone peroxide, cyclohexanone peroxide, tertbutylperoxyisopropyl carbonate, 2,2-bis-(tert-butylperoxy) butane, di-(2-ethylhexyl) peroxydicarbonate, and bis-(4-tert-butylcyclohexyl) peroxydicarbonate.

[0216] Non-limiting list of additive polymers

[0217] Additive polymers of the present invention include, but are not limited to, polyethylene glycol (PEG), Poly(ethylene glycol) methacrylate (PEGMMA), Poly(ethyleneglycol) dimethacrylate (PEGDMA), polydimethylsiloxane (PDMS), polycaprolactone (PCL), polyvinyl acetate (PVA), polypropylene glycol (PPG), polybutylene glycol (PBG), polyvinyl alcohol (PVA), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid); and / or non-crosslinkable hydrophilic polymer : Polyvinyl alcohol (PVA); Polyamidoamine; Polyamidoimide; Polylactone; N-vinylpyrrolidone homopolymer; N-vinylpyrrolidone and one or more hydrophilic vinyl groups Copolymer of comonomers; homopolymer of (meth) acrylamide; copolymer of (meth) acrylamide with one or more hydrophilic vinyl monomers; N-vinyl-N-methylethyl Homopolymer of fluorenamine; copolymer of N-vinyl-N-methylacetamide with one or more hydrophilic vinyl monomers; homopolymer of C2-C3 hydroxyalkyl (meth) acrylamide Polymers; copolymers of C2-C3 hydroxyalkyl (meth) acrylamide with one or more hydrophilic vinyl monomers; vinyl monomers containing phosphinocholine and one or more hydrophilic vinyl monomers Copolymer of monomers; poly (ethylene oxide) (PEO); poly(2-ethyloxazoline); heparin polysaccharides; polysaccharides; and / or non-crosslinkable hydrophobic polymers: Polystyrene (PS), Polypropylene (PP), Polyethylene (PE), Polytetrafluoroethylene (PTFE), Polyvinylidene difluoride (PVDF), Polymethyl methacrylate (PMMA), Polyacrylonitrile (PAN), Polybutylene terephthalate (PBT), Polyethylene terephthalate (PET).

[0218] Non-limiting list of additive solvents similar to vinyl acetate

[0219] Other additive solvents that are similar to vinyl acetate that may be used in the present invention include, but are not limited to, methyl acetate, ethyl acetate, propyl acetate, isobutyl acetate, butyl acetate, amyl acetate, isoamyl acetate, benzyl acetate, isopropyl acetate, ethylene glycol diacetate, propylene glycol diacetate, diethylene glycol diacetate, meth) acrylic ethoxyethylphosphonium choline, ((Meth) acryloxypropylphosphonium choline, 4-((meth) acryloxy) butyl-2 '-(trimethylamino) ethyl phosphate, 2-[(methyl ) Acrylamido] ethyl-2 '-(trimethylamino) -ethyl phosphate, 3-[(meth) acrylamido] propyl-2'-(trimethylamino) Ethyl phosphate, 4-[(meth) acrylamino] butyl-2 '-(trimethylamino) ethyl phosphate, 5-((meth) acrylamino) pentyl- 2 '-(trimethylamino) ethyl phosphate, 6-((meth) propenyloxy) hexyl-2'-(trimethylamino) -ethyl phosphate, 2-((methyl Propyl) propenyloxy) ethyl-2 '-(triethylamino) ethylphosphate, 2-((meth) propenyloxy) ethyl-2'-(tripropylamino) ) Ethyl phosphate, 2-((meth) acryloxy) ethyl-2 '-(tributylamino) ethyl phosphate, 2-((meth) acryloxy) propyl -2 '-(trimethylamino) -ethyl phosphate, 2-((meth) propenyloxy) butyl-2'-(trimethylamino) ethyl Acid ester, 2-((meth) propenyloxy) pentyl-2 '-(trimethylamino) ethyl phosphate, 2-((meth) propenyloxy) hexyl-2'- (Trimethylamino) ethyl phosphate, 2- (vinyloxy) ethyl-2 '-(trimethylamino) ethyl phosphate, 2- (allyloxy) ethyl-2 '- (Trimethylamino) ethyl phosphate Ester, 2- (vinyloxycarbonyl) ethyl-2 '- (trimethylamino) ethyl phosphate, 2- (allyloxycarbonyl) ethyl-2'-(trimethylamino) ) -Ethyl phosphate, 2- (vinylcarbonylamino) ethyl-2 '-(trimethylamino) -ethylphosphate, 2- (allyloxycarbonylamino) ethyl-2 '- (trimethylamino) ethyl phosphate, 2-(butenyloxy) ethyl-2'-(trimethylamino) ethyl phosphate.

[0220] Non-limiting list of active agents that can be used in ISCD

[0221] The list of potential active agents includes but is not limited to Antibiotics, Anti cancer Agents, Antiviral Agents, Antiretroviral Agents, Antifungal Agents, Anti-inflammatory Drugs, Peptides, Proteins, Nucleic Acid-based Agents (mRNA, siRNA, plasmid DNA, antisense oligonucleotides), Gene Editing Agents (e.g., CRISPR-Cas9, base editors), Aptamers, Vitamins, Nutrients, Chelating Agents, Ophthalmic Agents, Immunomodulatory Agents, Immunosuppressives, Hormones, Enzymes, Anticoagulants, Thrombolytics, Analgesics, Antianxiety Drugs, Sedatives, Anxiolytics, Antidepressants (water-soluble formulations), Cardiovascular Agents, Antiarrhythmics, Beta-Blockers, Antihypertensive Agents, Diuretics, Antidiabetic Agents, Hypoglycemics, Neuroprotective Agents, Antioxidants, Growth Factors, Vaccines, Antiparasitic Agents, Antiprotozoal Agents, Anticonvulsants, Antipsychotics (hydrophilic derivatives), Antiemetics, Antispasmodics, Bronchodilators, Antiallergic Agents, Antihistamines, Antipyretics, Antibacterials, Antiseptics, Disinfectants, Electrolyte Solutions, Detoxifying Agents, Mucolytics, Antidotes, Antiulcer Agents, Anticoagulant Reversal Agents, Antimetabolites, Dermatological Agents (e.g., water-soluble topical agents), Antiparkinsonian Agents, Antigout Agents, Hydrophilic Contrast Agents (for diagnostic imaging), Radioprotective Agents, Immune Checkpoint Inhibitors (in hydrophilic formulations), Biological Response Modifiers, Angiogenesis Inhibitors, Cholinesterase Inhibitors, Vasodilators, Anti-dementia Agents, Hydrophilic Carriers and Excipients, Hematopoietic Agents, Lipid-lowering Agents (water-soluble formulations), Cognitive Enhancers (Nootropics), Fibrinolytics, Inotropes, Nasal Decongestants, Corticosteroids, Cytotoxics, Muscle Relaxants, Barbiturates, Tranquilizers. Kit

[0222] A kit of the present invention makes it possible to make and use a ISCD of the present invention. A kit of the present invention may include any one of the above-described compounds or compositions. The kit may also include directions to perform any of the methods described herein, e.g., how to prepare an injectable composition using the components provided. The kit may further include directions for how to incorporate different active agents into the formulation for optimal encapsulation. In some embodiments, said directions may be a package insert. In some embodiments, the kit also includes at least one active agent.

[0223] Examples

[0224] Example 1 : Synthesis and Characterization of ISCD

[0225] The main component of engineered injectable is low molecular methacrylated PCL, which is biodegradable and biocompatible, and has been used previously in multiple FDA approved products. Drugs can be physically encapsulated in the polymer component by suspension or dissolution. This mixture prior to solidification has low enough viscosity for injection by conventional 18-25-gauge needles (FIG 1A). To prepare methcarylated PCL, PCL-diol and -triol were methacrylated via reaction with methacrylic anhydride (MAA) and triethylamine (TEA) to be polycaprolactone dimethylacrylate (PCLDMA) and polycaprolactone trimethylacrylate (PCLTMA) (FIG 1A, 2). Methacrylation was confirmed via1H NMR spectroscopy, with the ratio of integral areas under the protons of the double bond in the methacrylate group at 5 = 6.12 ppm (2H, olefinic, cis) to the methylene protons of the PEG segment at 5 = 3.70 ppm (4H, -OCH2CH2OCH2CH2O-) determined to be 1 :2. When mixed with the radical initiator - benzoyl peroxide (BPO) and the accelerator - N,N-dimethylptoluidine (DMT) which have been used clinically in the bone cements for past 50 years, radical polymerization of methcrylate PCL commences to create new carbon-carbon polymer chains, crosslinking the biodegradable polymer domains (FIG 1A). Over time, hydrolysis of the original biodegradable polymer ester bonds allows gradual erosion of the depot, obviating the need for surgical removal (FIG 3).

[0226] The time interval between the mixing of different components and the complete crosslinking of ISCD is a critical parameter for successful clinical application. Ideally, the crosslinking kinetics should provide sufficient time to mix and inject the formulation before it solidifies. Therefore, we investigated the cross-linking time of ISCD, focusing on the impact of initiator and accelerator concentrations on it. Using a rotational rheometer, we measured the cross-linking time as the point when the formulation's viscosity starts to rapidly increase. When using 0.1 wt% each of BPO and DMT (BPO / DMT), the formulation demonstrated complete solidification within approximately 9 minutes (FIG 4A). Increasing the concentrations of BPO / DMT from 0.1 to 0.3 wt% significantly reduced the PCLDMA crosslinking time to approximately 5 minutes, which further reduced to 2 minutes with BPO / DMT concentrations of 0.5 wt%. For subsequent experiments, we used 0.3 wt% concentrations of BMO / DMT.

[0227] Since radical polymerization is an exothermic process, we intended to confirm whether in situ crosslinking of ISCD would result in any thermal tissue damage. We employed two complementary techniques: differential scanning calorimetry (DSC) and infrared thermal imaging camera. The DSC analysis showed that the exothermic heat released during the polymerization of PCLDMA increased with increasing concentrations of BPO / DMT. Formulation with 0.5 wt% BMO / DMT showed <4 cal / g exothermic heat release (FIG 4B). To provide context, many common dietary carbohydrates and proteins provide about 4 cal / g of energy.41This indicates that the ISCD cross-linking reaction is relatively mild and releases minimal heat. Additionally, monitoring the temperature changes during the polymerization process via an infrared thermal imaging camera showed a temperature of 20.9 °C, confirming the absence of localized heating (FIG 5). These findings alleviate concerns about potential thermal tissue damage upon ISCD injection.

[0228] Another important property for the injectable formulations is the rheological properties because it can affect the ease of injection and ensure that the implant can be delivered effectively. We analyzed the viscosities of the ISCD pre-polymer solutions and calculated the force required to inject the solutions (1 mL in 30 seconds with a 23 G nozzle) indirectly using the Hagen-Poiseuille equation42. The calculated injection forces for ISCD solutions with and without 90 mg / mL TAF are 64.0 ± 3.7 and 44.4 ± 7.8 N (FIG 4C-E), respectively, showing an increase in viscosity and injection force with drug encapsulation. Both compositions exhibited values below 80 N, which is considered difficult for most people and therefore often considered the maximum acceptable injection force42, demonstrating their superior injectability. In summary, the comprehensive characterization of material properties in the ISCD system demonstrates its promising potential for long-acting drug delivery.

[0229] We demonstrated the sustained release of TAF, a hydrophilic drug with a water solubility of 5.63 mg / mL, over 200 days using either PCLDMA (Mn~630) or PCLTMA (Mn~1100), demonstrating the prolonged hydrophilic drug release capabilities of the methacrylated PCL-based long-acting injectables (FIG 4F). We encapsulated 30-150 mg / mL TAF in PCLDMA ISCDs and demonstrated that the release profiles from the ISCDs are similar over the range of drug loading (FIG 4G), demonstrating that the drug released is proportional to the drug loading (FIG 6).

[0230] The implants should maintain their structural integrity even when subjected to significant external forces within the patient's body, a critical consideration given the intricate and dynamic mechanical conditions present in the human body. Therefore, mechanical integrity is crucial for ensuring that the drug delivery system functions as intended throughout its lifecycle within the patient's body. To assess the mechanical properties of the PCLDMA ISCDs, we performed compression testing both with and without the presence of 90 mg / mL TAF to analyze their mechanical properties. The results showed that the modulus for ISCDs with and without TAF was 68.1 ± 10.3 and 67.2 ± 4.0 MPa, respectively, and the yield stress was 24.2 ± 1.1 and 25.9 ± 1 .5 MPa, respectively (FIG 4H , I) , indicating that the PCLDMA ISCDs exhibited a high level of structural rigidity. It is noteworthy that these mechanical properties align closely with those observed in clinically established solid implants28 43, which underscores the reliability and suitability of the ISCDs for drug delivery applications. Importantly, the inclusion of the drug does not compromise the structural integrity of the delivery system. This finding provides assurance that the drug remains adequately protected within the ISCDs and that these systems can efficiently deliver the drug without mechanical failure. We also demonstrated that ISCD can be molded and crosslinked ex vivo into various shapes, including cylinders, pipes, and disks, using PDMS molds. These forms maintain a similar drug release profile to the injectable version (FIG 4J ,7) , providing a versatile platform for both injectable and implantable drug delivery.

[0231] Example 2: Release kinetics and degradation of ISCD in vitro

[0232] Having demonstrated the ultra-long-term release of TAF from ISCD, we aimed to identify design parameters that can tailor the polymer network to fine-tune the drug release kinetics. For these experiments, TAF concentration in ISCD was maintained at 90 mg / ml. We hypothesized that modulating intrinsic factors of ISCD, including the molecular weight of PCLDMA, and the concentrations of BPO and DMT, could influence the cross-linking density of ISCD, thereby impacting drug release. To evaluate the impact of polymer molecular weight on drug release, TAF-loaded ISCDs (90 mg / mL TAF) were formulated using PCLDMA with two distinct molecular weights (630 Da and 2100 Da) (FIG 8A). As expected, depots with higher molecular weight polymer showed faster release of TAF. Previous reports have demonstrated that polymer molecular weight of LAIs impacts crosslinking density, which in turn affects drug release.44-45Similarly, release profiles of TAF from ISCDs with varying concentrations of BPO / DMT ranging between 0.1 to 0.4 wt% demonstrated that an increase in the concentrations of the initiator and accelerator reduces the burst release and the overall release rate (FIG 8B). To confirm if the effect of BMO / DMT concentration on drug release kinetics is attributed to the changes in the cross-linking density of the depot, we quantified the degree of cross-linking of different ISCD depots using a gravimetric approach. The depots were incubated in benzyl alcohol for a week to determine swelling and the Flory-Rehner equation31was used to estimate the degree of cross-linking based on the swelling data. Reducing BPO / DMT concentrations resulted in a reduction in the cross-linking density (FIG 8C) and an increase in swelling percentage (FIG 9), confirming our hypothesis.

[0233] Next, we hypothesized that incorporating hydrophilic polymer additives along with PCLDMA would modulate the hydrophilicity of the depot, influencing drug release kinetics. To test this, we added 25 wt% of different polymer additives with varying hydrophilicity but similar molecular weights: polyethylene glycol (PEG, MW 500), PCL-diol (MW 530), and poly(dimethylsiloxane) (PDMS, MW 500). PEG'S oxygen-rich polymer chain promotes strong interactions with water, making it highly hydrophilic,46while PCL-diol, with hydrocarbon chains and polar ester groups, displays moderate hydrophobicity.47PDMS, with its silicone-based structure and methyl-covered surface, is the most hydrophobic among the three polymers.48We chose non-methacryalted polymers to ensure that the effects observed on the release kinetics are purely due to the variation in hydrophilicity. Relase profiles of TAF from these ISCDs were compared to the release from unmodified ISCD formulated with pristine PCLDMA. The data support a positive correlation between the hydrophilicity of the additive and the drug release rate from ISCDs. ISCDs containing PEG exhibited the highest cumulative release (46.5 ± 4.5%) at 42 days post-incubation (FIG 8D, 10). This was nearly double the release observed for the unmodified ISCD (24.5 ± 2.3% on day 42). PCL-diol, with intermediate hydrophobicity, also led to a significantly increased release (38.4 ± 2.7%) compared to the unmodified ISCD. Conversely, PDMS, the most hydrophobic additive, showed minimal impact on drug release. These findings suggest that incorporating hydrophilic additives into the polymer network of ISCDs can enhance the drug release rate. Building upon our investigation into the influence of external polymer additives, we sought to explore if methacrylation of the hydrophilic polymer additives and the degree of methacrylation can further impact drug release. To study this, we incorporated 25 wt% of non methacrylated PEG (PEG), singly-methacrylated PEG (polyethylene glycol monomethyl ether mono-methacrylate, PEGMMA), or double-methacrylated PEG (polyethylene glycol dimethacrylate, PEGDMA) along with PCLDMA. All PEG derivatives had the same molecular weight of 500. Increasing the degree of methacrylation reduced both burst release and the overall release rate (FIG 8E). By 240 days, ISCDs with PEG, PEGMMA, and PEGDMA showed cumulative releases of 72.6 ± 5.3 %, 62.5 ± 6.3 %, and 54.2 ± 3.1 %, respectively, which were all significantly higher than the cumulative release of 42.4 ± 5.5 % observed for the unmodified ISCD (FIG 11 A). To elucidate the mechanism of increased drug release due to the incorporation of polymer additives, we performed scanning electron microscopy (SEM) based qualitative assessment of depots at week 1 post-incubation in PBS at 37°C and also determined their cross-linking density. Depot morphology aligned well with the observed drug release behavior (FIG 8G). ISCD containing PEG, the most hydrophilic additive, displayed a highly porous surface compared to the control depot consisting of PCLDMA alone, which showed a densely packed smooth surface. This suggests erosion of PEG-containing ISCD, which could be attributed to the efflux of the uncrosslinked PEG polymer due to its hydrophilicity, leading to enlarged pores, and hence faster drug release than the unmodified ISCD. ISCD with PCL-diol, exhibiting an intermediate hydrophilic character, also showed mild erosion with surface roughness, which explains faster drug release than the unmodified ISCD. Conversely, ISCD with PDMS, the most hydrophobic additive, displayed minimal erosion and a smooth surface, consistent with minimal difference in drug release compared to the unmodified ISCD. Interestingly, PEG-containing ISCD depots also showed significantly lower cross-linking density and swelling percentage compared to the unmodified ISCD (FIG 8C, 9). These observations support the conclusion that incorporating non-crosslinkable hydrophilic polymers as additives can significantly influence the drug release profile from ISCD by decreasing the cross-linking density and promoting erosion of the depot. Notably, unlike non- methacrylated PEG-containing ISCD, SEM images of PEGMMA or PEGDMA-containing ISCD didn’t show any signs of depot erosion (FIG 8G), despite their higher TAF release compared to the unmodified ISCD (FIG 8E). However, PEGMMA-containing ISCD showed a significantly lower crosslinking density and higher swelling percentage compared to the unmodified ISCD (FIG 8C,9). PEGDMA-containing ISCD on the other hand did not show significant changes in cross-linking density or swelling compared to the unmodified ISCD. These findings are consistent with the drug release kinetics, which was significantly faster for PEGMMA-containing depots compared to the ones with PEGDMA, and suggest that the increase in drug release observed with methacrylated hydrophilic polymers is correlated to their ability to reduce the cross-linking density of the network. Significantly increased drug release for PEGDMA-containing ISCD compared to the unmodified ISCD could be attributed to the inherent hydrophilic nature of PEGDMA, which can increase the overall hydrophilicity of the network, thereby increasing water permeability, leading to faster drug diffusion. The absence of depot erosion for PEGMMA and PEGDMA-containing ISCDs can be largely attributed to the ability of PEGMMA and PEGDMA to cross-link within the polymer network. Overall, our data shows that the incorporation of hydrophilic additives in ISCD can enhance drug release by promoting depot erosion, modulating the cross-linking density, or by simply increasing the overall hydrophilicity of the network.

[0234] The in vitro degradation of ISCD was also found to be dependent on the cross-linking density. Degradation was assessed by monitoring mass changes of ISCDs over time while incubated in PBS at 37 °C. After seven months, ISCDs with PEG and PEGMMA exhibited significantly faster degradation, reaching 28.9 ± 2.0% and 19.1 ± 3.0%, respectively, compared to the unmodified ISCD, 12.9 ± 1.3 % (FIG 8F, 11). In contrast, ISCD with PEGDMA showed a similar degradation profile as the control ISCD, reaching 11.1 ± 2.6 % at 7 months post-incubation, , which is consistent with their similar cross-linking densities.

[0235] Example 3: In vivo pharmacokinetics and biodegradation of ISCD

[0236] To assess the pharmacokinetics (PK) of ISCD systems in vivo, 500 pL of 90 mg / mLTAF loaded ISCDs were injected in Sprague Dawley rats (n = 3) and compared to the conventional ISFI systems, composed of PLGA 40 wt % dissolved in 55 wt % almond oil, 5wt % palm oil. Based on in vitro TAF release kinetics study, two ISCD compositions were selected, pristine PCLDMA and PCLDMA with 25 wt % PEGMMA. Plasma samples were collected at hour 4, day 1 , 2, 4, 7, 10, week 2, 3, 5, 6, 9, and then at three-week intervals postinjection through month 7. It should be noted that TAF is highly unstable in rodent plasma, and rapidly converts to tenofovir (TFV) due to high levels of plasma esterases expressed in rodent species which lead to hydrolytic cleavage of TAF.16 49Because of this reason, we couldn’t detect TAF levels in rat plasma, and therefore measured TFV levels to assess the PK. The in vivo release rate (pg / day) from ISCD formulations was determined using the area function method33by comparing the ISCD PK data (FIG 8H) with intravenous (IV) PK data of a single bolus of TAF in rats (FIG 12A). Additionally, we performed compartmental PK modeling to quantitatively characterize the in vivo PK profiles of ISCDs (FIG 12D, 13) (Tables 10, 11). This involved integrating an appropriate absorption kinetic model describing the in vivo release rate of ISCD (FIG 8I) with a two-compartment disposition kinetics model describing the IV PK data (FIG 12A). Burst release, as characterized by the peak plasma TFV concentration (Cmax) at 4 hours post-injection was almost 5-fold lower for both unmodified ISCD (133.7 ± 12.7 ng / mL) and PEGMMA-containing ISCD (146.7 ± 27.3 ng / mL), as compared to the ISFI formulation (668.0 ± 246.0 ng / mL) (FIG 8H). Following a minimal burst release (<6% of the total drug dose; Table 11), plasma TFV levels of rats injected with unmodified ISCDs reached less than 10 ng / mL within 10 days and maintained a sustained level of 1-10 ng / mL for at least 210 days (7 months). Based on our PK analysis, the unmodified ISCD maintained a steady daily release rate of 50-100 pg / day for at least 7 months (FIG 8I), translating to a cumulative drug release of 81 % at 7 months (FIG 8J). PEGMMA-containing ISCD displayed a distinct release profile compared to unmodified ISCD, characterized by a higher release rate and shorter duration (FIG 8H-J). After a minimal initial burst (<7% of the total dose), plasma TFV levels peaked below 20 ng / mL by day 10 and remained between 10-20 ng / mL until day 63. This was followed by a rapid decline, reaching undetectable levels (<1 ng / mL) after 120 days (4 months). Notably, PCLDMA / PEGMMA released TFV at a significantly higher rate (-200 pg / day) for 2 months compared to PCLDMA (FIG 8I), resulting in a cumulative release of 86% at 3 months (FIG 8J). These PK data are consistent with the trends observed in our in vitro release kinetics study. Importantly, both ISCD formulations demonstrated significantly longer sustained release compared to the conventional ISFI system, which exhibited a high initial burst release followed by a rapid decline in plasma TFV levels, falling below the detection limit after 2 months.

[0237] At the end of the study, we retrieved the depots from euthanized rats (FIG 8K), and the remaining drug load was quantified using CHN analysis (FIG 8L). Significantly higher amounts of remaining TAF were observed in the unmodified ISCD compared to PEGMMA-containing ISCD (22.8 ± 3.4% for PCLDMA and 9.1 ± 1 .8% for PCLDMA / PEGMMA). Notably, explants from ISFIs at month 2 post-injection exhibited minimal drug remaining (0.26 ± 0.20%) (FIG 14A). ISCDs could be extracted from tissue as a single, easily removable solid depot (FIG 8K), demonstrating superior retrievability, whereas ISFIs were observed as fragile solids, prone to fracture and difficult to extract from the tissue (FIG 14B). We also measured the mass of the remaining depots to assess the biodegradation of ISCD. The remaining mass of the extracted depots was found to be 67.1 ± 2.8% for the unmodified ISCD and 45.2 ± 12.1 % for PEGMMA-containing ISCD, which correspond to 33% and 55% in vivo degradation, respectively (FIG 8M). The trend is consistent with the in vitro degradation data and confirms that the addition of PEGMMA increases the degradation of ISCD. We also used magnetic resonance imaging (MRI) to evaluate the long-term morphological changes of ISCDs in vivo and to study the interactions between depots and host tissues at day 1 , month 1 , and month 7 after injection (FIG 8N). We confirmed that both unmodified and PEGMMA-containing ISCDs did not migrate to other sites or cause adverse tissue reactions. The size of the depots decreased over time, indicating biodegradation, but both depots maintained good structural integrity. Interestingly, at month 7 postinjection, we found that the PEGMMA-containing ISCD appeared white on MRI images, as did the tissue surrounding the depot, indicating that they had a higher water content than before, while the PCLDMA-only depots remained dark. Since PEGMMA is hydrophilic, higher water content could be attributed to the penetration of water into the depots, as the polymer network loosened over time.

[0238] It was critical to confirm if TAF encapsulated within the ISCDs didn’t degrade into TFV and maintained its chemical structure prior to release. To address this, we employed high-performance liquid chromatography (HPLC) analysis of explanted depots retrieved at 7 months post-injection. The HPLC data of the dissolved explants exhibited a single peak eluting at the same retention time as the freshly prepared TAF solution (FIG 15). This confirms the absence of degradation products within the retrieved depots, indicating that TAF remains stable during the crosslinking process and within the depot under in vivo conditions prior to its release.

[0239] Example 4: Biocompatibility and retrievability of ISCDs in vitro

[0240] In vivo biocompatibility is essential for LAIs to minimize adverse reactions and inflammation at the injection site, ensuring patient safety and efficacy.6 50We evaluated the biocompatibility of ISCD consisting of pristine PCLDMA without any drug. Depots were injected on day 0, followed by histological and immunohistochemistry (IHC) analysis of the local tissue explanted at week 1 , week 4, and month 7 post-injection. The H&E staining and IHC analysis revealed an initial inflammatory response at week 1 , characterized by the presence of immune cells, including CD3-positive T cells and CD68-positive macrophages, around the depot (FIG 16A-C). However, by week 4, there was a substantial decrease in the number of immune cells around the depot (FIG 16B), accompanied by a significant reduction in CD3 and CD68 positive populations (FIG 16C), a trend that persisted at month 7, resulting in negligible immune cells present around the depot. Importantly, the tissue samples exhibited no signs of fibrosis, a major complication associated with implant failure and typically identified by excessive collagen deposition.51These findings indicate successful integration of the depot with the surrounding tissue.

[0241] Although ISCD is biocompatible, the drug delivered via ISCD may exhibit adverse effects, necessitating prompt depot retrieval. In such cases, a rapid decrease in plasma drug levels following depot removal would be crucial to mitigate side effects. To confirm this, we subcutaneously administered TAF-loaded ISCD to rats and retrieved the depots two weeks later through a small incision near the injection site on the skin (FIG 12D). Plasma TFV levels were monitored after retrieval. Following the depot removal, plasma TFV concentrations showed an exponential decline, decreasing more than four-fold within a day to less than 10 ng / mL (FIG 12E). By day 10 post-retrieval, TFV plasma concentrations had dropped below the limit of detection (1 ng / ml) for two out of three rats, and the plasma level went below detection for all three rats by day 14, confirming the safety of ISCD.

[0242] Example 5: Compatibility of ISCD with a wide range of therapeutics and combination therapy options

[0243] Next, we wanted to understand the versatility of the ISCD platform for delivering a broad spectrum of hydrophilic drugs with water solubilities in the range of 3-112 mg / mL (FIG 17A, Table 9; see Table 8 for chemical structures). We chose drugs representing diverse therapeutic classes: antiretrovirals, including emtricitabine (FTC), abacavir (ABC) and lamivudine (LAM), an opiate antagonist, naltrexone (NAL), and antibiotics, including vancomycin (VAN) and amoxicillin (AMX). We also evaluated a hydrophobic drug - tacrolimus - a clinically used immunosuppressant. All drugs were encapsulated at a concentration of 90 mg / mL for direct comparison of the release profiles between different drugs. In vitro, ISCD demonstrated ultra-long-term release of all the therapeutics over at least 150-360 days (FIG 17A). Our analysis revealed a positive correlation between a drug's hydrophilicity and its cumulative release on day 1 . Drugs with higher water solubility (more hydrophilic) showed a higher initial release compared to drugs with lower water solubility (FIG 17B, Table 9). This can be explained by the stronger interaction between hydrophobic drugs and the hydrophobic polymer backbone of PCLDMA. However, ISCD successfully minimized the overall burst release for all the drugs. Even for the drugs with water solubilities as high as 100 mg / mL, such as NAL and FTC, day 1 cumulative release was -20%, which is significantly lower than the cumulative release reported for hydrophilic drugs from injectable systems developed previously.52-53

[0244] We also demonstrated the potential of ISCD for co-delivery of combination therapies. We encapsulated two clinically used combination regimens for HIV therapy: Epzicom (FTC and LAM) and Descovy (ABC and TAF). We compared the release kinetics of these drugs from ISCD when encapsulated individually versus in combination. Release profiles of drugs encapsulated individually were almost identical to those drugs encapsulated in combination (FIG 17C,D). This suggests that ISCD can be formulated to co-deliver multiple drugs while maintaining their independent release characteristics, making it a promising platform for long-acting combination therapy.

[0245] Building on the in vitro demonstration of ISCD’s versatility, we conducted a PK study to validate the ultra-long-term release of drugs with different water solubilities in vivo. \Ne selected two drugs with contrasting water solubilities compared to TAF, which showed ultra-long-term release in vivo. These included TAC, which has lower water solubility than TAF, and NAL, a drug with higher water solubility than TAF (Table 9). Rats were subcutaneously injected with 500 pL of PCLDMA ISCDs containing either 45 mg / mL of TAC or 90 mg / mL of NAL. Since our in vitro data showed a faster release of NAL compared to TAC, we used a higher concentration of NAL than TAC. Blood samples were collected at intervals up to 6 or 7 months post-injection to analyze whole-blood concentration of TAC and plasma concentration of NAL. Similar to TAF, we performed PK analysis for TAC and NAL- loaded ISCDs by utilizing both non-compartmental and compartmental methods. For compartment modeling of the PK profiles (FIG 12,13, Table 10), we established the disposition kinetics by two- compartmental analysis of PK data obtained from experimental bolus intravenous injection of TAC and NAL in rats. Consistent with the in vitro release data, ISCD showed a higher initial burst release for NAL in vivo (27%) compared to TAC (5.8%) (FIG 17G,H, Table 9), with Cmax values of 64.9 ± 13.0 ng / mL and 150.7 ± 80.5 ng / mL for NAL and TAC, respectively. Following the initial burst, the ISCD established sustained drug release. As expected based on the water solubilities of NAL and TAC, the systemic concentration of NAL was maintained within a range of 5-15 ng / mL, while TAC concentrations were lower, ranging from 0.5-1 .5 ng / mL. This translated to a cumulative release of approximately 92% for NAL and 35% for TAC in 6 months (FIG 17G,H). These findings demonstrate the versatility of the ISCD platform to enable ultra-long-term release of drugs with a wide range of water solubility. The data also establish a clear relationship between a drug's hydrophilicity and its PK, when delivered using an ISCD system.

[0246] Convolution analysis was utilized to predict the human PK of NAL- and TAC-loaded ISCDs. Human disposition kinetics information was obtained by the analysis of previously reported human PK data for NAL IV bolus38and TAC administered orally37(Table 12). These human disposition kinetics were then integrated with the release function derived from the PK analysis of NAL and TAC-loaded ISCDs in rats, assuming simple allometry on release rate constants between species. The predicted human PK profiles for NAL and TAC from ISCDs (FIG 171-J) were compared with the PK of their clinically available formulations, including oral TAC37, oral NAL38, and an intramuscular (IM) LAI of NAL (Vivitrol®)54. Notably, a single dose of NAL-loaded ISCD, when injected at 20 mg / kg is predicted to maintain prolonged steady plasma concentrations for at least 6 months, well within the established peak and trough levels observed with once-daily oral doses of NAL tablets at 50 mg (FIG 171).55Compared to Vivitrol®, the projected PK profile of NAL-loaded ISCD showed 4.6-fold lower burst release.54Interestingly, while Vivitrol®, with a NAL dose of 380 mg, requires a once-monthly injection, the ISCD, with only ~4 times the dose of NAL compared to Vivitrol®, maintains steady plasma concentrations for at least 6 months, suggesting the potential to improve the dosing schedule from once a month to once every 6 months. Similarly, the predicted PK profile of TAC from 3, 4 and 5 mg / kg TAC-loaded ISCDs showed sustained release for at least 6 months (FIG 18), with blood levels maintained within the therapeutic concentration range of twice-daily oral doses of TAC capsules (0.038 mg / kg) for 2-3 months (FIG 17J).37Overall, our data clearly suggest that ISCD has the potential to enable ultra-long-term release of both hydrophilic and hydrophobic drugs in humans. Table 9 Initial burst release against water solubility of therapeutics

[0247] Table 10 The estimated PK parameters of disposition and release kinetics were obtained from the concentration -time profiles following IV administration and SC implants of ISCDs in rats. aCoefficient of Variability (CV)%;bFixed parameters

[0248] Table 11 Drug release kinetic parameters for drug-loaded ISCD formulations.

[0249] Calculated as %Total drug mass released in vivo by the end of the study -%Release by initial and intermediate phases

[0250] Table 12 The disposition PK parameter values obtained from the concentration-time profiles of TAC (0.075 mg / kg / day) and NAL (1 mg) following oral or IV administration in humans.

[0251] Example 6: Addition of small vinyl molecules to ISCD to improve the drug release behavior

[0252] The addition of a small vinyl molecule to the polycaprolactone component lower burst release in vitro, provides easier injectability and easier homogenization of drug. By addition vinyl acetate, which acts as both a chain extender and a non-water-miscible organic solvent, we were able to allow long curing formulations (up to 8 minutes) of 75% PCLDMA, 25% vinyl acetate to restrain burst volumes to be similar to that of fast-curing formulations (3-minute curing) (FIG 19). Conversely, similarly long curing formulations of 100% PCLDMA observe an increase in burst compared to fastcuring formulations. An additional benefit to the addition of vinyl acetate is that it further decreases the formulation viscosity (FIG 20), allowing it to be delivered through smaller needles. Finally, vinyl acetate was also observed to aid dissolution of TAF and led to a more homogeneous drug distribution in the final suspension. It should be noted that vinyl acetate is poorly miscible in water and so does not cause the solvent efflux effects that occur in in situ forming implants. We further explored other small vinyl molecules in addition to vinyl acetate. By addition of 25% ethyl / propyl / butyl acetate molecule to 75 % PCLDMA ISCD, ISCD increase the curing time along with slight increase in TAF release rate than 75 % PCLDMA with 25% vinyl acetate (FIG 21).

[0253] Example 7: Delivery of biologies using ISCD

[0254] The in situ cross-linked depots (ISCD) can be utilized for ultra-long-term delivery of biologies, including peptides, proteins, antibodies, and nucleic acids, as was demonstrated by studies done on the release of semaglutide, a glucagon-like peptide-1 (GLP-1) agonist.

[0255] In one study, prolonged delivery of semaglutide was demonstrated in rats. A single subcutaneous injection of 500 pL of ISCD, composed of polycaprolactone dimethylacrylate (PCLDMA) and polyethylene glycol dimethacrylate (PEGDMA) at a 70:30 weight ratio, and loaded with 10 mg / mL of semaglutide, achieved over 150 days of sustained semaglutide release, as evidenced by the consistent plasma concentrations (Figure 22). Adjusting the ratio of PCLDMA to PEGDMA allowed for modulation of both the initial burst release and the steady-state plasma concentrations. Notably, increasing the PEGDMA content from 30 wt% to 50 wt% resulted in higher burst release and elevated steady-state plasma levels (Figure 23).

[0256] Distinguishing features of the disclosed embodiments

[0257] 1 . ISCD can exhibit sustained release of hydrophilic drugs for at least six months. This is a significant advancement, as this allows for thelong-term delivery of hydrophilic drugs with injectable delivery systems.

[0258] 2. ISCD formulation is injectable, but also retrievable and maintains drug stability in vivo: ISCD depots are retrievable from rats as a single clean piece and retained -60% drug after 2 months and -40 % drug after 7 months purely as TAF with no detectable levels of degradation products. This suggests the ISCD formation process does not adversely affect the drug structure, and the formed depot has an excellent ability to protect stored drug from the outside environment.

[0259] 3. ISCD's release rate can be tuned to achieve desired plasma levels of drugs: We have demonstrated that using 1) higher molecular weight PCLDMA, 2) lower concentrations of BPO and DMT, 3) higher concentration of non-crosslinkable polymers (spacers), and 4) higher concentration of hydrophilic polymers in ISCD can increase the release rate. This might be helpful in case a faster release rate is required in large animal models / humans to achieve higher plasma levels of the encapsulated drugs.

[0260] 4. A small vinyl monomer can be added to the ISCD formulation to tune burst release and crosslinking time: By blending the ISCD polycaprolactone component with 25% of vinyl acetate, a small vinyl molecule, ISCD formulations with lower BPO / DMT and prolonged curing times up to 8 minutes may be used without increase in burst relative to faster-curing formulations. This can help to make the ISCD practically usable in a clinical setting, where the pre-ISCD formulation will have a wider window of time for injection after mixing without premature solidification or unwanted burst release.

[0261] 5. ISCD can also deliver hydrophobic drugs including tacrolimus and darunavir with minimal release burst release.

[0262] 6. ISCD can encapsulate various types of therapeutics, including small molecules, peptides and biologies.7 7. ISCD can encapsulate multiple therapeutics at the same time, enabling combination therapy in a single injection.

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[0317] Enumerated Embodiments

[0318] E1 . A long-acting injectable composition comprising:

[0319] (i) at least one crosslinkable low molecular weight hydrophobic polymer with a molecular weight of less than 10000 Da;

[0320] (ii) at least one crosslinking initiator and accelerator pair; and

[0321] (iii) at least one therapeutic, prophylactic or diagnostic agent.

[0322] E2. A long-acting injectable composition comprising:

[0323] (i) at least one hydrophobic methacrylated or acrylated polymer with a molecular weight of less than 5000 Da;

[0324] (ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking; and

[0325] (iii) at least one therapeutic, prophylactic or diagnostic agent.

[0326] E3. The composition of claim E1 or E2, wherein the hydrophobic liquid polymer comprises polycaprolactone dimethylacrylate (PCLDMA) or polycaprolactone tri methacrylate (PCLTMA) or both.

[0327] E4. The composition of any of claims E1 to E3, wherein the organic peroxide is selected from benzoyl peroxide, parachlorobenzoyl peroxide, 2,4-dichlorobenzoyl peroxide, acetyl peroxide, lauroyl peroxide, tertiary butyl peroxide, cumene hydroperoxide, 2,5-dimethylhexane, 2,5- dihydroperoxide, methyl ethyl ketone peroxide, and tertiary butyl peroxybenzoate, or a combination of them.

[0328] E5. The composition of any of the claims E1 to E4, wherein the secondary or tertiary amine is selected from N,N-dimethyl-p-toluidine, N,N-dimethylaniline, N-p-hydroxyethyl-aniline, N,N-di(p- hydroxyethyl)-aniline, N,N-di(p-hydroxyethyl)-p-toluidine, N-methyl-aniline, and N-methyl-p-toluidine, or a combination of them.

[0329] E6. The composition of any of claims E1 to E5, wherein the hydrophobic liquid polymer comprises a low molecular weight (MW) polymer, e.g. MW less than 2000 Da.

[0330] E7. The composition of any of claims E1 to E6, wherein the hydrophobic liquid polymer comprises a low molecular weight (MW) polymer, e.g. MW less than 1000 Da.

[0331] E8. The composition of any of claims E1 to E7, wherein the hydrophobic liquid polymer comprises a low molecular weight (MW) polymer, e.g. MW less than 600 Da.

[0332] E9. The composition of any of claims E1 to E8, wherein the concentration of crosslinking initiator and accelerator is between 0.025-1% w / v each.

[0333] E10. The composition of any of claims E1 to E9, wherein the concentration of crosslinking initiator and accelerator is between 0.05%-0.5% w / v each.

[0334] E11. The composition of any of claims E1 to E10, wherein the concentration of crosslinking initiator and accelerator is between 0.1%-0.3% w / v each.

[0335] E12. The composition of any of the claims E1 to E11 , wherein the composition is injectable with a viscosity between 0.1-1 Pa.sa and crosslinks to form a solid monolithic depot, with a curing time ranging between 1-10 min.

[0336] E13a. A long-acting injectable composition comprising:

[0337] (i) at least one hydrophobic methacrylated or acrylated polymer with a molecular weight of less than 5000 Da;

[0338] (ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking;

[0339] (iii) at least one therapeutic, prophylactic, or diagnostic agent; and

[0340] (iv) at least one polymeric additive to tune the release kinetics of the encapsulated agent.

[0341] E13b. The composition of any of the claims E1-12, wherein the additive is either a low molecular weight non-crosslinking hydrophobic polymer or a low molecular weight hydrophilic polymer, with molecular weight less than 5000 Da.

[0342] E14. The composition of any of the claims E1-13, wherein the additive is selected from polyethylene glycol (PEG), Poly(ethylene glycol) methacrylate (PEGMMA), Poly(ethylene glycol) dimethacrylate (PEGDMA), vinyl acetate, polydimethylsiloxane (PDMS), polycaprolactone (PCL) or their derivates.

[0343] E15. The composition of any of the claims E1-14, wherein the additive is present at a concentration between 5-90% w / v.

[0344] E16. The composition of any of the claims E1-15, wherein the additive is present at a concentration between 25-75% w / v.

[0345] E17. A long-acting injectable composition comprising:

[0346] (i) at least one hydrophobic methacrylated or acrylated polymer with a molecular weight of less than 5000 Da;

[0347] (ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking;

[0348] (iii) at least one therapeutic, prophylactic or diagnostic agent; and

[0349] (iv) a solvent additive to prolong the curing time of the polymer / initiator / accelerator mixture.

[0350] E18. The composition of any of the claims E1-17, wherein the additive is selected from vinyl acetate, ethyl acetate, propyl acetate, butyl acetate or a combination thereof.

[0351] E19. The composition of any of the claims E1-18, wherein the solvent is present at a concentration between 5-50% w / v.

[0352] E20. The composition of any of the claims E1-19, wherein the solvent is present at a concentration between 15-30% w / v.

[0353] E21. The composition of any of the claims E1 to E20, wherein the therapeutic or prophylactic agent is selected from the group consisting of but not limited to Analgesics, Antianxiety Drugs, Antiarrhythmics, Antibacterials, Antibiotics, Anticoagulants and Thrombolytics, Anticonvulsants, Antidepressants, Antiemetics, Antifungals, Antihistamines, Antihypertensives, Anti-Inflammatories, Antineoplastics, Antipsychotics, Antipyretics, Antiretrovirals, Antivirals, Barbiturates, Beta-Blockers, Bronchodilators, Corticosteroids, Cytotoxics, Diuretics, Hormones, Hypoglycemics Immunosuppressives, Muscle Relaxants, Sedatives, Tranquilizer, and Vitamins or a combination thereof.

[0354] E22. The composition of any of the claims E1 to E21 , wherein the active agent comprises of a small molecule or a biologic including protein, peptide, enzyme and nucleic acid. E23. The composition of any of claims E1 to E22, wherein the drug or active agent is selected from the group consisting of but not limited to tenofovir alafenamide, emtricitabine, abacavir sulfate, lamivudine, zidovudine, tacrolimus, naltrexone, or a combination thereof.

[0355] E24. The composition of any of the claims E1 to E23, wherein the extended release comprises a substantially sustained release of the drug or active agent over months, optionally at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 15 months, or more.

[0356] E25. The composition of any of the claims E1 to E24, wherein the composition is injectable with a viscosity between 0.1-1 Pa.sa and crosslinks to form a solid monolithic depot, with a curing time ranging between 1-20 min.

[0357] E26. The composition of any of the claims E1 to E25, wherein the composition can be injected subcutaneously or intramuscularly into a subject and cures to form an implant in situ.

[0358] E27. The composition of any of the claims E1 to E26, wherein the composition is cured to form an implant, which is then implanted subcutaneously or intramuscularly into a subject.

[0359] E28. The composition of any of the claims E1 to E27, wherein the composition is suitable for treatment, prevention, or diagnosis of a disease in a subject.

[0360] E29. The composition of any of the claims E1 to E28, wherein the subject is a mammal.

[0361] E30. The composition of any of the claims E1 to E29, wherein the subject is a human, dog, cat, or farm animal.

[0362] E31. The composition of any of the claims E1 to E30, wherein the cured implant is biodegradable.

[0363] Other Embodiments

[0364] It will be appreciated by those skilled in the art that while the disclosed subject matter is described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto. Each reference cited herein is incorporated by reference in its entirety.

[0365] Various features and advantages of the invention are set forth in the following claims.

Claims

What is claimed:Claims1 . An injectable composition comprising:(i) at least one crosslinkable low molecular weight hydrophobic pre-polymer with a molecular weight of less than 10000 Da;(ii) at least one crosslinking initiator and accelerator pair; and(iii) at least one active agent.

2. An injectable composition comprising:(i) at least one hydrophobic methacrylated or acrylated pre-polymer with a molecular weight of less than 10000 Da;(ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking; and(iii) at least one active agent.

3. The injectable composition of any one of claims 1 or 2, wherein the composition is substantially solvent-free.

4. An injectable composition comprising:(i) at least one hydrophobic methacrylated or acrylated pre-polymer with a molecular weight of less than 10000 Da;(ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking;(iii) at least one active agent; and(iv) a solvent additive to dissolve the pre-polymer and / or to prolong the curing time of the pre- polymer / initiator / accelerator mixture.

5. The composition of claim 4, wherein the solvent additive is selected from vinyl acetate, ethyl acetate, propyl acetate, butyl acetate or a combination thereof.

6. The composition of claim 4 or 5, wherein the solvent additive is present at a concentration between 5-50% w / v.

7. The composition of any of the claims 4-6, wherein the solvent additive is present at a concentration between 15-30% w / v.

8. The composition of any one of claims 1-7, wherein the active agent is a therapeutic agent.

9. The composition of any one of claims 1 -7, wherein the active agent is a prophylactic agent.

10. The composition of any one of claims 1 -7, wherein the active agent is a diagnostic agent.11 . The composition of any one of claims 1-10, wherein the composition comprises of one active agent.

12. The composition of any one of claims 1-10, wherein the composition comprises of at least two active agents.

13. The composition of any one of claims 1-12, wherein the active agent is suspended in the prepolymer using ultra-sonication.

14. The composition of any one of claims 1-12, wherein the active agent is suspended in the prepolymer using micronization.

15. The composition of any one of claims 1-12, wherein the active agent is dissolved in the prepolymer using a co-solvent.

16. A depot formed from the injectable composition of any one of claims 1-15, wherein the composition crosslinks to form the depot with a curing time ranging between 1-10 min.

17. The depot of claim 16, wherein the depot is a solid monolithic depot.

18. The depot of claim 16, wherein the active agent is incorporated into the depot by physical entrapment.

19. The depot of claim 16, wherein the active agent is incorporated into the depot by covalent conjugation with the polymer.

20. The depot of any one of claims 16-19, wherein the active agent is distributed throughout the depot and released from the depot.21 . The depot of claim 20, wherein the depot releases at least 80% of the active agent in a time 2.5 times greater than tso in the post-injection environment.

22. The depot of claim 20 or 21 , wherein the time during which 10% of the active agent is released is equal to or greater than of tso in the post-injection environment.

23. The depot of any one of claims 20-22, wherein tso is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or at least 15 months.

24. The composition or the depot of any one of claims 1 -22, wherein the injectable composition comprises at least one polymeric additive to tune the release kinetics of the encapsulated active agent.

25. The composition or the depot of claim 24, wherein the polymeric additive is either a low molecular weight non-crosslinking hydrophobic polymer or a low molecular weight crosslinking or non- cross-inking hydrophilic polymer, with molecular weight less than 10000 Da.

26. The composition or the depot of claim 24 or 25, wherein the polymeric additive is selected from polyethylene glycol (PEG), Poly(ethylene glycol) methacrylate (PEGMMA), Poly(ethylene glycol) dimethacrylate (PEGDMA), vinyl acetate, polydimethylsiloxane (PDMS), or polycaprolactone (PCL).

27. The composition or the depot of any of the claims 24-26, wherein the polymeric additive is present at a concentration between 5-90% w / v.

28. The composition or the depot of any of the claims 24-26, wherein the polymeric additive is present at a concentration between 25-75% w / v.

29. The composition or the depot any one of claims 1-28, wherein the hydrophobic pre-polymer comprises a polymer with a molecular weight less than 2000 Da.

30. The composition or the depot of any one of claims 1-28, wherein the hydrophobic pre-polymer comprises a polymer with a molecular weight less than 1000 Da.

31. The composition or the depot of any one of claims 1-28, wherein the hydrophobic pre-polymer comprises a polymer with a molecular weight less than 600 Da.

32. The composition or the depot of any one of claims 1-31 , wherein the hydrophobic pre-polymer is a liquid below 45 °C.

33. The composition or the depot of any one of claims 1-32, wherein the hydrophobic pre-polymer comprises polycaprolactone dimethylacrylate (PCLDMA) or polycaprolactone tri methacrylate (PCLTMA) or both.

34. The composition or the depot of any of claims 1-33, wherein the initiator is an organic peroxide or a combination of organic peroxides selected from a group consisting of: benzoyl peroxide, parachlorobenzoyl peroxide, 2,4-dichlorobenzoyl peroxide, acetyl peroxide, lauroyl peroxide, tertiary butyl peroxide, cumene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, methyl ethyl ketone peroxide, and tertiary butyl peroxybenzoate.

35. The composition or the depot of any of claim 1 -34, wherein the initiator is benzoyl peroxide.

36. The composition or the depot of any one of claims 1-35, wherein the accelerator is a secondary or tertiary amine or a combination of amines selected from a group consisting of: N,N- dimethyl-p-toluidine, N,N-dimethylaniline, N-p-hydroxyethyl-aniline, N,N-di(p-hydroxyethyl)-aniline, N,N-di(p-hydroxyethyl)-p-toluidine, N-methyl-aniline, and N-methyl-p-toluidine.

37. The composition or the depot of any one of claims 1-36, wherein the accelerator is N,N- dimethyl-p-toluidine.

38. The composition or the depot of any one of claims 1-37, wherein the concentration of crosslinking initiator and accelerator is between 0.025-1% w / v each.

39. The composition or the depot of any one of claims 1-38, wherein the concentration of crosslinking initiator and accelerator is between 0.05%-0.5% w / v each.

40. The composition or the depot of any one of claims 1-39, wherein the concentration of crosslinking initiator and accelerator is between 0.1%-0.3% w / v each.

41. The composition or the depot of any one of claims 1-40, wherein the injectable composition has a viscosity between 0.01-1 Pa.sa.

42. The composition or the depot of any one of claims 1-41 , wherein the composition comprises between 1% and 50% w / w of the active agent.

43. The composition or the depot of any one of claims 1-42, wherein the composition comprises between 2% and 12% w / w of the active agent.

44. The composition or the depot of any one of claims 1-42, wherein the composition comprises at least 10 mg / mL of active agent.

45. The composition or the depot of any one of claims 1-42, wherein the composition comprises 30 to 150 mg / mL of active agent.

46. The composition or the depot of any of the claims 1-45, wherein the active agent is hydrophobic.

47. The composition or the depot of any of the claims 1-45, wherein the active agent is hydrophilic.

48. The composition or the depot of any of the claims 1-47, wherein the active agent is selected from the group consisting of: Analgesics, Antianxiety Drugs, Antiarrhythmics, Antibacterials, Antibiotics, Anticoagulants and Thrombolytics, Anticonvulsants, Antidepressants, Antiemetics, Antifungals, Antihistamines, Antihypertensives, Anti-Inflammatories, Antineoplastics, Antipsychotics, Antipyretics, Antiretrovirals, Antivirals, Barbiturates, Beta-Blockers, Bronchodilators, Corticosteroids, Cytotoxics, Diuretics, Hormones, Hypoglycemics Immunosuppressives, Muscle Relaxants, Sedatives, Tranquilizer, and Vitamins or a combination thereof.

49. The composition or the depot of any of the claims 1-48, wherein the active agent comprises of a small molecule, or a biologic selected from a group consisting of: protein, peptide, enzyme and nucleic acid.

50. The composition or the depot of any one of claims 1-49, wherein the active agent is selected from the group consisting of: tenofovir alafenamide, emtricitabine, abacavir sulfate, lamivudine, zidovudine, tacrolimus, naltrexone, and a combination thereof.

51. The composition or the depot of any of the claims 1-50, wherein the injectable composition can be injected subcutaneously or intramuscularly into a subject and cures to form an implant in situ.

52. The composition of any of the claims 1-50, wherein the composition is cured to form an implant, which is then implanted subcutaneously or intramuscularly into a subject.

53. A solvent-free long-acting injectable composition comprising:(i) at least one hydrophobic methacrylated or acrylated pre-polymer with a molecular weight of less than 5000 Da;(ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking;(iii) at least one therapeutic, prophylactic, or diagnostic agent; and(iv) at least one polymeric additive to tune the release kinetics of the encapsulated agent.

54. An injectable composition comprising:(i) at least one hydrophobic methacrylated or acrylated pre-polymer with a molecular weight of less than 10000 Da;(ii) at least one organic peroxide (initiator) and a secondary or tertiary amine (accelerator) redox pair for cross linking;(iii) at least one active agent; and(iv) a solvent additive to dissolve the pre-polymer and / or to prolong the curing time of the pre- polymer / initiator / accelerator mixture.(v) at least one polymeric additive to tune the release kinetics of the encapsulated agent.

55. A kit comprising of the components needed to make an injectable composition or depot according to any one of claims 1-54.

56. The kit of claim 55, the kit comprising of:(i) a pre-polymer or a pre-polymer and active agent mixture; and(ii) a separate mixture comprising BPO and DMT.

57. The kit of claim 55 or 56, the kit further comprising instructions for preparing an injectable composition with different active agents.

58. A method of treating a subject in need thereof, the method comprising the steps:(i) prepare an injectable composition according to any one of claims 1-54;(ii) administer the injectable composition to a subject;(iii) the composition crosslinks to form a depot in vivo; and(iv) the depot formed releases the active agent to the subject over time.

59. The method of claim 58, wherein step (i) comprises using a kit of claim 55, 56, or 57.

60. The method of claim 58 or 59, wherein the subject is a mammal.61 . The method of any one of claims 58-60, wherein the subject is a human, dog, cat, or farm animal.

62. The method of any one of claims 58-61 , wherein step (ii) comprises administration of the injectable composition subcutaneously or intramuscularly to the subject.

63. The method of any one of claims 58-62, wherein the depot is biodegradable.

64. The method of any one of claims 58-63, wherein step (iv) comprises the release of at least 80% of the active agent in a time 2.5 times greater than tso.

65. The method of any one of claims 58-64, wherein the time during which 10% of the active agent is released is equal to or greater than — of tso.

66. The method of any one of claims 58-65, wherein tso is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or at least 15 months.

67. A method of treating a subject in need thereof, the method comprising the steps:(i) prepare an injectable composition according to any one of claims 1-54;(ii) the composition crosslinks to form an implant;(iii) the implant is then implanted subcutaneously or intramuscularly into a subject; and(iv) the implant releases the active agent to the subject over time.

68. The method of claim 64, wherein step (i) comprises using a kit of claim 55, 56, or 57.

69. The method of claim 65 or 66, wherein the subject is a mammal.

70. The method of any one of claims 67-69, wherein the subject is a human, dog, cat, or farm animal.71 . The method of any one of claims 67-70, wherein the implant is biodegradable.

72. The method of any one of claims 67-71 , wherein step (iv) comprises the release of at least80% of the active agent in a time 2.5 times greater than tso.

73. The method of any one of claims 67-72, wherein the time during which 10% of the active agent is released is equal to or greater than of tso.

74. The method of any one of claims 67-73, wherein tso is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or at least 15 months.

75. A method of diagnosing a disease in a subject, the method comprising the steps:(i) prepare an injectable composition according to any one of claims 1-74, wherein the injectable composition comprises of a diagnostic agent;(ii) administer the injectable composition to a subject;(iii) the composition crosslinks to form a depot in vivo; and(iv) the depot formed releases the diagnostic agent to the subject over time.

76. The method of claim 75, wherein the diagnostic agent is a fluorescent marker.

77. The method of claim 75, wherein the diagnostic agent is a small molecule or biologic.

78. The method of claim 75, wherein step (i) comprises using a kit of claim 55, 56, or 57.

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