Antiviral prodrugs and their nanoparticle preparations

Abstract

The present application relates to antiviral prodrugs and nanoformulations thereof. The present invention provides prodrugs and methods of use thereof, and in particular provides prodrugs of integrase inhibitors. In a specific embodiment, the prodrug comprises an integrase inhibitor modified with an ester, wherein the ester comprises an aliphatic group or a hydrocarbon group. In a specific embodiment, the integrase inhibitor is selected from the group consisting of: cabotegravir, raltegravir, elvitegravir, dolutegravir and bictegravir.

Description

[0001] This application is a divisional application of an application filed on October 22, 2019, with application number 201980085100.X, and invention name “Antiviral prodrugs and nanoformulations thereof”.

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 748,798, filed on October 22, 2018. The foregoing application is incorporated herein by reference. Field of the Invention

[0003] The present invention relates generally to the delivery of therapeutic agents. More specifically, the present invention relates to compositions and methods for delivering therapeutic agents to a patient to treat a disease or disorder. Background of the Invention

[0004] Significant progress has been made in the development of effective diagnosis and treatment for human immunodeficiency virus type 1 (HIV-1). Antiretroviral therapy (ART) has significantly reduced the morbidity and mortality associated with the disease, allowing infected people to have a near-normal quality of life (Vittinghoff et al. (1999) J. Infect Dis., 179(3): 717-720; Lewden et al. (2007) J. Acquir. Immune Defic. Syndr., 46(1): 72-77). However, ART requires lifelong treatment in order to suppress viral replication and prevent the onset of AIDS. In addition, the effectiveness of ART may be limited by HIV-1 resistance, drug toxicity, and poor patient adherence (Wensing et al. (2014) Top. Antivir. Med., 22(3): 642-650; Siliciano et al. (2013) Curr. Opin. Virol., 3(5): 487-494; Prosperi et al. (2012) BMC Infect. Dis., 12: 296-296; Van den Berk et al. (2016) Abstract No.: 948, in Conference on Retroviruses and Opportunistic Infections, 22-25; Siefried et al. (2017) PLoS One 12(4): e0174613). Treatment fatigue, lack of financial and social support, coexisting psychiatric symptoms and / or substance abuse may lead to failure to adhere to critical ART regimens (Tucker et al. (2017) EBioMedicine, 17: 163-171).

[0005] Long-acting parenteral (LAP) antiretroviral drugs have improved regimen compliance (Spreen et al. (2013) Curr. Opin. HIV AIDS, 8(6):565-571). Reducing the treatment schedule from daily to monthly or even less frequent administration provides greater patient privacy and satisfaction and improves regimen compliance (Sangaramoorthy et al. (2017) J. Assoc. Nurses AIDS Care, 28(4):518-531; Carrasco et al. (2017) Afr. J. AIDS Res. 16(1):11-18; Williams et al. (2013) Nanomed. Lond. 8(11):1807-1813). However, only a few antiretroviral drugs have been successfully reformulated as LAP.

[0006] Cabotegravir (CAB) is an integrase inhibitor or integrase strand transfer inhibitor (INSTI) with low aqueous solubility, high melting point, high potency, long half-life and slow metabolic clearance (Karmon et al. (2015) J. Acquir. Immune Defic. Syndr., 68(3): 39-41; Trezza et al. (2015) Curr. Opin. HIV AIDS 10(4): 239-245). These properties enable CAB to be formulated as a 200-mg / mL suspension (CAB LAP) and administered intramuscularly monthly or even less frequently (Margolis et al. (2017) Lancet 390(10101): 1499-1510; Spreen, WW (2014) J. Acquir. Immune Defic. Syndr., 67(5): 481-486). In particular, CAB plus rilpivirine (RPV) is the first long-acting combination ART regimen in which monthly or every other month CAB and RPV LAP formulations have shown antiretroviral activity comparable to daily oral three-drug combinations for maintenance therapy (Margolis et al. (2017) Lancet 390(10101):1499-1510).

[0007] Advantageously, CABs are primarily metabolized by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and have a low potential for interaction with other antiretroviral drugs (Trezza et al. (2015) Curr. Opin. HIV AIDS 10(4):239-245; Bowers et al. (2016) Xenobiotica 46(2):147-162). CAB LAP is highly protective against rectal, vaginal, and intravenous simian / human immunodeficiency virus (SHIV) transmission in non-human primates and has entered clinical trials for HIV prevention (NCT02720094) (Andrews et al. (2014) Science 343(6175): 1151-1154; Andrews et al. (2015) Sci. Transl. Med., 7(270) 270ra4; Radzio et al. (2015) Sci. Transl. Med., 7(270) 270ra5-270ra5; Andrews et al. (2016) AIDS 2016: 461-467). However, the administration mode of CAB LAP has limitations. Specifically, split injections in 2 mL volumes need to be given, which leads to treatment interruptions due to intolerable injection site reactions (Margolis et al. (2017) Lancet 390(10101):1499-510; Markowitz et al. (2017) Lancet HIV 4(8):331-340). In addition, the maximum dosing interval is only 8 weeks. Recently, CAB LAP administration every 12 weeks has been tested with the goal of maintaining plasma CAB concentrations above 4 times the protein binding-regulated 90% inhibitory concentration (4×PA-IC 90, 660 ng / mL), a concentration that has been shown to be protective against new infection in macaques (Spreen, WW (2014) J. Acquir. Immune Defic. Syndr., 67(5):481-486; Andrews et al. (2014) Science 343(6175):1151-1154; Andrews et al. (2015) Sci. Transl. Med., 7(270)270ra4; Radzio et al. (2015) Sci. Transl. Med., 7(270)270ra5-270ra5; Andrews et al. (2016) AIDS 2016:461-467; Markowitz et al. (2017) Lancet HIV 4(8):331-340; Spreen et al. (2014) J. Acquir. Immune Defic. Syndr., 67(5):487-492). However, two-thirds of the participants had faster-than-expected drug absorption, resulting in plasma drug concentrations less than 4×PA-IC at 12 weeks. 90 Therefore, there is a great need to extend the dose interval to more than 8 weeks and reduce the injection volume to improve regimen compliance (Boyd et al. (2017) Lancet 390(10101):1468-1470). SUMMARY OF THE INVENTION

[0008] According to the present invention, a prodrug of an integrase inhibitor is provided. In a specific embodiment, the prodrug includes an integrase inhibitor modified with an ester, the ester comprising an aliphatic group or a hydrocarbyl group (e.g., an aliphatic group or a hydrocarbyl group comprising about 3 to about 30 carbons). In a specific embodiment, the aliphatic group or the hydrocarbyl group is a hydrocarbyl chain or a saturated linear aliphatic chain of a fatty acid optionally substituted with at least one heteroatom. In a specific embodiment, the integrase inhibitor is selected from the group consisting of: cabotegravir (CAB), raltegravir (raltegravir) (RAL), elvitegravir (EVG), dolutegravir (dolutegravir) (DTG) and bictegravir (bictegravir) (BIC). The present invention also includes a composition comprising at least one prodrug of the present invention and at least one pharmaceutically acceptable carrier.

[0009] According to another aspect of the present invention, a kind of nanoparticle is provided, and the nanoparticle comprises at least one prodrug of the present invention and at least one polymer or surfactant.In a specific embodiment, the prodrug is crystalline.In a specific embodiment, the polymer or surfactant is an amphiphilic block copolymer, such as an amphiphilic block copolymer (e.g., poloxamer 407) comprising at least one poly (ethylene oxide) block and at least one poly (propylene oxide) block. The nanoparticle may comprise a polymer or surfactant connected to at least one targeting ligand. A single nanoparticle may comprise a targeted surfactant and an untargeted surfactant. In a specific embodiment, the nanoparticle has a diameter of about 100nm to 1 μm. The present invention also includes a composition comprising at least one nanoparticle of the present invention and at least one pharmaceutically acceptable carrier.

[0010] According to another aspect of the invention, a method for treating, suppressing and / or preventing a disease or disorder in a subject in need thereof is provided. The method comprises administering to the subject at least one prodrug or nanoparticle of the present invention, optionally in a composition comprising a pharmaceutically acceptable carrier. In a specific embodiment, the disease or disorder is a viral infection (e.g., a retroviral infection). In a specific embodiment, the method further comprises administering at least one other therapeutic agent or therapy for the disease or disorder, such as at least one other anti-HIV compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A Images of NM2CAB (top) and NCAB (bottom) nanoparticles determined by scanning electron microscopy are provided. Figure 1B A graph of the stability of NM2CAB nanoparticles over the indicated time periods at 25° C. In particular, the nanoparticle zeta potential (top), polydispersity index (middle), and particle diameter (bottom) were determined by dynamic light scattering.

[0012] Figure 2A Provided are graphs of drug uptake by human monocyte-derived macrophages (MDM) measured by UPLC-UV / Vis (top) and drug retention by MDM collected at indicated time points for intracellular drug analysis (bottom). Figure 2B Provides graphs of HIV-1 reverse transcriptase activity at indicated drug concentrations (top) and in vitro activity following drug treatment and in vitro incubation with HIV-1 ADA Graphs of HIV-1 reverse transcriptase activity in MDM measured at the indicated time points after challenge and treatment (bottom). Figure 2C Provided are images of p24-stained MDM cells after challenge.

[0013] Figure 3AA graph is provided of plasma CAB levels following a single intramuscular (IM) dose of NCAB, NMCAB, or NM2CAB in female NSG mice (NOD scidγ mice). The dose administered was 45 mg CAB equivalent (eq) / kg. The top bold dashed line indicates a plasma CAB 4×PA-IC of 664 ng / ml. 90 , and the bottom dotted line shows 166 ng / ml of plasma CAB 1×PA-IC 90 . Figure 3B A graph is provided of plasma CAB levels following a single intramuscular (IM) dose of NM3CAB in female NSG mice (NOD scidγ mice). The dose administered was 45 mg CAB equivalent (eq) / kg. The top bold dashed line indicates a plasma CAB 4×PA-IC of 664 ng / ml. 90 , and the bottom dashed line shows 166 ng / ml plasma CAB 1×PA-IC 90 .

[0014] Figure 4 Provided are images of the antiretroviral dose-response of NM2CAB at concentrations of 10 μM, 50 μM or 100 μM by immunocytochemistry against HIV-1 p24 antigen.

[0015] Figure 5 Figures of prodrug levels are provided. Tissue and blood biodistribution of prodrugs (MCAB or M2CAB) are evaluated at 14 days, 28 days, 42 days and 364 days after a single IM injection of NMCAB or NM2CAB. Prodrug levels are measured in spleen, liver, intestine, lung, brain, rectal tissue, kidney, lymph node dissection-related tissues and blood. Prodrug levels are determined by LC-MS / MS. Data are expressed as mean ± SEM. For the 14th, 28th and 42nd day groups, the number of animals in each group is N=5, and for the 364th day group, the number of animals is N=3 (NMCAB), N=4 (NM2CAB). Single-factor ANOVA is followed by Tukey's post hoc test for comparison of drug levels in tissues of the three treatments (*P<0.05, **P<0.01, ***P<0.001).

[0016] Fig. 6A and Figure 6B Provide the pharmacokinetics and biodistribution figure of NM2CAB in rhesus monkey.Four rhesus monkeys are administered NM2CAB of 45mg / kg CAB equivalent dosage by single IM injection.Collect plasma samples, and measure CAB (top line) and M2CAB (bottom line) levels until the 393rd day ( Fig. 6A). Rectal, lymph node, and adipose tissue biopsies were collected on day 204 after drug administration, and both CAB (top) and M2CAB (bottom) concentrations were determined ( Figure 6B ). Both plasma and tissue drug concentrations were determined by LCMS / MS. DETAILED DESCRIPTION OF THE INVENTION

[0017] Limiting the viral load at the site of tissue infection to the greatest extent possible can contribute to viral eradication strategies. This can be achieved by generating effective lipophilic and hydrophobic antiretroviral prodrug nanocrystals stabilized by surfactants. Hydrophobicity, drug hydrolysis rate, and antiretroviral potency must be balanced in order to obtain the best therapeutic effect. Herein, it is demonstrated that manipulation of the size of the hydrophobic and lipophilic carbon chain length of the prodrug can optimize therapeutic efficacy, particularly in long-acting slow effective release antiretroviral therapy (LASER ART). LASER ART refers to a long-acting antiretroviral drug with a lipid tail generated by a nanocrystal prodrug. Myristoylated CAB prodrug nanocrystals provide PA-IC in rhesus monkeys after a single 45 mg / kg CAB equivalent intramuscular injection dose. 90 Sustained plasma CAB concentrations last for 4 months. Herein, further chemical modifications are unexpectedly used to enhance CAB lipophilicity and hydrophobicity, improve drug efficacy, and slow down prodrug hydrolysis, thereby widely extending the half-life of the parent drug. The novel cabotegravir prodrug (M2CAB) enhances drug encapsulation with appropriate excipients and stabilizers such as poloxamer 407 (P407). M2CAB nanoformulations (NM2CAB) provide sustained drug release and site-specific antiretroviral drug delivery. Prodrugs include native drugs conjugated to hydrophobic moieties via hydrolyzable covalent bonds. NM2CAB nanoformulations are easily taken up by human monocyte-derived macrophages (MDMs) and have sustained drug retention for 30 days in vitro; while the parent drug nanoformulations (NCAB) or the first generation myristoylated cabotegravir (NMCAB) show HIV-1 breakthrough in MDMs within 1 day or 20 days of treatment, respectively. Notably, after a single drug treatment, MDM treated with NM2CAB exhibited sustained antiretroviral activity after HIV-1 challenge for up to 30 days. HIV-1 p24 was not detected at 5-day increment time points for up to or beyond 30 days in the NM2CAB treated group. In addition, a single intramuscular (IM) injection of NM2CAB at 45 mg CAB equivalent / kg in female NSG mice (NOD scidγ mice) demonstrated zero-order controlled release kinetics of active CAB and provided at or above 4 times the PA-IC 90Drug levels were sustained for more than 5 months. The NM2CAB nanoformulation proposed herein improves current combination ART regimens that require multiple daily administrations by reducing pill burden, lowering the risk of viral rebound, limiting toxicity, and / or allowing drug penetration into viral reservoirs. Importantly, NM2CAB also facilitates dosing intervals of once every six months (or even less frequently) to maximize the effectiveness of pre-exposure prophylaxis or treatment regimens.

[0018] Long-acting slow effective release ART (LASER ART) formulations can extend dosing intervals, reduce systemic toxicity, and improve pharmacokinetic (PK) and pharmacodynamic (PD) profiles (Sillman et al., Nat. Commun. (2018) 9: 443; Zhou et al., Biomaterials (2018) 151: 53-65; McMillan et al., Antimicrob. Agents Chemother. (2018) 62: e01316-17). Herein, novel integrase inhibitor prodrugs, long-acting slow effective release formulations thereof, and synthesis methods and uses thereof are provided. Integrase inhibitors (integrase strand transfer inhibitors (INSTIs)) are a class of antiretroviral drugs designed to block the action of integrase (e.g., HIV integrase), which is a viral enzyme that inserts the viral genome into the DNA of the host cell. Examples of integrase inhibitors include, but are not limited to, cabotegravir (CAB, GSK1265744), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG, GSK1349572), bictegravir (BIC, GS-9883), BI 224436 (Boehringer Ingelheim, Ingelheim, Germany) and MK-2048 (Merck, Kenilworth, NJ). Compared to the parent integrase inhibitors, hydrophobic and lipophilic prodrugs and their slow effective release formulations exhibit enhanced potency and efficacy, increased cell and tissue penetration, and extended half-life. The prodrugs of the present invention and their formulations and combinations thereof can be used to manage viral (e.g., retroviral) infections.

[0019] Currently available treatments for viral infections, particularly HIV infection, include inhibitors of viral entry, nucleoside reverse transcriptase, nucleotide reverse transcriptase, integrase, and protease. Resistance is associated with shortened drug half-life, viral life cycle, and rapid mutations leading to high genetic variability. Combination therapies, such as antiretroviral therapy (ART), considered to be "cocktail" therapies, have gained a great deal of attention. Benefits include reduced viral resistance, limited toxicity, improved compliance with treatment regimens, and sustained antiretroviral efficacy. Combination therapy minimizes potential drug resistance by inhibiting viral (e.g., HIV) replication, thereby reducing spontaneous resistant mutants. Treatment failure is partly attributed to short drug half-lives. In addition, failure can also be partly attributed to limited drug entry into tissues and cellular viral reservoirs, thereby hindering viral eradication efforts. For this reason, the development of cell and tissue-targeted nanoformulated prodrug (nanoparticle) platforms has aroused great interest in the management of viral (e.g., HIV) infections. Pre-exposure prophylaxis (PrEP) is another strategy for managing viral (e.g., HIV) transmission. For example, (tenofovir / emtricitabine) is already approved for pre-exposure prophylaxis of HIV infection. In addition, the combination of lamivudine and zidovudine It has been used as both pre-exposure prophylaxis and post-exposure prophylaxis.

[0020] The prodrugs and nano-prepared prodrugs (nanoparticles) provided herein unexpectedly extend the drug half-life, increase hydrophobicity and lipophilicity, and improve antiretroviral efficacy. This will be beneficial to people who must receive high doses every day or even several doses a day, because lower doses and lower dosing frequencies not only reduce side effects, but are also convenient for patients. The prodrugs and nano-prepared prodrugs (nanoparticles) provided herein can also be used as post-exposure treatment and / or pre-exposure prevention (for example, for people at high risk of infection with HIV-1). In other words, the prodrugs and nanoparticles of the present invention and combinations thereof can be used to prevent viral infection (for example, HIV infection) and / or treat or inhibit acute or long-term viral infection (for example, HIV infection). Although the prodrugs and nanoparticles of the present invention are generally described as anti-HIV agents, the prodrugs and nanoformulations of the present invention are also effective for other viral infections, and the other viral infections include, but are not limited to: retroviruses (for example, slow viruses), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T-cell leukemia virus (HTLV), particularly retroviruses.

[0021] The present invention describes novel, effective, broad-spectrum prodrugs having improved biological activity relative to the parent drug. Also provided is a method for encapsulating the prodrug into a long-acting slow-effective formulation for effective intracellular and tissue delivery and extended drug half-life. The long-acting slow-effective release (LASER) compositions described herein exhibit enhanced efficacy and can be used as effective therapeutic interventions or preventive interventions for viral infections (e.g., retroviral infections).

[0022] The prodrug of the present invention allows effective intracellular delivery of integrase inhibitors. Herein, a prodrug is provided, which is a derivative of an integrase inhibitor, wherein a chemical moiety, particularly an oxygen-containing moiety such as a hydroxyl group, has been replaced with an ester moiety comprising a hydrophobic and lipophilic cleavable moiety (e.g., a therapeutic fatty alcohol). A hydrophobic and lipophilic cleavable moiety (e.g., a therapeutic fatty alcohol) can exhibit antiviral activity against enveloped viruses (Katz et al., Ann. NY Acad. Sci. (1994) 724: 472-88). It is noteworthy that the synergistic interaction between therapeutic fatty alcohols and nucleoside analogs can significantly enhance the antiviral efficacy of nucleosides (Marcelletti et al., Antiviral Res. (2002) 56: 153-66).

[0023] As described herein, prodrugs can include unstable therapeutic fatty alcohols to improve drug efficacy, accelerate intracellular and tissue permeability, protein binding and bioavailability. The hydrophobic nature of the synthetic prodrugs helps to be encapsulated into long-acting slow-release drug nanocrystals with improved biopharmaceutical characteristics. The nanoformulations of the present invention can include prodrug particles dispersed in a sterile aqueous suspension and stabilized by a polymer excipient, a lipid and / or a surfactant or a polymer. Without being bound by theory, the mechanism of drug release includes the dissolution of the prodrug from the nanoparticles, followed by effective cleavage to generate two bioactive agents, i.e., an integrase inhibitor and a broad-spectrum antiviral fatty alcohol.

[0024] The benefit of system described herein includes but is not limited to improved drug efficacy, bioavailability and the half-life of extension to facilitate the patient. In fact, the nano formulation described in the present invention demonstrates the drug uptake of the macrophage (MDM) of monocyte origin that significantly increases. In addition, the modified medicine and nanoparticle present the effectiveness of enhancement by the inhibition of increased and prolonged viral replication. Therefore, nano formulation of the present invention allows the drug delivery of the acceleration of antiviral efficacy and to the anatomical reservoir of infection.

[0025] According to the present invention, a prodrug of an integrase inhibitor is provided. In a particular embodiment, the prodrug comprises an integrase inhibitor wherein a chemical moiety such as a hydroxyl group is replaced with an ester comprising an optionally substituted aliphatic or hydrocarbon group. In a particular embodiment, the ester comprises a hydrocarbon chain, particularly a hydrocarbon chain having a length of 16-20 carbon atoms or a length of 18 carbon atoms (the numbering herein includes the carbon in the C=O of the ester).

[0026] In a specific embodiment, the integrase inhibitor is selected from the group consisting of: cabotegravir (CAB), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), bictegravir (BIC), BI 224436 and MK-2048. In a specific embodiment, the integrase inhibitor is selected from the group consisting of: cabotegravir (CAB), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG) and bictegravir (BIC). Examples of the chemical structures of these integrase inhibitors are:

[0027]

[0028]

[0029] In a specific embodiment, the prodrug of the present invention is selected from the following group or a pharmaceutically acceptable salt thereof:

[0030]

[0031]

[0032] Wherein R is an optionally substituted aliphatic or hydrocarbyl group. The aliphatic or hydrocarbyl group can be unsaturated or saturated and can be substituted by at least one heteroatom (e.g., O, N or S). In a specific embodiment, R can include an aromatic moiety that can be substituted by at least one heteroatom (e.g., O, N or S).

[0033] In a specific embodiment, the alkyl group or aliphatic group is hydrophobic. In a specific embodiment, R is an optionally substituted hydrocarbon chain, particularly saturated. In a specific embodiment, R is a saturated linear aliphatic chain. In a specific embodiment, the alkyl group or aliphatic group comprises about 3 to about 30 carbons (for example, in the main chain of the alkyl group or aliphatic group), and the alkyl group or aliphatic group can be replaced by at least one heteroatom (for example, O, N or S). In a specific embodiment, R is a C14-C21 unsaturated or saturated alkyl group or aliphatic group, and the alkyl group or aliphatic group can be replaced by at least one heteroatom (for example, O, N or S). In a specific embodiment, R is a C14-C19 unsaturated or saturated alkyl group or aliphatic group, and the alkyl group or aliphatic group can be replaced by at least one heteroatom (for example, O, N or S). In a specific embodiment, R is a C14-C17 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C15-C21 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C15-C19 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C15-C17 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C16-C21 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C16-C19 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C17-C21 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C17-C19 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S). In a specific embodiment, R is a C17 unsaturated or saturated hydrocarbon group or aliphatic group, which may be substituted with at least one heteroatom (e.g., O, N, or S).

[0034] In particular embodiments, R is a hydrocarbon chain of a fatty acid (saturated or unsaturated), particularly a C16-C22 fatty acid, a C16-C20 fatty acid, a C16-C18 fatty acid, a C18-C22 fatty acid, a C18-C20 fatty acid or a C18 fatty acid (the numbering here includes the carbon in the C=O of the fatty acid).

[0035] In specific embodiments, R is a saturated linear aliphatic or hydrocarbon chain of at least 14 carbons (e.g., 14 to 21 carbons in length, 14 to 19 carbons in length, 14 to 17 carbons in length, 15 to 21 carbons in length, 15 to 19 carbons in length, 15 to 17 carbons in length, or 17 carbons in length). In specific embodiments, R is a saturated linear aliphatic or hydrocarbon chain of 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length, particularly 14, 15, 16, 17, 18, or 19 carbons in length, 15, 16, or 17 carbons in length, or 17 carbons in length. In specific embodiments, R is a saturated linear aliphatic or hydrocarbon chain of 17 carbons in length.

[0036] In a specific embodiment, the prodrug of the invention is:

[0037]

[0038] (M2CAB) or a pharmaceutically acceptable salt thereof.

[0039] The present invention also includes nanoparticles (sometimes referred to herein as nanoformulations) comprising the prodrugs of the present invention. The nanoparticles can be used to deliver compounds to cells or hosts (e.g., in vitro or in vivo). In specific embodiments, the nanoparticles are used to deliver antiretroviral therapy to a subject. The nanoparticles of the present invention comprise at least one prodrug and at least one surfactant or polymer. In specific embodiments, the nanoparticles comprise a surfactant / polymer: drug ratio that maintains optimal targeting of the drug nanoparticles to maintain spectroscopic determination of the macrophage reservoir. These components of the nanoparticles are described below along with other optional components.

[0040] The method for synthesizing the nanoparticles of the present invention is known in the art. In a specific embodiment, the method generates nanoparticles comprising a prodrug (e.g., crystalline or amorphous prodrug) coated with a polymer and / or a surfactant (partially or completely). Examples of synthetic methods include, but are not limited to, milling (e.g., wet milling), homogenization (e.g., high pressure homogenization), particle replication in non-wetting template (Particle Replication in Nonwetting Template) (PRINT) technology and / or ultrasonic technology. For example, U.S. Patent Application Publication No. 2013 / 0236553 (incorporated herein by reference) provides a method suitable for synthesizing the nanoparticles of the present invention. In a specific embodiment, the polymer or surfactant is first chemically modified with a targeting ligand, and then directly used or mixed with a non-targeted polymer or surfactant in a certain molar ratio to be coated on the surface of the prodrug suspension, for example, by using a nanoparticle synthesis process (for example, a crystalline nanoparticle synthesis process), such as milling (for example, wet milling), homogenization (for example, high pressure homogenization), particle replication in a non-wetting template (PRINT) technology and / or ultrasound technology, thereby preparing a targeted nano formulation. Nanoparticles can be used with or without further purification, although it is desirable to avoid further purification in order to produce nanoparticles faster. In a specific embodiment, milling and / or homogenization are used to synthesize nanoparticles. Targeted nanoparticles (for example, using ligands (optionally with high molecular weight ligands)) can be developed by physically or chemically coating and / or combining on the surface of a polymer or surfactant and / or drug nano suspension.

[0041] In a specific embodiment, the nanoparticles of the present invention are synthesized by adding a prodrug (e.g., crystal) to a polymer or surfactant solution, and then generating nanoparticles (e.g., by wet milling or high pressure homogenization). The prodrug and polymer or surfactant solution can be stirred before wet milling or high pressure homogenization.

[0042] The nanoparticles of the present invention can be used to deliver at least one prodrug of the present invention to cells or subjects (including non-human animals). The nanoparticles of the present invention may also contain at least one other agent or compound, particularly a bioactive agent, particularly a therapeutic agent (e.g., an antiviral compound) or a diagnostic agent, particularly at least one antiviral agent or antiretroviral agent. In a specific embodiment, the nanoparticles of the present invention contain at least two therapeutic agents, particularly at least one of which is a prodrug of the present invention. For example, the nanoparticles may contain an integrase inhibitor prodrug of the present invention and at least one other therapeutic agent (e.g., an anti-HIV agent).

[0043] In certain embodiments, the nanoparticles of the present invention are submicron colloidal dispersions of nanosized prodrug crystals (e.g., surfactant-coated drug crystals; nanoformulations) stabilized by polymers or surfactants. In certain embodiments, the prodrug may be crystalline (a solid having crystalline properties), amorphous, or a solid nanoparticle of a prodrug formed as a crystal comprising a drug and a polymer or surfactant. In certain embodiments, the prodrug is crystalline. As used herein, the term "crystalline" refers to an ordered state (i.e., non-amorphous) and / or a substance that exhibits long-range order in three-dimensional space. In certain embodiments, a majority (e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or more) of the prodrug and optionally the hydrophobic portion of the surfactant are crystalline.

[0044] In a specific embodiment, the diameter (e.g., z-average diameter) or its longest dimension of the nanoparticles of the present invention is up to about 2 μm or 3 μm, particularly up to about 1 μm (e.g., about 100 nm to about 1 μm). For example, the diameter or longest dimension of the nanoparticles can be from about 50 nm to about 800 nm. In a specific embodiment, the diameter or longest dimension of the nanoparticles is from about 50 nm to about 750 nm, from about 50 nm to about 500 nm, from about 200 nm to about 500 nm, from about 200 nm to about 400 nm, or from about 250 nm to about 350 nm. The nanoparticles can be, for example, rod-shaped, elongated rod-shaped, irregularly shaped, or rounded. The nanoparticles of the present invention can be neutral or charged. The nanoparticles can be positively charged or negatively charged.

[0045] As stated above, the nanoparticles of the present invention comprise at least one polymer or surfactant. "Surfactant" refers to a surface-active agent, including substances commonly referred to as wetting agents, detergents, dispersants or emulsifiers. Surfactants are generally amphiphilic organic compounds.

[0046] Examples of polymers or surfactants include, but are not limited to, synthetic or natural phospholipids, PEGylated lipids (e.g., PEGylated phospholipids), lipid derivatives, polysorbates, amphiphilic copolymers, amphiphilic block copolymers, poly(ethylene glycol)-co-poly(lactide-co-glycolide) (PEG-PLGA), derivatives thereof, ligand-conjugated derivatives thereof, and combinations thereof. Other polymers or surfactants and combinations thereof that can form stable nanosuspensions and / or can chemically / physically bind to targeting ligands of CD4+T cells, macrophages, and dendritic cells that can be infected / infected with HIV can be used in the present invention.Additional examples of polymers or surfactants include, but are not limited to: 1) nonionic surfactants (e.g., pegylated and / or polysaccharide-conjugated polyesters and other hydrophobic polymer blocks such as poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), other polyesters, poly(propylene oxide), poly(1,2-butylene oxide), poly(n-butylene oxide), poly(tetrahydrofuran), and poly(styrene); 2) glycerides, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, dehydrated sorbitan fatty acid esters, polyoxyethylene ... sorbitol esters, glyceryl monostearate, polyethylene glycol, polypropylene glycol, cetyl alcohol, cetearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohol, polyoxyethylene-polyoxypropylene copolymer, poloxamine, cellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polysaccharides, starch and its derivatives, hydroxyethyl starch, polyvinyl alcohol (PVA), polyvinyl pyrrolidone and combinations thereof); and 2) ionic surfactants (e.g., phospholipids, amphiphilic lipids, 1,2-dialkylglycerol-3-alkylphosphocholine, 1,2 -Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol) (DSPE-PEG), dimethylaminoethanecarbamoylcholesterol (DC-Chol), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), alkylpyridinium halides, quaternary ammonium compounds, lauryldimethylbenzylammonium, acylcarnitine hydrochloride, dioctadecyldimethylammonium (DDAB), n-octylamine, oleylamine, benzalkonium (benzalkonium), cetyl trimethylammonium, chitosan, chitosan salts, poly (ethyleneimine) (PEI), poly (N-isopropylacrylamide) (PNIPAM) and poly (allylamine) (PAH), poly (dimethyldiallylammonium chloride) (PDDA), alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, sodium dodecyl sulfate, alkyl polyoxyethylene sulfates, alginic acid, alginate, hyaluronic acid, hyaluronate, gelatin, sodium dioctyl succinate sulfonate (dioctyl sodium sulfosuccinate), sodium carboxymethyl cellulose, cellulose sulfate, dextran sulfate and carboxymethyl cellulose, chondroitin sulfate, heparin, synthetic poly(acrylic acid) (PAA), poly(methacrylic acid) (PMA), poly(vinyl sulfate) (PVS), poly(styrene sulfonate) (PSS), bile acids and their salts, cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, derivatives thereof, and combinations thereof).

[0047] The polymers or surfactants of the present invention may be charged or neutral. In certain embodiments, the polymers or surfactants are neutral or negatively charged (eg, poloxamers, polysorbates, phospholipids and derivatives thereof).

[0048] In a specific embodiment, the polymer or surfactant is an amphiphilic block copolymer or a lipid derivative. In a specific embodiment, at least one polymer or surfactant of the nanoparticle is an amphiphilic block copolymer, particularly a copolymer comprising at least one poly(ethylene oxide) block and at least one poly(propylene oxide) block. In a specific embodiment, the polymer or surfactant is a triblock amphiphilic block copolymer. In a specific embodiment, the polymer or surfactant is a triblock amphiphilic block copolymer comprising the following: a central hydrophobic block of polypropylene glycol, flanked by two hydrophilic blocks of polyethylene glycol. In a specific embodiment, the surfactant is poloxamer 407.

[0049] In a particular embodiment, the amphiphilic block copolymer is a copolymer comprising at least one poly(oxyethylene) block and at least one poly(oxypropylene) block. In a particular embodiment, the amphiphilic block copolymer is a poloxamer. Examples of poloxamers include, but are not limited to In a specific embodiment, the poloxamer is poloxamer 407 ( F127).

[0050] In a specific embodiment of the present invention, the polymer or surfactant is present in the nanoparticle and / or solution in a concentration ranging from about 0.0001% to about 10% or 15% by weight to synthesize the nanoparticle (as described herein). In a specific embodiment, the concentration of the polymer or surfactant is in a range of from about 0.01% to about 15%, about 0.01% to about 10%, about 0.1% to about 10%, or about 0.1% to about 6% by weight. In a specific embodiment, the nanoparticle contains at least about 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more of the therapeutic agent (prodrug) by weight. In a specific embodiment, the nanoparticle contains a determined drug: polymer / surfactant ratio. In a specific embodiment, the drug: polymer / surfactant ratio (e.g., by weight) is from about 10:6 to about 1000:6, about 20:6 to about 500:6, about 50:6 to about 200:6 or about 100:6.

[0051] As stated above, the polymer or surfactant of the present invention can be connected to a targeting ligand. Compared with free drugs or untargeted nanoparticles, the targeting of nanoparticles (e.g., targeting macrophages) can provide excellent targeting, reduced excretion rate, reduced toxicity and extended half-life. A targeting ligand is a compound that specifically binds to a particular type of tissue or cell type (e.g., with a desired target: cell ratio). For example, a targeting ligand can be used to engage or bind to a target cell (e.g., macrophage) surface marker or receptor, which can contribute to the targeting ligand being taken into the cell (e.g., in a protected subcellular organelle that is not affected by metabolic degradation). In a specific embodiment, a targeting ligand is a ligand of a cell surface marker / receptor. A targeting ligand can be an antibody or an antigen binding fragment thereof that is immunospecific for a cell surface marker (e.g., a protein or carbohydrate), which preferentially or exclusively expresses on a targeted tissue or cell type. A targeting ligand can be directly or via a linker to a polymer or surfactant. Typically, a linker is a chemical moiety comprising a covalent bond or atom chain that covalently attaches a ligand to a polymer or surfactant. A linker can be connected to any synthetically feasible position of a ligand and a polymer or surfactant. An exemplary linker can include at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic aliphatic group, alkyl group or optionally substituted aryl group. A linker can be a low alkyl group or an aliphatic group. A linker can also be a polypeptide (e.g., from about 1 to about 10 amino acids, particularly about 1 to about 5 amino acids). In a specific embodiment, a targeting moiety is connected to one or both ends of a polymer or surfactant. A linker can be non-degradable and can be a covalent bond or any other chemical structure that is substantially or completely incapable of being cleaved under physiological environments or conditions.

[0052] Nanoparticles / nanoformulations of the present invention may include targeted and / or untargeted polymers or surfactants. In specific embodiments, the molar ratio of targeted and untargeted polymers or surfactants in the nanoparticles / nanoformulations of the present invention is from about 0.001% to 100%, about 1% to about 99%, about 5% to about 95%, about 10% to about 90%, about 25% to about 75%, about 30% to about 60% or about 40%. In specific embodiments, nanoparticles only include targeted polymers or surfactants. In specific embodiments, nanoparticles / nanoformulations of the present invention include folic acid-targeted polymers or surfactants and untargeted forms of the polymers or surfactants. In specific embodiments, nanoparticles / nanoformulations of the present invention include folic acid-poloxamer 407 (FA-P407) and / or poloxamer 407.

[0053] Examples of targeting ligands include, but are not limited to, macrophage targeting ligands, CD4+T cell targeting ligands, dendritic cell targeting ligands, and tumor targeting ligands. In a specific embodiment, the targeting ligand is a macrophage targeting ligand. The targeted nanoformulations of the present invention may include targeting ligands for directing nanoparticles to HIV tissue and cell sanctuaries / reservoirs (e.g., central nervous system, gut-associated lymphoid tissue (GALT), CD4+T cells, macrophages, dendritic cells, etc.). Macrophage targeting ligands include, but are not limited to, folate receptor ligands (e.g., folate (folic acid) and folate receptor antibodies and fragments thereof (see, e.g., Sudimack et al. (2000) Adv. Drug Del. Rev., 41: 147-162)), mannose receptor ligands (e.g., mannose), formyl peptide receptor (FPR) ligands (e.g., N-formyl-Met-Leu-Phe (fMLF)), and tuftsin (tetrapeptide Thr-Lys-Pro-Arg). Other targeting ligands include, but are not limited to, hyaluronic acid, gp120 and peptide fragments thereof, and ligands or antibodies specific to CD4, CCR5, CXCR4, CD7, CD111, CD204, CD49a, CD29, CD19, CD20, CD22, CD171, CD33, Leis-Y, WT-1, ROR1, MUC16, MUC1, MUC4, estrogen receptor, transferrin receptor, EGF receptor (e.g., HER2), folate receptor, VEGF receptor, FGF receptor, androgen receptor, NGR, integrin, and GD2. In a specific embodiment, the targeting ligand is folic acid.

[0054] As stated above, the nanoparticles of the present invention may contain additional therapeutic agents. The present invention also includes methods of treatment in which the prodrugs and / or nanoparticles of the present invention are co-administered with another therapeutic agent. In specific embodiments, the therapeutic agent is a hydrophobic, water-insoluble compound or a poorly water-soluble compound, particularly when contained in a nanoparticle. For example, in a pH range of 0-14, preferably between pH 4 and pH 10, particularly at 20° C., the therapeutic agent may have a solubility of less than about 10 mg / ml, less than 1 mg / ml, more particularly less than about 100 μg / ml, and more particularly less than about 25 μg / ml in water or an aqueous medium.

[0055] In certain embodiments, the therapeutic agent is an antiviral or antiretroviral agent. Antiretroviral agents can be effective against slow viruses or specific to slow viruses. Slow viruses include, but are not limited to, human immunodeficiency virus (HIV) (e.g., HIV-1, HIV-2), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIA). In certain embodiments, the therapeutic agent is an anti-HIV agent. Anti-HIV compounds or anti-HIV agents are compounds that inhibit HIV (e.g., inhibit HIV replication and / or infection). Examples of anti-HIV agents include, but are not limited to:

[0056] (I) Nucleoside analog reverse transcriptase inhibitors (NRTIs). NRTIs refer to nucleosides and nucleotides and their analogs that inhibit the activity of reverse transcriptase, especially HIV-1 reverse transcriptase. NRTIs contain sugars and bases. Examples of nucleoside analog reverse transcriptase inhibitors include, but are not limited to, adefovir dipivoxil, adefovir, lamivudine, telbivudine, entecavir, tenofovir, stavudine, abacavir, didanosine, emtricitabine, zalcitabine, and zidovudine.

[0057] (II) Non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIs are allosteric inhibitors that reversibly bind to the non-substrate binding site of reverse transcriptase, especially HIV reverse transcriptase, thereby changing the shape of the active site or blocking polymerase activity. Examples of NNRTIs include, but are not limited to, delavirdine (DLV, BHAP, U-90152; ), efavirenz (EFV, DMP-266, ), nevirapine (NVP, ), PNU-142721, capravirine (S-1153, AG-1549), emivirine (+)-calanolide A (NSC-675451) and B, etravirine (ETR, TMC-125, ), rilpivirine (RPV, TMC278, Edurant TM )、DAPY(TMC120)、doravirine(Pifeltro TM), BILR-355BS, PHI-236 and PHI-443 (TMC-278).

[0058] (III) Protease inhibitors (PI). Protease inhibitors are inhibitors of viral proteases, especially HIV-1 protease. Examples of protease inhibitors include, but are not limited to, darunavir, amprenavir (141W94, ), tipranivir (PNU-140690, ), indinavir (MK-639; ), saquinavir Fosamprenavir Lopinavir (ABT-378), ritonavir (ABT-538, ), atazanavir Nelfinavir (AG-1343, ), lasinavir (BMS-234475 / CGP-61755), BMS-2322623, GW-640385X (VX-385), AG-001859 and SM-309515.

[0059] (IV) Fusion or entry inhibitors. Fusion or entry inhibitors are compounds, such as peptides, that block HIV from entering cells (e.g., by binding to HIV envelope proteins and blocking the structural changes necessary for the virus to fuse with the host cell). Examples of fusion inhibitors include, but are not limited to, CCR5 receptor antagonists (e.g., maraviroc ( Celsentri)), enfuvirtide (INN, )、T-20(DP-178, ) and T-1249.

[0060] (V) Integrase inhibitors (except for the prodrugs of the present invention). Integrase inhibitors are a class of antiretroviral drugs designed to block the action of integrase (e.g., HIV integrase), which is a viral enzyme that inserts the viral genome into the DNA of the host cell. Examples of integrase inhibitors include, but are not limited to, raltegravir, elvitegravir, GSK1265744 (cabotegravir), GSK1349572 (dolutegravir), GS-9883 (bictegravir), and MK-2048.

[0061] Anti-HIV compounds also include maturation inhibitors (e.g., bevirimat). Maturation inhibitors are generally compounds that bind to HIV gag during the maturation of the virus and disrupt its processing. Anti-HIV compounds also include HIV vaccines, such as, but not limited to HIV(vCP1521), B / E (gp120) and combinations thereof. Anti-HIV compounds also include HIV antibodies (eg, antibodies against gp120 or gp41), particularly broadly neutralizing antibodies.

[0062] More than one anti-HIV agent may be used, particularly when the agents have different mechanisms of action (as outlined above). For example, an anti-HIV agent that is not a NNRTI may be combined with a NNRTI prodrug of the invention. In a particular embodiment, the anti-HIV therapy is highly active antiretroviral therapy (HAART).

[0063] The present invention includes a composition (e.g., a pharmaceutical composition) comprising at least one prodrug and / or nanoparticle of the present invention and at least one pharmaceutically acceptable carrier. As stated above, the nanoparticles may contain more than one therapeutic agent. In a specific embodiment, the pharmaceutical composition comprises a first nanoparticle containing a first prodrug and a second nanoparticle containing a second prodrug, wherein the first prodrug and the second prodrug are different. In a specific embodiment, the first prodrug is a prodrug of the present invention, and the second prodrug is a prodrug of a non-nucleoside reverse transcriptase inhibitor (NNRTI), particularly rilpivirine (RPV). The composition (e.g., a pharmaceutical composition) of the present invention may also contain other therapeutic agents (e.g., other anti-HIV compounds (e.g., those described herein)).

[0064] The present invention also includes methods for preventing, inhibiting and / or treating a disease or disorder. The method comprises administering a prodrug and / or nanoparticle of the present invention (optionally in a composition) to a subject in need thereof. In a specific embodiment, the disease or disorder is a viral (e.g., retroviral) infection. Examples of viral infections include, but are not limited to, HIV, hepatitis B, hepatitis C, and HTLV. In a specific embodiment, the viral infection is a retroviral infection or a lentiviral infection, particularly an HIV infection (e.g., HIV-1).

[0065] The prodrugs and / or nanoparticles of the present invention (optionally in a composition) can be administered to animals, particularly mammals, more particularly humans, to treat / suppress / prevent diseases or disorders (e.g., retroviral infections such as HIV infections). The pharmaceutical compositions of the present invention may also include at least one other therapeutic agent, such as an antiviral agent, particularly at least one other anti-HIV compound / agent. Additional anti-HIV compounds may also be administered in a pharmaceutical composition separate from the prodrugs or compositions of the present invention. The pharmaceutical composition may be administered at the same time or at different times (e.g., sequentially).

[0066] The dosage ranges for administration of the prodrugs, nanoparticles and / or compositions of the present invention are those dosage ranges large enough to produce the desired effect (e.g., cure, alleviation, treatment and / or prevention of a disease or disorder (e.g., HIV infection), a symptom thereof (e.g., AIDS, ARC), or susceptibility thereto). In specific embodiments, the pharmaceutical compositions of the present invention are administered to a subject in an amount from about 5 μg / kg to about 500 mg / kg. In specific embodiments, the pharmaceutical compositions of the present invention are administered to a subject in an amount greater than about 5 μg / kg, greater than about 50 μg / kg, greater than about 0.1 mg / kg, greater than about 0.5 mg / kg, greater than about 1 mg / kg, or greater than about 5 mg / kg. In specific embodiments, the pharmaceutical compositions of the present invention are administered to a subject in an amount from about 0.5 mg / kg to about 100 mg / kg, about 10 mg / kg to about 100 mg / kg, or about 15 mg / kg to about 50 mg / kg. The dose should not be so large as to cause significant adverse side effects, such as undesirable cross-reactions, allergic reactions, and the like. Generally, the dosage will vary with the patient's age, condition, sex and degree of disease, and can be determined by one skilled in the art. In the event of any counter indications, the dosage can be adjusted by the individual physician.

[0067] The prodrugs and nanoparticles described herein will generally be administered to a patient as a pharmaceutical composition. As used herein, the term "patient" refers to a human or animal subject. These prodrugs and nanoparticles can be used therapeutically under the guidance of a physician.

[0068] The pharmaceutical composition comprising the prodrug of the present invention and / or nanoparticle can be conveniently formulated for use with any pharmaceutically acceptable carrier. For example, the complex can be formulated with an acceptable medium, such as water, buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), dimethyl sulfoxide (DMSO), oil, detergent, suspending agent or its suitable mixture, particularly aqueous solution. The concentration of prodrug and / or nanoparticle in the selected medium can vary, and the medium can be selected based on the desired route of administration of the pharmaceutical composition. Except that any conventional medium or agent is incompatible with the nanoparticle to be administered, the purposes of nanoparticle in the pharmaceutical composition are expected.

[0069] The dosage and dosage regimen of the prodrug and / or nanoparticle according to the present invention suitable for administration to a specific patient can be determined by a physician taking into account the patient's age, sex, weight, general medical condition, and the specific condition and severity of the nanoparticle being administered. The physician can also consider the biological activity of the route of administration, pharmaceutical carrier, and nanoparticle.

[0070] The selection of suitable pharmaceutical composition will also depend on the selected mode of administration. For example, the nanoparticles of the present invention can be administered by direct injection or intravenous administration. In this case, the pharmaceutical composition comprises prodrugs and / or nanoparticles dispersed in a medium compatible with the injection site.

[0071] Prodrug of the present invention and / or nanoparticle can be used by any method.For example, prodrug of the present invention and / or nanoparticle can be not limited to parenteral, subcutaneous, oral, local, pulmonary, rectal, vaginal, intravenous, intraperitoneal, intrathecal, intracerebral, epidural, intramuscular, intradermal or intracarotid administration.In a specific embodiment, prodrug and / or nanoparticle are administered parenterally.In a specific embodiment, prodrug and / or nanoparticle are administered orally, intramuscularly, subcutaneously or are administered to blood flow (for example, intravenous administration).In a specific embodiment, prodrug and / or nanoparticle are administered intramuscularly or subcutaneously.Injection pharmaceutical composition is known in the art.If selection injection is used as the method for administering prodrug and / or nanoparticle, measures must be taken to ensure that sufficient amount of molecules or cells arrive at its target cell to play a biological effect. Dosage forms for oral administration include, but are not limited to, tablets (e.g., coated and uncoated, chewable), gelatin capsules (e.g., soft or hard gelatin capsules), lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders / granules (e.g., reconstructible or dispersible powders / granules), gums, and effervescent tablets. Dosage forms for parenteral administration include, but are not limited to, solutions, emulsions, suspensions, dispersions, and powders / granules for reconstruction. Dosage forms for topical administration include, but are not limited to, creams, gels, ointments, salves, patches, and transdermal delivery systems.

[0072] Pharmaceutical compositions containing prodrugs and / or nanoparticles of the present invention as active ingredients intimately mixed with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical formulation techniques. Depending on the form of the pharmaceutical composition desired for administration (e.g., intravenous, oral, direct injection, intracranial and intravitreal), the carrier can take a variety of forms.

[0073] The pharmaceutical composition of the present invention can be formulated into a dosage unit form that is easy to use and dosage uniformity. The dosage unit form as used herein refers to the physically discrete unit of the pharmaceutical composition suitable for the patient undergoing treatment. Each dosage should contain a certain amount of active ingredient, and this certain amount of active ingredient is calculated to produce the desired effect associated with the selected pharmaceutical carrier. The program for determining a suitable dosage unit is well known to those skilled in the art. In a specific embodiment, the prodrug and / or nanoparticle of the present invention can be applied once or even less frequently every 1 to 12 months due to its long-acting therapeutic effect. For example, the nano formulation of the present invention can be applied once every 0.5 month, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months or more months. In a specific embodiment, the prodrug and / or nanoparticle of the present invention are less than once every two months. In specific embodiments, the prodrug and / or nanoformulation of the prodrug is administered once a month, once every two months, particularly once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, once every twelve months, or less frequently.

[0074] The dosage unit may be proportionally increased or decreased based on the patient's body weight. As known in the art, the appropriate concentration for alleviating a particular pathological condition can be determined by dose concentration curve calculations.

[0075] According to the present invention, the suitable dosage unit for administering nanoparticles can be determined by evaluating the toxicity of molecules or cells in animal models. The nanoparticles in the pharmaceutical composition of various concentrations can be administered to mice, and the minimum dose and maximum dose can be determined based on the beneficial results and side effects observed as the treatment results. Suitable dosage units can also be determined by assessing the effectiveness of the nanoparticle treatments combined with other standard drugs. The dosage unit of nanoparticles can be determined individually or in combination with each treatment according to the effect detected.

[0076] The pharmaceutical composition comprising nanoparticles can be administered at appropriate intervals until the pathological symptoms are reduced or alleviated, after which the dosage can be reduced to a maintenance level. The appropriate interval in a particular case will generally depend on the patient's condition.

[0077] The present invention includes methods for treating diseases / disorders, comprising administering a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising a prodrug and / or nanoparticle of the present invention, and preferably at least one pharmaceutically acceptable carrier. The present invention also includes methods in which the subject is treated via ex vivo therapy. In particular, the method includes removing cells from the subject, exposing the cells to / contacting the nanoparticles of the present invention in vitro, and returning the cells to the subject. In a specific embodiment, the cells include macrophages. Other methods for treating diseases or disorders may be combined with the methods of the present invention, or may be co-administered with the pharmaceutical compositions of the present invention.

[0078] The invention also includes delivering the nanoparticles of the invention to cells in vitro (eg, in culture).The nanoparticles may be delivered to cells in at least one carrier.

[0079] definition

[0080] The following definitions are provided to aid in understanding the present invention.

[0081] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0082] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0083] "Carrier" refers to, for example, a diluent, adjuvant, preservative (e.g., thimerosal, benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), antimicrobial, filler (e.g., lactose, mannitol), excipient, auxiliary agent, or vehicle with which the active agent of the invention is administered. Pharmaceutically acceptable carriers can be sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," by EW Martin (Mack Publishing Co., Easton, PA); Gennaro, AR, Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0084] The term "prodrug" refers to a compound that is metabolized or otherwise converted into a biologically active or more active compound or drug, typically after administration. A prodrug is chemically modified relative to a drug in such a way that the prodrug is less active, substantially inactive, or inactive relative to the drug. However, the chemical modification allows the corresponding drug to be produced by metabolic or other biological processes, typically after administration of the prodrug.

[0085] As used herein, the term "treatment" refers to any type of treatment that confers a benefit to a patient suffering from a disease, including improving the patient's condition (e.g., one or more symptoms), delaying the progression of the condition, etc. In specific embodiments, treating a retroviral infection results in at least inhibiting / reducing the number of infected cells and / or detectable virus levels.

[0086] As used herein, the term "prevention" refers to prophylactic treatment of a subject at risk of developing a condition (eg, HIV infection), resulting in a reduced likelihood that the subject will develop the condition.

[0087] A "therapeutically effective amount" of a compound or pharmaceutical composition refers to an amount effective to prevent, inhibit, treat or alleviate the symptoms of a particular disorder or disease. Treating a microbial infection (e.g., HIV infection) herein may refer to curing, alleviating and / or preventing a microbial infection, its symptoms or susceptibility to it.

[0088] As used herein, the term "therapeutic agent" refers to a chemical compound or biological molecule, including but not limited to nucleic acids, peptides, proteins, and antibodies, that can be used to treat a condition, disease, or disorder or to reduce the symptoms of a condition, disease, or disorder.

[0089] As used herein, the term "small molecule" refers to a substance or compound having a relatively low molecular weight (e.g., less than 4,000, less than 2,000, and particularly less than 1 kDa or 800 Da). Typically, small molecules are organic but not proteins, polypeptides, or nucleic acids, although they may be amino acids or dipeptides.

[0090] The term "antimicrobial agent" as used herein indicates a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, viruses or protozoa.

[0091] As used herein, the term "antiviral agent" refers to a substance that destroys viruses and / or inhibits viral replication (reproduction). For example, an antiviral agent can inhibit and / or prevent: the production of viral particles, the maturation of viral particles, viral attachment, viral uptake into cells, viral assembly, viral release / budding, viral integration, etc.

[0092] As used herein, the term "highly active antiretroviral therapy" (HAART) refers to HIV therapy that employs a combination of therapeutic agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, HIV protease inhibitors, and fusion inhibitors.

[0093] As used herein, the term "amphiphilic" means the ability to dissolve in both water and lipid / non-polar environments. Typically, amphiphilic compounds comprise a hydrophilic portion and a hydrophobic portion. "Hydrophobicity" indicates a preference for non-polar environments (e.g., a hydrophobic substance or hydrophobic portion is more easily dissolved in or wetted by a non-polar solvent, such as a hydrocarbon, than water). Most "hydrophobic" compounds are insoluble in water. As used herein, the term "hydrophilicity" means the ability to dissolve in water.

[0094] As used herein, the term "polymer" refers to a molecule formed by the chemical combination of two or more repeating units or monomers. The term "block copolymer" most simply refers to a conjugate of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same type.

[0095] "Antibody" or "antibody molecule" is any immunoglobulin that binds to a specific antigen, including antibodies and fragments thereof (e.g., scFv). As used herein, antibodies or antibody molecules contemplate complete immunoglobulin molecules, immunologically active portions of immunoglobulin molecules, and fusions of immunologically active portions of immunoglobulin molecules.

[0096] As used herein, the term "immunospecific" refers to a protein / polypeptide, particularly an antibody, that binds to one or more epitopes or compounds of a protein of interest in a sample containing a mixed population of antigenic biomolecules, but does not substantially recognize and bind other molecules.

[0097] As used herein, the term "targeting ligand" refers to any compound that specifically binds to a particular type of tissue or cell type, and in particular any compound that does not substantially bind to other types of tissue or cell types. Examples of targeting ligands include, but are not limited to, proteins, polypeptides, peptides, antibodies, antibody fragments, hormones, ligands, carbohydrates, steroids, nucleic acid molecules, and polynucleotides.

[0098] The term "aliphatic" refers to a non-aromatic hydrocarbon-based portion. Aliphatic compounds can be acyclic (e.g., linear or branched) or cyclic portions (e.g., cycloalkyl), and can be saturated or unsaturated (e.g., alkyl, alkenyl, and alkynyl). Aliphatic compounds can contain a main chain that is mainly carbon (e.g., 1 to about 30 carbons), and contain heteroatoms and / or substituents (see below). As used herein, the term "alkyl" includes saturated or unsaturated straight or branched hydrocarbons containing 1 to about 30 carbons in the normal chain / main chain. The hydrocarbon chain of the alkyl group can be interrupted by one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur). The alkyl (or aliphatic group) can be optionally substituted (e.g., substituted by less than about 8, less than about 6, or 1 to about 4 substituents). The term "lower alkyl" or "lower aliphatic group" refers to a alkyl or aliphatic group containing 1 to 3 carbons in the hydrocarbon chain, respectively. Hydrocarbyl substituents or aliphatic substituents include, but are not limited to, alkyl (e.g., lower alkyl), alkenyl, halogen (such as F, Cl, Br, I), haloalkyl (e.g., CCl3 or CF3), alkoxy, alkylthio, hydroxy, methoxy, carboxyl, oxo, epoxy, alkoxycarbonyl, alkylcarbonyloxy, amino, carbamoyl (e.g., NH2C(=O)- or NHRC(=O)-, where R is alkyl), urea (-NHCONH2), alkylurea, aryl, ether, ester, thioester, nitrile, nitro, amide, carbonyl, carboxylate, and thiol. Aliphatic and hydrocarbyl groups having at least about 5 carbons in the backbone are generally hydrophobic without extensive substitution with hydrophilic substituents.

[0099] The term "aryl" as used herein refers to monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion. Examples of aryl groups include, but are not limited to, phenyl or naphthyl, such as 1-naphthyl and 2-naphthyl, or indenyl. The aryl group may optionally include one to three other rings fused to a cycloalkyl ring or a heterocycle. The aryl group may optionally be substituted by, for example, 1, 2 or 3 groups through available carbon atoms, the group being selected from hydrogen, halogen, alkyl, polyhaloalkyl, alkoxy, alkenyl, trifluoromethyl, trifluoromethoxy, alkynyl, aryl, heterocyclic radical, aralkyl, aryloxy, aryloxyalkyl, aralkyloxy, arylthio, arylazo, heterocyclic radical oxygen base, hydroxyl, nitro, cyano, sulfonyl anion, amino or substituted amino. The aryl group may be a heteroaryl. "Heteroaryl" refers to an optionally substituted monocyclic, bicyclic, tricyclic or other polycyclic aromatic ring system containing at least one, and preferably from 1 to about 4 sulfur, oxygen or nitrogen heteroatom ring members. Heteroaryl groups can have, for example, from about 3 to about 50 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 4 to about 10 carbons being preferred.

[0100] The following examples provide illustrative methods of practicing the invention and are not intended to limit the scope of the invention in any way.

[0101] Example 1

[0102] By developing long-acting reservoir-targeted drugs, it is possible to minimize residual HIV-1 in tissue sanctuary sites of HIV-1, including the brain, lymph nodes, bone marrow, gut-associated lymphoid tissue, and reproductive tract. In addition to the benefits of infrequent dosing intervals, long-acting injectable drug formulations can be designed to exploit receptor-mediated processes to achieve improved cellular targeting, extended drug half-life, and enhanced tissue biodistribution. CAB is a potent viral integrase inhibitor and has been formulated as LAP (CAB-LAP), which exhibits sustained plasma drug levels in humans after a single intramuscular dose. Long-acting injectable nanoformulations of rilpivirine and CAB-LAP have been able to be injected once a month for HIV suppression and prevention (Andrews et al. (2014) Science 343 (6175): 1151-1154; Cohen, J. (2014) Science 343 (6175): 1067; Spreen et al. (2013) Curr. Opin. HIV AIDS, 8 (6): 565-571). The main limitations of existing nanoformulations include the need for high doses and high injection volumes. To this end, long-acting slow effective release antiretroviral therapy (LASERART) has been developed by synthesizing lipophilic and hydrophobic prodrug nanocrystals that allow rapid drug penetration across physiological barriers. LASER ART maximizes drug loading through limited excipient use while maintaining scalability and long-term storage. Myristoylated prodrugs have been formulated with poloxamer surfactants. By increasing the drug lipophilicity, which maintains plasma CAB concentrations at PA-IC in rhesus monkeys after a single 45 mg / kg CAB equivalent intramuscular injection 90 Improved efficacy, bioavailability and tissue distribution of CABs were demonstrated over 4 months.Here, improved prodrugs and nanoformulations have been synthesized that reduce dosing frequency while improving viral reservoir targeting and drug activity.

[0103] Provided herein are potent ester prodrugs of CABs having physicochemical properties that allow for infrequent administration (such as once every 6 to 12 months or even less frequently) using LASER ART formulations. Criteria evaluated in selecting optimal CAB prodrug candidates include drug potency, lipophilicity profile, efficient conversion to CAB in vivo with minimal systemic prodrug circulation, and greater than 4-fold PA-IC sustained for 6 months or longer following a single intramuscular injection of the prodrug formulation. 90Surprisingly, the present invention has demonstrated that changes in the hydrocarbon chain length of fatty ester prodrugs significantly improve the release and retention of active drug. This culminated in the identification of M2CAB, an 18-carbon fatty ester prodrug of CAB, which has unexpectedly superior controlled release kinetics for CAB when compared to MCAB or other fatty acid hydrocarbon chain lengths.

[0104] The prodrug of the present invention is a derivative of an integrase inhibitor, such as a CAB conjugated to a hydrophobic cleavable portion. Therefore, the hydrophobic parent compound is converted into a more hydrophobic ester derivative. This is achieved by attaching a fatty acid portion, which can improve drug protein binding and bioavailability. The ester bond between the integrase inhibitor (e.g., CAB) and the derivative portion is susceptible to enzymatic cleavage or hydrolytic cleavage. The mechanism of drug release from the particle includes the dissolution of the prodrug from the excipient, followed by effective ester degradation to produce the active parent compound. The developed NM2CAB significantly improved the drug uptake of MDM, with sustained drug retention over a 30-day observation period; while the NCAB or NMCAB formulations were eliminated from macrophages after 1 day or 20 days of treatment, respectively. Similarly, after a single drug treatment of up to 30 days, when HIV-1 attack continued, MDM treated with NM2CAB presented an enhanced and sustained antiretroviral activity compared to NCAB or NMCAB. HIV-1 p24 was not detected in the NM2CAB treatment group at any of these time points. Benefits of this system include unexpectedly improved drug bioavailability and extended half-life. NM2CAB's prolonged plasma and tissue CAB concentrations demonstrate that an effective once-every-six-month dosing interval can be achieved.

[0105] Synthesis of M2CAB

[0106] The synthesis of the M2CAB prodrug was performed as depicted:

[0107]

[0108] Briefly, the preparation of M2CAB prodrugs is carried out by: 1) deprotonation of the phenol functionality with a suitable base such as N,N-diisopropylethylamine; and 2) reaction with an acyl chloride or an activated carboxylic acid of a hydrocarbyl fatty acid.

[0109] Step 1 and step 2 are both performed in a single container. Specifically, the hydroxyl group is deprotonated using a suitable reagent. The alcohol anion is then coupled with a fatty acid chloride or an activated carboxylic acid to generate a prodrug. Examples of coupling reagents that can be used to activate carboxylic acids include, but are not limited to, urea salts, carbodiimide reagents, and phosphonium salts. Examples of bases that can be used for coupling reactions are, but are not limited to, N,N-diisopropylethylamine (DIEA). Examples of polar aprotic solvents that can be used include, but are not limited to, N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and acetonitrile. The reagents are mixed at 0°C and gradually heated within 12-24 hours. The final compound is purified by silica gel column chromatography and characterized by nuclear magnetic resonance spectroscopy and high performance liquid chromatography coupled to mass spectrometry.

[0110] Deprotonation of hydroxyl groups and coupling to fatty acids

[0111] A solution of CAB (2 g, 4.93 mmol, 1.0 equiv) in anhydrous dimethylformamide (20 mL) was cooled to 0 °C under argon. N,N-diisopropylethylamine (1.7 mL, 9.86 mmol, 2.0 equiv) was then added dropwise to the pre-cooled solution of the drug. Stearoyl chloride (3.3 mL, 9.86 mmol, 2.0 equiv) was then added to the deprotonated phenol solution. The mixture was gradually warmed to room temperature over 16 hours with stirring, concentrated, and purified by flash chromatography eluting with 80% to 90% EtOAc / Hex to give the prodrug in 90% chemical yield. M2CAB 1 The H-NMR spectrum showed the presence of broad peaks at 1.20-1.50 ppm, as well as those corresponding to aliphatic protons on the fatty acid moieties.

[0112] Preparation synthesis

[0113] M2CAB nanocrystals were coated with poloxamer 407 (P407), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)-2000 (DSPE-PEG) and / or polyvinyl alcohol (PVA). The nanocrystals can also be stabilized with polysorbate and polyethylene glycol surfactants. Based on proton NMR spectroscopy data, a drug to surfactant ratio of 100:6 by weight was used to make nanoformulated M2CAB, MCAB and CAB. Briefly, 1%-5% (w / v) of M2CAB, MCAB or CAB and 0.06%-0.3% (w / v) of P407 were mixed in endotoxin-free water. Premixed suspensions were prepared by wet milling or high pressure homogenization at 20,000 psi until the desired size and polydispersity index (PDI) were obtained. The particle size, polydispersity index (PDI) and zeta potential ( Figure 1B This was performed using a Malvern Zetasizer, Nano series Nano-ZS (Malvern Instruments Inc, Westborough, MA). Nanoparticle morphology was determined by scanning electron microscopy (SEM). UPLC MS / MS was used for drug quantification.

[0114] Macrophage uptake and retention

[0115] Human monocytes were obtained from HIV-1 / 2 and hepatitis B seronegative donors by leukapheresis and then purified by countercurrent centrifugal elutriation (Balkundi et al., Intl. J. Nanomed. (2011) 6:3393-3404; Nowacek et al., Nanomed. (2009) 4(8):903-917). Human monocytes were cultured at 1.0×10 per well using DMEM supplemented with 10% heat-inactivated pooled human serum, 1% glutamine, 10 μg / mL ciprofloxacin, and 50 μg / mL gentamicin. 6The cells were plated in 12-well plates at a density of . The cells were maintained at 37°C in a 5% CO2 incubator. After 7-10 days of differentiation in the presence of 1000U / mL recombinant human macrophage colony stimulating factor (MCSF), the MDMs were treated with a series of nanoformulations and natural drugs. The uptake of the drug was evaluated by measuring the intracellular drug concentration at different time points after treatment. For drug retention studies, cells were treated for 8 hours, then washed with PBS and maintained with half-medium replacement every other day until collected at the designated time points. For both studies, adherent MDMs were washed with PBS (3×1 mL), then scraped into 1 mL of fresh PBS, and Countess was used at the designated time points. TM Automated cell counter (Invitrogen, Carlsbad, CA) was used for counting. Cells were pelleted by centrifugation at 950×g for 8 minutes at 4°C. The cell pellet was reconstituted in 200 μl of high performance liquid chromatography (HPLC) grade methanol and probe sonicated followed by centrifugation at 20,000×g for 20 minutes. The supernatant was analyzed for drug content using HPLC.

[0116] like Figure 2A As can be seen in the Figure 2, NM2CAB was taken up by MDM to statistically higher levels than NMCAB. In addition, MDM retained NM2CAB at statistically higher levels for a longer period of time than NMCAB ( Figure 2A ).

[0117] Antiretroviral activity

[0118] Antiretroviral efficacy is determined by measuring HIV reverse transcriptase (RT) activity. Figure 2B and Figure 2C ). To evaluate the antiretroviral efficacy, MDM were treated with 100 μM CAB-LAP, NMCAB or NM2CAB for 8 hours. After treatment, cells were washed with PBS to remove excess free drug and nanoparticles, and cells were cultured with fresh medium, with half of the medium replaced every other day. HIV-1 was used at an MOI of 0.01 infectious virus particles / cell. ADAThe MDMs were challenged for up to 30 days. The production of progeny virions was measured by RT activity in the culture medium (Kalter et al. (1992) J. Clin. Microbiol., 30(4): 993-995). HIV-1p24 protein antigen expression was assessed (Guo et al. (2014) J. Virol., 88(17): 9504-9513). The MDMs were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were blocked with 10% BSA containing 1% Triton X-100 in PBS for 30 minutes at room temperature. After blocking, the cells were incubated with HIV-1p24 mouse monoclonal antibody (1:50; Dako, Carpinteria, CA, USA) at 4°C overnight, followed by incubation at room temperature for 1 hour. HRP-labeled polymer anti-mouse secondary antibody (Dako System) (one drop / well). Hematoxylin was added to counterstain the cell nuclei, and images were captured using a Nikon TE300 microscope with a 20× objective. Figure 2B and Figure 2C As can be seen in Figure 5, unexpectedly superior antiretroviral efficacy was observed for nanoformulated M2CAB compared to nanoformulated CAB or MCAB.

[0119] Pharmacokinetic / biodistribution studies

[0120] Female NSG mice were administered a single IM 45 mg / kg CAB equivalent dose of NM2CAB, NMCAB or NCAB. Plasma and tissue drug levels were determined by UPLC-MS / MS. Drug levels in plasma were monitored weekly. When compared to NMCAB or NCAB, a single IM injection of NM2CAB exhibited zero-order controlled release kinetics of active CAB, which remained above 4-fold PA-IC 90 ( Figure 3A ). At day 364 after injection, plasma CAB levels were 345.2 ng / ml for NM2CAB, 8.5 ng / ml for NMCAB, and undetectable values ​​for NCAB (detection limit of 0.5 ng / ml).

[0121] Mice were also administered a single IM 45 mg / kg CAB equivalent dose of NM3CAB (C22). Drug levels in plasma were monitored weekly. On day 28 after injection, plasma CAB levels were 233.2 ng / ml ( Figure 3B ). Therefore, it is clear that NM2CAB (C18) is statistically superior to the shorter NMCAB (C14) and the longer NM3CAB (C22) in maintaining long-term, effective release of the drug.

[0122] After being derived into M2CAB prodrug, the hydrophobicity of CAB was greatly improved. The improved hydrophobicity of M2CAB helps to produce a stable formulation with a high drug load. In addition, the conversion of CAB to a more hydrophobic M2CAB and the formation of nanoparticles significantly improved the intracellular accumulation of the drug compared to nanoformulated CAB or MCAB. Significant improvements in MDM retention and antiretroviral efficacy were observed for nanoformulated M2CAB compared to nanoformulated CAB or MCAB. A single IM injection of NM2CAB at 45 mg CAB equivalent / kg in female NSG mice also unexpectedly demonstrated a PA-IC higher than 4 times that lasted for more than five months. 90 The plasma CAB concentration was significantly greater than that of nanoformulated CAB or MCAB.

[0123] Example 2

[0124] Current antiretroviral (ARV) treatment regimens are effective and well tolerated, enabling sustained, lifelong suppression of human immunodeficiency virus type 1 (HIV-1) (Fauci et al., JAMA (2019) 321: 844-845). However, such control of viral replication must be linked to regimen compliance, which is in turn affected by concurrent comorbidities, social stigma, behavior, concurrent illicit drug use, and costs (Fauci et al., JAMA (2019) 321: 844-845). However, even strict compliance with daily dosing often leads to the emergence of drug toxicity, drug-drug interactions, and viral resistance. All drug regimens exclude viral elimination (Dash et al., Nat. Commun. (2019) 10: 2753). This emphasizes the fact that all therapies require lifelong adherence to maintain HIV-1 suppression and alleviate disease. These have led scientists to develop long-acting (LA) treatments. All research focuses on improving regimen compliance and drug efficacy (Gendelman et al., Trends Microbiol. (2019) 27: 593-606). The two most active and close to clinical approval by the US Food and Drug Administration are ARV LA injectables, while implantable drug devices are still under development (Margolis et al., Lancet (2017) 390: 1499-1510; Taylor et al., Topics Antiviral Med. (2019) 27: 50-68).

[0125] To date, studies conducted have shown considerable promise for LA injectables for widespread human use (Margolis et al., Lancet (2017) 390: 1499-1510; Taylor et al., Topics Antiviral Med. (2019) 27: 50-68; Kerrigan et al., PloS One (2018) 13: e0190487). Therefore, an injectable nanoformulation of LA as a combination of cabotegravir (CAB) and rilpivirine (RPV) as a two-drug combination will likely be approved by the end of 2019 for monthly administration (Margolis et al., Lancet (2017) 390: 1499-1510; Taylor et al., Topics Antiviral Med. (2019) 27: 50-68; Kerrigan et al., PloS One (2018) 13: e0190487). Both "Antiretroviral therapy as long-acting suppression" (ATLAS) and "the first long-acting injectable regimen" (FLAIR) have demonstrated promising safety, efficacy, and tolerability (Taylor et al., Topics Antiviral Med. (2019) 27: 50-68). When the treatment was compared with the standard oral three-drug regimen, the combination therapy demonstrated non-inferiority (Taylor et al., Topics Antiviral Med. (2019) 27: 50-68). Similarly, drug implants have also shown promise (Kovarova et al., Nat. Commun. (2018) 9:4156; Gunawardana et al., Antimicrob. Agents Chemother. (2015) 59:3913-3919; Flexner, C., Curr. Opin. HIV AIDS (2018) 13:374-380; Barrett et al., Antimicrob. Agents Chemother. (2018) 62(10):e01058-18.). However, both approaches have limitations, including injection site reactions, large injection volumes, dosing frequency, and limited penetration into viral reservoirs (Margolis et al., Lancet (2017) 390: 1499-1510; Markowitz et al., Lancet HIV (2017) 4: e331-e340; Zhou et al., Biomaterials (2018) 151: 53-65). In addition, these newer treatments require frequent professional health care services, either to provide the injections themselves or to perform the insertion and removal of the implants.The measurement of tissue and plasma drug levels is related to the efficacy of LAARV, including the penetration of drugs into mucosa, lymphoid tissue and central nervous system and long-term safety. Based on these unknown extents, there is an urgent need for further improvement of LA ARV regimen. Any future LA ARV needs to be administered in a reduced volume without systemic toxicity. If achieved, the ARV regimen can also be performed in a manner that reflects ARV vaccine mimics. Success will prevent new infections and reduce new transmissions, and functional HIV-1 cure can be achieved in this way.

[0126] To this end, LA ARV libraries have been established for a range of antiretroviral agents (Zhou et al., Biomaterials (2018) 151:53-65; Hilaire et al., J. Control Release (2019) 311-312:201-211; Ibrahim et al., Int. J. Nanomedicine (2019) 14:6231-6247; Lin et al., Chem. Commun. (Camb) (2018) 54:8371-8374; McMillan et al., Antimicrob. Agents Chemother. (2017) 62: e01316-17; McMillan et al., AIDS (2019) 33: 585-588; Sillman et al., Nat. Commun. (2018) 9: 443; Smith et al., Biomaterials (2019) 223: 119476; Soni et al., Biomaterials (2019) 222: 119441). Herein, the purpose of producing CAB prodrugs is to extend the apparent half-life and antiretroviral activity of the drug while strictly controlling hydrolysis. The synthesis and comprehensive physicochemical characterization of three prodrugs of CAB with 14-carbon, 18-carbon and 22-carbon linker arms (MCAB, M2CAB and M3CAB, respectively) are reported, as well as a complete evaluation of their respective nanoformulations (NMCAB, NM2CAB and NM3CAB). These analyses extend previous testing of the first-generation CAB prodrug MCAB (Zhou et al., Biomaterials (2018) 151: 53-65; McMillan et al., AIDS (2019) 33: 585-588). The C18 nanoformulation NM2CAB enhanced the uptake and retention of CAB in mononuclear macrophages and showed long-term monthly protection against HIV-1 infectious challenge. NM2CAB produced a protein-associated 90% inhibitor concentration (PA-IC) above 166 ng / mL for 52 weeks. 90) of CAB plasma concentration. This is related to the obvious lymph, mucosal and intestinal biodistribution levels after a single parenteral injection. There are no recorded systemic adverse events. Parallel drug plasma concentrations in normal and immunodeficient mice and rhesus monkeys injected with drugs have confirmed the long-term sustained drug release of prevention and treatment regimens. These results comprehensively show that prodrugs can be used for the same purpose of preventive vaccines against HIV-1.

[0127] Materials and methods

[0128] Reagents

[0129] CAB was purchased from BOC sciences (Shirley, NY). Pyridine, dimethylformamide (DMF), N,N-diisopropylethylamine (DIEA), myristoyl chloride, stearyl chloride, behenic acid, poloxamer 407 (P407), ciprofloxacin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), and 3,3'-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St. Louis, MO). Ether, ethyl acetate, hexane, acetonitrile (ACN), methanol, LC-MS grade water, cell culture grade water (endotoxin-free), gentamicin, potassium dihydrogen phosphate (KH2PO4), bovine serum albumin (BSA), Triton TM X-100 and Reagents were purchased from Fisher Scientific (Hampton, NH). Monoclonal mouse anti-human HIV-1 p24 (clone Kal-1) and polymer-based HRP-conjugated anti-mouse EnVision TM Secondary antibodies were purchased from Dako (Carpinteria, CA). Heat-inactivated pooled human serum was purchased from Innovative Biologics (Herndon, VA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Corning Life Sciences (Tewksbury, MA).

[0130] Synthesis and characterization of CAB prodrugs

[0131] A series of three prodrugs were synthesized by esterifying the 10-hydroxyl group on CAB, resulting in lipophilic prodrugs with 14-carbon, 18-carbon and 22-carbon linkers, named MCAB, M2CAB and M3CAB. Initially, CAB was dried from anhydrous pyridine and then suspended in anhydrous DMF. The mixture was cooled to 0°C under argon. DIEA (2 equivalents) was used to deprotonate the 10-hydroxyl group of CAB, and then reacted with 2 equivalents of myristoyl chloride or stearyl chloride for 24 hours to obtain MCAB or M2CAB, respectively. The synthesis of M3CAB is a two-step process. In the first step, behenoyl chloride is synthesized by chlorinating the carboxylic acid group of behenic acid using 4 equivalents of thionyl chloride in anhydrous chloroform. CAB dried from anhydrous pyridine and resuspended in anhydrous DMF in the presence of 4 equivalents of triethylamine was further added to behenoyl chloride in DMF at 0°C under argon followed by heating the reaction at 50°C for 24 hours. All the obtained prodrugs were purified by silica gel column chromatography using an initial mobile phase of 4:1 ethyl acetate:hexane for the initial fractions and then 9:1 ethyl acetate:hexane for the remainder. Finally, the prodrug was precipitated and washed in ether and dried under vacuum to obtain a white powder in an average yield of 85%-95%. The prodrugs were characterized by proton and carbon nuclear magnetic resonance ( 1 H and 13 The successful synthesis of the prodrug was confirmed by C NMR) spectroscopy, which was recorded by a Bruker Avance-III HD (Billerica, MA) operating at 500 MHz and a magnetic field strength of 11.7 T. Fourier transform infrared analysis (FT-IR) was performed on a universal attenuated total reflectance (UATR) Spectrum Two (PerkinElmer, Waltham, MA). Comparative crystallographic analysis of CAB and prodrug by powder X-ray diffraction (XRD) was performed at 40 kV, 45 mA using a 400 nm spectral spectroscopy setup with Cu-Kα radiation. The diffractometer was used at a rate of 1° / s in the 2θ range of 2°-70° on a PANalytical Empyrean diffractometer (PANalytical Inc., Westborough, MA). Molecular masses were determined by direct infusion into a Waters TQD mass spectrometer.

[0132] UPLC-UV / Vis Quantification of CAB, MCAB, M2CAB and M3CAB

[0133] Waters ACQUITY ultra-high performance liquid chromatography (UPLC) H-Class system with TUV detector and Empower3 software (Milford, MA) were used to measure drug concentration. CAB, MCAB, M2CAB and M3CAB samples were separated on Phenomenex Kinetex 5μm C18 columns (150×4.6mm) (Torrance, CA). CAB was detected at 254nm using a mobile phase consisting of 65% 50mM potassium dihydrogen phosphate (KH2PO4) at pH3.2 and 35% acetonitrile (ACN) and a flow rate of 1.0mL / min. MCAB, M2CAB and M3CAB were detected at 230nm using a mobile phase consisting of 90% ACN and 10% water, 95% ACN and 5% water, 98% ACN and 2% water and a flow rate of 1.0mL / min, respectively. Drug content was determined relative to the peak area of ​​drug standards (0.05 μg / mL-50 μg / mL) in methanol.

[0134] Measurement of water solubility of pharmaceutical preparations

[0135] The water solubility of CAB, MCAB, M2CAB and M3CAB in optima water was determined by adding excess drug or prodrug powder to 1 mL of water to prepare a saturated aqueous solution. The mixture was stirred at room temperature for 24 hours. In addition, the solution was centrifuged at 14,000 rpm for 10 minutes to precipitate undissolved powder. The supernatant was collected, lyophilized and dissolved in methanol for drug content analysis by UPLC UV / Vis.

[0136] Chemical stability

[0137] The stability of MCAB, M2CAB and M3CAB under acidic (pH 1), basic (pH 11) and neutral (pH 7) conditions at room temperature and elevated temperature (37°C) was determined. A stock solution of each prodrug was prepared in DMSO at a concentration of 1 mg / mL. For acidic, basic and neutral assays, 100 μL of the stock solution of each prodrug was added to 1900 μL of 0.1 M HCl, 0.1 M NaOH or optima grade water (pH adjusted to 7), respectively. The samples were then incubated at room temperature and 40°C under shaking conditions ( 42 shaking incubator, 150 rpm). Samples were taken out at 0, 2, 4, 8 and 24 hours and stored at -80°C. Afterwards, the drug content of the samples was analyzed by UPLC-UV / Vis.

[0138] Plasma cleavage drug hydrolysis kinetics

[0139] The hydrolysis kinetics of MCAB, M2CAB and M3CAB and the relative release of active drugs were determined in the plasma of different species (mouse, rat, rabbit, monkey, dog and human).MCAB, M2CAB or M3CAB (1 μM) 100 μL plasma were incubated at 37 ° C.At different time points, 1 mL of acidified methanol (0.1% formic acid and 25 mM ammonium formate to avoid further prodrug hydrolysis) was added to each sample and vortexed for 3 minutes to stop the reaction.For 0 minute time point, 100 μL ice-cold plasma was mixed with prodrug stock solution, and 1 ml of ice-cold acidic methanol was added immediately.After adding methanol, the sample was centrifuged at 15,000 g for 10 minutes, and the collected supernatant was analyzed for drug content by UPLC-MS / MS (Waters Xevo TQ-XS).

[0140] Nanoparticle synthesis and characterization

[0141] Nanoformulations of parent CAB (NCAB) and nanoformulations of its prodrug (NMCAB, NM2CAB, and NM3CAB) were made by high pressure homogenization using poloxamer surfactant 407 (P407). Drug / prodrug and P407 were premixed (10:1 w / w) in endotoxin-free water for 24 hours in a concentration range of 2%-20% w / v drug / prodrug and 0.2%-2% w / v P407. The premix was further homogenized using an Avestin EmulsiFlex-C3 high pressure homogenizer (Ottawa, ON, Canada) at ~18,000 psi to generate homogenous nanocrystals of the desired particle size. The particle size (D) of the nanoparticles was characterized by dynamic light scattering (DLS) using a Malvern Nano-ZS (Worcestershire, UK). eff), polydispersity index (PDI) and zeta potential. The stability of the nanoformulations was monitored at 4°C, 25°C and 37°C for 3 months. The drug / prodrug content in the nanoformulation was measured by dissolving the nanoformulation in methanol (dilution factor range: 1000-10000) and analyzing by UPLC UV / Vis. The following equation is used to calculate the encapsulation efficiency. Encapsulation efficiency (%) = (weight of drug in the formulation / initial weight of the drug added) × 100. The morphology of the nanoparticles was evaluated by scanning electron microscopy (SEM). The nanoparticles were fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde in 0.1M sorenson phosphate buffer (pH 7.2) at 4°C for 24 hours and processed for imaging. In brief, the nanosuspension was air-dried on a glass coverslip mounted on a SEM sample holder and sputter-coated with about 50nm of gold / palladium alloy. Samples were measured using a FEI Quanta 200 scanning electron microscope (Hillsboro, OR) operating at 5.0 kV (Sillman et al., Nat. Commun. (2018) 9:443).

[0142] Human monocyte-derived macrophages (MDM)

[0143] Human monocytes were obtained from HIV-1 / 2 and hepatitis B seronegative donors by leukocyte separation, and then purified by countercurrent centrifugal elutriation (Gendelman et al., J.Exper.Med. (1988) 167: 1428-1441). Monocytes were cultured in DMEM culture medium containing 4.5 g / L glucose, L-glutamine and sodium pyruvate, supplemented with 10% heat-inactivated human serum, 50 μg / mL gentamicin and 10 μg / mL ciprofloxacin. Cells were maintained in a 5% CO2 incubator at 37°C. Recombinant human macrophage colony stimulating factor (MCSF, 1000 U / mL) was added to the culture medium in the first 7 days to promote the differentiation of monocyte-derived macrophages (MDM). Half of the culture medium was replaced with fresh culture medium every other day. After differentiation, MDM was used for subsequent in vitro assays.

[0144] Cytotoxicity assay

[0145] The viability of cells after treatment with nanoparticles was assessed by performing an MTT assay. Cells were treated with 10 μM, 25 μM, 50 μM, 100 μM, 200 μM, or 400 μM of NCAB, NMCAB, NM2CAB, or NM3CAB at a concentration of 0.08 × 10 cells / well. 6Human MDM were plated in 96-well plates at a density of 100 cells for 24 hours. Untreated cells were used as controls. For each group, samples were in quadruplicate. The cells were washed with PBS and incubated with 100 μL / well of MTT solution (5 mg / mL) at 37°C for 45 minutes. After incubation, the MTT solution was removed and the cells were washed with PBS. Then, 200 μL of DMSO was added to each well and the concentration was measured at 490 nm on a Molecular Devices The absorbance was measured on an M3 plate reader using SoftMax Pro 6.2 software (Sunnyvale, CA).

[0146] Study on the uptake, retention and release of drug particles MDM

[0147] MDM uptake and retention studies were performed in clear flat-bottom 12-well plates at 1.0 × 10 6 The density of cells was carried out, and each treatment group was completed in triplicate. For cellular uptake studies, MDM was treated with 100 μM NCAB, NMCAB, NM2CAB or NM3CAB. MDM was collected 2 hours, 4 hours, 8 hours and 24 hours after treatment to measure intracellular drug and prodrug levels. For retention studies, MDM was treated with 100 μM NCAB, NMCAB, NM2CAB or NM3CAB for 8 hours, and then washed twice with phosphate buffered saline (PBS). Fresh culture medium was added every other day and half of the culture medium was replaced. MDM was collected on the 1st day, the 5th day, the 10th day, the 15th day, the 20th day, the 25th day and the 30th day to determine intracellular drug and prodrug concentrations. For these two studies, at the specified time point, the adhered MDM was washed twice with PBS. The cells were then scraped into PBS and Invitrogen Countess was used. TMAutomatic cell counter (Carlsbad, CA) counts. The suspension of cells in PBS was centrifuged at 3,000rpm for 8 minutes at 4°C. The obtained cell pellet was sonicated in 200 μL methanol using a probe sonicator to extract intracellular drugs. The resulting product was centrifuged at 20,000g for 10 minutes at 4°C to separate cell debris from the supernatant containing the drug. The drug and prodrug content of the sample was further analyzed by UPLC-UV / Vis. For release studies, culture medium was collected at a time point similar to that in the retention study to quantify the drugs and prodrugs released by MDM. The culture medium was mixed with methanol to precipitate the insoluble components in the culture medium, and the drugs and prodrugs were extracted. The mixture was centrifuged at 17,000g for 10 minutes at 4°C to separate the insoluble precipitate. The supernatant was transferred to a new tube to be dried in a rapid vacuum. The dried contents were suspended in methanol to be further analyzed by UPLC-UV / Vis.

[0148] Particle characterization by transmission electron microscopy (TEM)

[0149] MDM is treated with NCAB, NMCAB, NM2CAB or NM3CAB of the concentration of 100 μM for 8 hours, and then washed twice with PBS.Add fresh culture medium every other day and replace half of the culture medium.After drug particle treatment, collect MDM culture supernatant fluid on the 0th day, the 15th day and the 30th day, and analyze by TEM to image intracellular nanoparticles.For the 0th day, collect cells immediately after 8 hours of treatment duration.At the time point specified, wash cells, scrape into PBS, precipitate at 3000rpm for 8 minutes at room temperature, and fix in the solution of 2% glutaraldehyde, 2% paraformaldehyde in 0.1M Sorenson phosphate buffer (pH 6.2).A drop of fixed cell suspension is placed on formvar / silicon monoxide 200 mesh copper grid, and allowed to stand for 2 minutes.Suck off excess solution and allow to dry.A drop of NanoVan vanadium negative stain is placed on the grid for 1 minute, then suck off and allow to dry. Grids were examined on a FEITecnai G2 Spirit TWIN transmission electron microscope (Hillsboro, OR) operated at 80 kV. Images were acquired digitally with an AMT digital imaging system (Woburn, MA) (Sillman et al., Nat. Commun. (2018) 9:443).

[0150] Measurement of reverse transcriptase (RT) activity in HIV-1 infected and infected cytosolic fluids

[0151] MDM was added at 0.8×10 6The density of each cell / well is plated in a transparent flat-bottom 24-well plate. NCAB, NMCAB, NM2CAB or NM3CAB of 100 μM are used to process MDM for 8 hours. After treatment, cells are washed with PBS, and cultured in fresh culture medium, and half of the culture medium is replaced every other day. After treatment, 1 day, 5 days, 10 days, 15 days, 20 days, 25 days and 30 days, HIV-1ADA (a kind of macrophage virus strain) is used to infect cells with the multiplicity of infection (MOI) of 0.1 infectious particles per cell for 16 hours. After the infection period, MDM is washed with PBS, and supplemented with fresh culture medium. In the next ten days, cells are cultured, and half of the culture medium is replaced every other day, and full culture medium is replaced at the 8th day. Culture medium was collected on day 10 after infection for measurement of HIV-1 RT activity (Kalter et al., J. Immunol. (1991) 146: 3396-3404; Nowacek et al., J. Neuroimmune Pharm. (2010) 5: 592-601). The degree of infection was expressed as a percentage of the RT activity of untreated infected MDM. Cells were fixed in 2% PFA and the expression of HIV-1 p24 antigen was determined by immunocytochemistry (Nowacek et al., J. Neuroimmune Pharm. (2010) 5: 592-601).

[0152] The half-maximal effective concentration (EC 50 ) determination

[0153] MDM were plated in a transparent flat-bottom 96-well plate (0.08 × 10 6 Cells were treated with a range of drug concentrations (0.01 nM-1000 nM) of CAB, MCAB, M2CAB, M3CAB, NCAB, NMCAB, NM2CAB or NM3CAB for 1 hour and then inoculated with HIV-1 ADA (MOI of 0.1 infectious particles per cell) infection lasted 4 hours. After 4 hours of viral challenge, cells were washed and given fresh medium containing drugs (0.1 nM-1000 nM). Subsequently, cell supernatants were collected after 10 days and HIV-1 RT activity was determined as described above.

[0154] Nanoparticle uptake by CD4+ T cells using CEM-ss cells as a standard

[0155] CEM-ss CD4+T cells were suspended in RPMI supplemented with 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin. Cell suspension (1mL / well) was added to a transparent flat-bottom 12-well plate pre-coated with poly-L-lysine solution (500μg / mL in distilled water) for 1 hour. After attachment to the well surface, cells were treated with 25μM NCAB, NMCAB, NM2CAB or NM3CAB. At 2 hours, 4 hours and 8 hours, cells were washed and scraped into PBS. Cell suspension was centrifuged at 200g for 5 minutes, and intracellular drug and prodrug concentrations were quantified by Waters TQD mass spectrometer. After 8 hours of treatment at 25μM concentration, the uptake of nanoparticles in CEM-ss CD4+T cell lines was confirmed by TEM imaging. Sample processing for TEM imaging is described above.

[0156] Pharmacokinetic (PK) and biodistribution (BD) studies in mice

[0157] NSG mice (female, 6 weeks-8 weeks, Jackson Labs, Bar Harbor, ME) were administered 45 mg / kg CAB equivalents of NCAB, NMCAB, NM2CAB or NM3CAB by a single intramuscular (IM, tail thigh muscle) injection of 40 μL / 25g mice. After injection, blood samples were collected into heparinized tubes on the 1st day after administration and then collected weekly until the 364th day by cheek puncture (submandibular vein, MEDIpoint, Inc., Mineola, NY). The collected blood (25 μL) was immediately diluted into 1 mL ACN and stored at -80 ° C until drug measurement. The remaining blood sample was centrifuged at 2,000 g for 8 minutes for plasma collection. Plasma was collected and stored at -80 ° C for analysis of drug content. Animals were artificially euthanized by isoflurane inhalation and subsequent cervical dislocation on the 14th, 28th, 42nd and 364th days after administration. Spleen, lymph nodes, liver, lung, intestine, kidney, brain, vaginal tissue and rectal tissue were collected for quantitative CAB and prodrug concentration. CAB, MCAB, M2CAB and M3CAB in mouse plasma, blood and tissue were quantitatively analyzed by UPLC-MS / MS using Waters ACQUITY H-class UPLC (Waters, Milford, MA, USA) connected to Xevo TQ-S micro mass spectrometer. All solvents for sample processing and UPLC-MS / MS analysis were LCMS grade (Fisher). For plasma and blood samples, 25 μL of sample were added to 1mL of acetonitrile (ACN) mixed with 10 μL internal standards (IS). d3-dolutegravir (d3-DTG), myristoylated dolutegravir (MDTG), and stearoyl darunavir (SDRV) were used as IS for CAB, MCAB, and M2CAB / M3CAB analysis at final concentrations of 200 ng / mL, 20 ng / mL, and 20 ng / mL, respectively. The samples were vortexed and centrifuged at 17,000 × g for 10 min at 4°C. The supernatant was collected and used Dry, and reconstitute in 100 μL 80% methanol; inject 10 μL for MCAB, M2CAB and M3CAB UPLC-MS / MS analysis. Standard curves were prepared in blank mouse plasma / blood in the range of 0.2ng / mL-2000ng / mL for CAB, MCAB, M2CAB and M3CAB. For tissue drug quantification, 3mg-200mg of sample was homogenized in 4-29 volumes of 90% methanol containing 0.1% v / v formic acid and 2.5mM ammonium formate. 280μl methanol, 80% methanol (10μL) and IS (10μL) containing 0.1% formic acid and 2.5mM ammonium formate were added to 100μL of tissue homogenate, followed by vortexing for 3min and centrifugation at 17,000×g for 15 minutes. For MCAB, M2CAB and M3CAB analysis, 85 μl of supernatant was mixed with 15 μl of water. For CAB analysis, 20 μl of supernatant was mixed with 80 μl of 50% ACN. These aliquots were vortexed, centrifuged at 17,000 × g for 10 minutes, and 10 μl of supernatant was used for LC-MS / MS analysis. Standards were similarly prepared using a blank homogenate containing 10 μL of spiked solution (CAB / MCAB / M2CAB / M3CAB, 5 ng / mL-20,000 ng / mL in 50% ACN). For CAB quantification, chromatographic separation of 10 μL CAB samples was performed on a Waters ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 mm×100 mm) using a 10-minute gradient of mobile phase A (7.5 mM ammonium formate in water, adjusted to pH 3 using formic acid) and mobile phase B (100% ACN) at a flow rate of 0.25 mL / min. For the first 3.5 min, the mobile phase composition was 35% B and increased to 95% B within 0.5 min and kept constant for 1.5 minutes. Mobile phase B was then reset to 35% within 0.5 min, and the column was equilibrated for 1 minute before the next injection. For MCAB and M2CAB quantification, chromatographic separation was achieved on a shorter 30 mm column (PN, using an 8-min gradient method at a flow rate of 0.28 mL / min). The initial mobile phase composition was 80% B for the first 2 min and increased to 95% B within 4 min, kept constant for 0.75 min, reset to 80% within 0.25 min, and the column was equilibrated for 1 min before the next injection. For M3CAB quantification, chromatographic separation was also achieved on a shorter 30 mm column using an 8 min gradient at a flow rate of 0.35 mL / min.The initial mobile phase composition was 88% B for the first 5 min and increased to 95% B in 0.25 min, kept constant for 1.5 min, reset to 88% in 0.25 min, and the column was equilibrated for 1 min before the next injection. CAB, MCAB, M2CAB and M3CAB were detected in positive ionization mode at cone voltages of 10 V, 24 V, 2 V and 20 V, respectively, and collision energies of 24 V, 18 V, 24 V and 26 V, respectively. The multiple reaction monitoring (MRM) transitions for CAB, MCAB, M2CAB, M3CAB, d3-DTG, MDTG and SDRV were 406.04>126.93, 616.28>406.09, 672.34>406.07, 728.47>406.09, 422.84>129.99, 630.20>420.07 and 814.52>658.44, respectively. The spectrum was analyzed and quantified by MassLynx software version 4.1. All quantifications were determined using the ratio of the analyte peak area to the internal standard peak area. After PK and BD analysis in NSG mice, the excellent NM2CAB in all preparations was evaluated again in another mouse strain BALB / cJ. (Male, 6 weeks-8 weeks, Jackson Labs). NCAB was used as a control. Mice injected with NM2CAB were manually euthanized on day 280 after administration. Quantification of drug and prodrug in collected plasma and tissues was similar to above. For studies in NSG mice, non-compartmental PK analysis of plasma CAB was performed using Phoenix WinNonlin-8.0 (Certara, Princeton, NJ).

[0158] PK and BD in rhesus monkeys (RM)

[0159] Four male RM (4.4kg-6.7kg; PrimeGen) were anesthetized with ketamine (10mg / kg) and then administered 45mg / kg CAB equivalents of NM2CAB and laboratory-generated RPV prodrugs by IM injection (quadriceps, 0.5mL / kg). Blood samples were collected in potassium-EDTA coated tubes for complete blood counts (CBC), metabolic profiles. Plasma was separated for drug measurement. Tissue biopsies of lymph nodes, adipose, and rectal tissues were collected on day 204 after injection for drug quantification. The quantification methods of drugs and prodrugs in plasma and tissues are similar to those described in the mouse study above.

[0160] Statistical analysis

[0161] Statistical analysis was performed using GraphPad Prism 7.0 software (La Jolla, CA). The data from the in vitro studies were expressed as mean ± SEM, with a minimum of 3 biological replicates, while the results from the in vivo studies were expressed as mean ± SEM, with a minimum of 3 biological replicates. For comparisons between the two groups, Student's t test (bilateral) was used. One-way ANOVA followed by Tukey's test was used to compare three or more groups. Statistical significance was expressed as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0162] Study Approval

[0163] All animal studies were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC) according to the standards contained in the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academy of Sciences, 2011). Human mononuclear cells were isolated from HIV-1 / 2 and hepatitis B seronegative donors by leukapheresis under an exempt protocol approved by the UNMC Institutional Review Board.

[0164] result

[0165] Prodrug synthesis and characterization

[0166] CAB was chemically modified by attaching fatty acid chains of variable carbon length (14, 18, and 22) to produce the esters MCAB, M2CAB, and M3CAB, respectively. The prodrugs were further characterized by nuclear magnetic resonance (NMR) to confirm the synthesis. 1 The H NMR spectrum showed triplet peaks in the ranges of 0.86ppm-0.89ppm, 1.77ppm-1.78ppm and 2.66ppm-2.70ppm and a broad singlet in the range of 1.24ppm-1.25ppm, corresponding to the terminal methyl and repeating methylene protons of the fatty acid alkyl chain. The disappearance of the phenolic proton peak at 11.5ppm confirmed that the hydroxyl protons of CAB were replaced by fatty acyl moieties. 13 C NMR spectroscopy confirmed the carbon atoms of the conjugated aliphatic chains. Electrospray ionization mass spectrometry (ESI-MS) confirmed the molecular weights of all prodrugs. The ESI-MS infusion of MCAB generated a strong signal at 616.28 m / z, the ESI-MS infusion of M2CAB generated a strong signal at 672.34 m / z, and the ESI-MS infusion of M3CAB generated a strong signal at 728.47 m / z. Fourier transform infrared spectroscopy (FTIR) prodrug characterization produced a strong signal at 2908 cm -1 -2935cm -1 and 1748cm -1-1775cm -1 The XRD spectra in the range of 2θ=2°-70° show the crystalline characteristics of the prodrugs MCAB, M2CAB and M3CAB. The nanoparticles of M2CAB retain the crystalline properties of the prodrug. Compared with CAB (12.12±0.41μg / mL), a significant continuous decrease in the aqueous solubility of MCAB (2.83±1.37μg / mL), M2CAB (1.91±0.23μg / mL) and M3CAB (1.51±0.7μg / mL) was observed. In addition, a parallel significant increase in 1-octanol solubility was observed for MCAB (2.31 ± 28.15 mg / mL), M2CAB (2.64 ± 0.02 mg / mL), and M3CAB compared to CAB (0.09 ± 0.002 mg / mL). These results confirm the increase in hydrophobicity and lipophilicity of the prodrugs due to chemical modification with fatty acid conjugates compared to CAB.

[0167] Prodrugs are pharmacologically inactive compounds that require enzymatic activation or hydrolytic activation to bioconvert to active drugs under physiological conditions. Therefore, the hydrolysis kinetics of MCAB, M2CAB and M3CAB and the subsequent CAB formation were evaluated in the plasma of different species (mice, rats, rabbits, monkeys, dogs and humans). After incubation in the plasma of all tested species, MCAB showed more than 85% cleavage within 30 minutes, M2CAB showed an average of 75% cleavage by 2 hours, and 80% cleavage by 6 hours. M3CAB showed the slowest cleavage rate, and after incubation in plasma for 24 hours, about 50% of the prodrug remained. These studies show that MCAB is quickly converted to CAB, while M3CAB shows the least conversion to CAB. By 6 hours, the CAB formed by hydrolysis of M2CAB was an average of 71.1%, indicating that M2CAB is the best prodrug in terms of stability and active drug formation under physiological conditions. There are certain differences in the rate of prodrug hydrolysis in the plasma of various animal species. Variability in plasma CAB concentrations among test species may be due to differences in body fat distribution, muscle mass, physical activity, and plasma carboxylesterases effective for prodrug hydrolysis (Trezza et al., Curr. Opin. HIV AIDS (2015) 10: 239-245; Bahar et al. (2012) J. Pharm. Sci. (2012) 101: 3979-3988; Wang et al., Acta Pharmaceutica Sinica. B (2018) 8: 699-712). In addition, the chemical stability of M2CAB was determined at room temperature and elevated temperature (37°C) in acidic conditions (pH 1), neutral conditions (pH 7), and alkaline conditions (pH 11) for 24 hours to determine the long-term stability of prodrug nanoformulations under various storage conditions. At room temperature, at 24 hours, 19% hydrolysis in acidic conditions and 24% hydrolysis in neutral conditions were determined. However, under alkaline conditions, M2CAB was completely hydrolyzed and was not detected. At 37°C, about >95% of the prodrug was hydrolyzed under acidic conditions in 24 hours, and 28% of the prodrug was hydrolyzed under neutral conditions. However, under alkaline conditions, M2CAB was completely hydrolyzed within 2 hours. The half maximal effective concentration (EC) of the prodrugs MCAB, M2CAB, and M3CAB was 50 ) values ​​were compared with those of CAB to determine changes, if any, in antiretroviral activity due to chemical modification. ADA HIV-1 RT activity measurements in the culture medium of MDMs showed that the EC values ​​of CAB (28.39 nM), MCAB (34.19 nM), M2CAB (44.71 nM) and M3CAB (51.15 nM) were 50 The values ​​were comparable, indicating no significant effect on drug activity.

[0168] Nanoformulation fabrication and characterization

[0169] Nanoformulations of CAB (NCAB), MCAB (NMCAB), M2CAB (NM2CAB) and M3CAB (NM3CAB) were manufactured by top-down synthesis using high pressure homogenization. Encapsulation efficiency of drug or any prodrug>85%. Poloxamer 407 (P407) surfactant provides particle surface stabilization. Nanoparticle size, polydispersity index (PDI) and zeta potential were determined by DLS at room temperature (RT), 4°C and 37°C. All formulations remained stable for up to 98 days, indicating that these formulations can maintain their physical integrity under a range of storage conditions. In addition, temperature changes did not affect the physicochemical properties of any formulation during the study. On the day of manufacture, the average particle size, PDI, and zeta potential were 294.5 ± 1.8 nm, 0.23 ± 0.01, -28.3 ± 0.21 mV for NCAB; 302.1 ± 10.8 nm, 0.23 ± 0.01, -31.1 ± 1.1 mV for NMCAB; 359.7 ± 3.63 nm, 0.23 ± 0.02, -31.3 ± 0.1 mV for NM2CAB; and 268.47 ± 6.9 nm, 0.23 ± 0.03, -31.1 ± 0.06 mV for NM3CAB. At 98 days after manufacture, the physicochemical parameters were 327.5 ± 4.9 nm, 0.21 ± 0.04, -18.2 ± 0.25 mV for NCAB; 388.7 ± 6.4 nm, 0.22 ± 0.03, -24.8 ± 2.87 mV for NMCAB; 369.5 ± 2.5 nm, 0.19 ± 0.03, -19.6 ± 0.53 mV for NM2CAB; and 245.6 ± 2.6 nm, 0.27 ± 0.06, -25.7 ± 0.56 mV for NM3CAB. SEM images show that NCAB, NMCAB, NM2CAB and NM3CAB nanoparticles have uniform rod-shaped morphology. In order to confirm reproducibility, NM2CAB preparations were manufactured in 11 different batches (Table 1). The nanoparticle sizes varied from 243.00±2.48 nm to 378.00±1.90 nm with narrow PDI (0.18±0.03 to 0.33±0.03).

[0170] Manufacturing batch number Size ± SEM (nm) PdI±SEM 1 320.13±6.81 0.23±0.02 2 322.10±2.99 0.21±0.01 3 328.67±1.60 0.18±0.03 4 311.43±4.53 0.33±0.03 5 287.17±0.94 0.22±0.01 6 317.57±2.83 0.27±0.01 7 378.00±1.90 0.26±0.01 8 244.63±5.85 0.19±0.01 9 268.97±7.66 0.19±0.02 10 259.77±2.09 0.23±0.01 11 243.00±2.48 0.22±0.01

[0171] Table 1: Reproducibility of NM2CAB synthesis.

[0172] The antiretroviral activity of the nanoformulation for the prodrug was determined (EC 50The antiviral activity of NCAB, NMCAB, NM2CAB or NM3CAB was determined in MDM at a concentration range (0.01 nM-1000 nM) and with HIV-1 ADA The activity of HIV-1 reverse transcriptase was measured after viral challenge at an MOI of 0.1. Compared with non-nanoformulated drugs or prodrugs, EC 50 This may be due to the need for nanoparticle dissolution before prodrug cleavage. 50 The values ​​were comparable among NCAB (39.83 nM), NMCAB (89.67 nM), and NM2CAB (37.02 nM). However, the EC 50 The values ​​were significantly increased (~1.78E+06nM). This may be the effect of the slower cleavage of the prodrug and the subsequent generation of active CABs and the intracellular stability of the nanoformulation. In order to further characterize the prodrug nanoformulation and determine the treatment concentration for in vitro studies, cell viability was assessed in MDM and CEM-ssCD4+T cells by MTT assay. In MDM, no cytotoxicity was observed for all nanoformulations in the tested concentration range (10μM-400μM). In CEM-ss CD4+T cells, cytotoxicity was determined to be 50μM and higher concentrations. Therefore, the treatment concentrations of all nanoformulations were 100μM for the assay in MDM and 25μM for the study in CEM-ss CD4+T cells.

[0173] In vitro screening of MDM and CEM-ss CD4+T cells

[0174] Macrophages can be successfully used as cellular drug depots and carriers. Because of their phagocytic properties and ability to migrate throughout the body (Zhou et al., Biomaterials (2018) 151: 53-65; Darville et al., J. Pharm. Sci. (2014) 103: 2072-2087; Aderem et al., Ann. Rev. Immunol. (1999) 17: 593-623; Dou et al., Blood (2006) 108: 2827-2835), MDM can be used as a drug delivery system for viral reservoirs. Therefore, MDM has been used as an in vitro system for evaluating nanoformulations (Zhou et al., Biomaterials (2018) 151:53-65; Hilaire et al., J. Control Release (2019) 311-312:201-211; Ibrahim et al., Int. J. Nanomedicine (2019) 14:6231-6247; Lin et al., Chem. Commun. (2018) 54:8371-8374; Sillman et al., Nat. Commun. (2018) 9:443; Smith et al., Biomaterials (2019) 223:119476; Soni et al., Biomaterials (2019) 222:119441).

[0175] Uptake assays in MDMs were performed by measuring drug and prodrug levels up to 24 hours after treatment with 100 μM NCAB, NMCAB, NM2CAB, or NM3CAB. The intracellular prodrug levels measured at 24 hours were 61.69 ± 0.78 nmol / 10 for NMCAB, NM2CAB, and NM3CAB, respectively. 6 cells, 84.07±5.82 nanomoles / 10 6 cells and 73.34±13.59 nmol / 10 6 cells; and the intracellular CAB levels of NCAB, NMCAB, NM2CAB, or NM3CAB were 0.58±0.11 nanomoles / 10 6 cells, 12.31±0.46 nanomoles / 10 6 cells, 17.79±2.92 nanomoles / 10 6 cells and 7.97±1.76 nmol / 10 6cells. The ability of macrophages to retain intracellular drugs and prodrugs was then evaluated over a 30-day period after a single treatment. MDMs were treated with 100 μM of NCAB, NMCAB, NM2CAB, or NM3CAB for 8 hours. After a single treatment, intracellular prodrug levels persisted for up to 30 days. Specifically, the amount of intracellular prodrug measured for NMCAB, NM2CAB, and NM3CAB on day 30 was 0.41 ± 0.09 nanomoles / 10 6 cells, 14.21±2.28 nanomoles / 10 6 cells and 26.70±3.29 nmol / 10 6 Similarly, the intracellular CAB levels formed by prodrug cleavage were measured for up to 30 days. The amount of intracellular CAB levels measured for NM2CAB and NM3CAB on day 30 was 1.71 ± 0.35 nanomoles / 10 6 cells and 2.05±0.10 nmol / 10 6 However, within 24 h after NCAB treatment, intracellular CAB levels decreased to below the limit of quantification; and intracellular CAB concentrations after NMCAB treatment were measured until day 25 (0.09 ± 0.04 nmol / 10 6 In parallel with the retention assay, CAB release into the culture medium was measured over 30 days. NM2CAB showed the most sustained CAB release, with 1.0±0.10 nmole / 10 cells at day 30. 6 The drug levels in each cell were plotted. No CAB was detected in the NCAB treatment and NM3CAB treatment. No prodrug was detected in the medium of any treatment, indicating biotransformation of the prodrug.

[0176] Next, TEM images of MDM were taken on days 0, 15, and 30 after treatment with the nanoformulation for 8 hours to assess the presence of nanoparticles in cytoplasmic vesicles. TEM images confirmed that the prodrug nanoformulations (NM2CAB and NM3CAB) were present in MDM until day 30, indicating the drug reservoir properties of MDM; and the uptake, retention, and release results were verified. After treatment at a concentration of 25 μM, the uptake of NCAB, NMCAB, NM2CAB, or NM3CAB was determined in CD4+T cells, reflecting what was observed in MDM. After a single treatment of NCAB, NMCAB, NM2CAB, or NM3CAB for 8 hours, TEM imaging of CEM-ss CD4+T cells confirmed the presence of nanoparticles in the cytoplasmic compartment.

[0177] To examine whether sustained drug retention in MDMs would protect against HIV-1 infection, cells were treated with 100 μM NCAB, NMCAB, or NM2CAB for 8 h and then inoculated with HIV-1 ADA Challenge was performed up to 30 days later and HIV-1 RT activity was measured quantitatively and HIV-1 p24 antigen expression was measured qualitatively. NM3CAB was not selected for this study because it has a low level of expression in the expected EC 50 The values ​​did not show a clear protective effect. NM2CAB treatment suppressed HIV-1RT activity for up to day 30 and was confirmed by the lack of HIV-1p24 expression. In contrast, complete viral breakthrough occurred on day 1 after NCAB treatment and on day 20 after NMCAB treatment. Therefore, the enhanced MDM drug retention presented by NM2CAB provides excellent protection against HIV-1 challenge compared to NCAB and NMCAB. In addition, the dose-response antiretroviral activity of NM2CAB was determined ( Figure 4 MDM were treated with 10 μM, 50 μM or 100 μM NM2CAB for 8 hours and infected with HIV-1 ADA Similar to above, antiretroviral activity was determined to persist for up to 30 days. After 30 days, complete viral inhibition was observed in 50 μM and 100 μM NM2CAB treatments, while 57% protection was observed in 10 μM treatments and was verified by HIV-1 p24 expression ( Figure 4 ).

[0178] Pharmacokinetics (PK) and biodistribution

[0179] To evaluate PK and biodistribution profiles, female NSG mice were injected IM with a single dose of 45 mg / kg CAB equivalent of NCAB or NMCAB or NM2CAB. Immunodeficient female NSG mice were used to evaluate immune-compensated simulated disease pathology and to evaluate biodistribution in the urogenital tract. NCAB is an effective equivalent formulation of CAB-LA, as both formulations produced similar plasma CAB levels up to day 49 after administration to BALB / cJ mice.

[0180] On day 1 post-injection, NCAB treatment produced higher plasma CAB concentrations compared to both NMCAB and NM2CAB and showed faster decay kinetics over the study period compared to NM2CAB. In the case of NCAB treatment, plasma CAB concentrations were maintained above the 90% inhibitory concentration of 4-fold protein regulation (4×PA-IC 90 ; 664 ng / mL) until day 35 (792.7 ng / mL), and then rapidly decreased to below PA-IC by day 49 (75 ng / mL). 90(166 ng / mL), and then decreased to below the limit of quantification (0.5 ng / mL) by day 126. NMCAB treatment showed a slower decay and maintained plasma CAB levels above 4×PA-IC 90 Until day 91 (673.8 ng / mL), and higher than PA-IC 90 Until day 168 (186.7 ng / mL). At day 364 after NMCAB treatment, CAB levels were quantified at 8.5 ng / mL. In sharp contrast, until day 364, NM2CAB treatment provided slower plasma decay kinetics compared to both NCAB and NMCAB, maintaining sustained plasma CAB concentrations above 4×PA-IC 90 Until day 231 (702 ng / mL), and higher than PA-IC 90 Until the 364th day (354.2ng / mL). For all treatment groups, non-compartmental analysis was used to determine the plasma pharmacokinetic parameters of CAB. Quantitative display of PK parameters showed that the apparent CAB half-life after NM2CAB (131.56 days) treatment was 17 times and 3 times that of NCAB (7.80 days) and NMCAB (44.40 days). Similarly, the CAB mean residence time (MRT) of NM2CAB was 21 times that of NCAB (201.94 days relative to 9.79 days, respectively), and was 7 times that of NMCAB (201.94 days relative to 30.76 days, respectively).

[0181] NM2CAB also causes significantly higher CAB tissue levels that last up to one year. Tissue biodistribution of CAB in vaginal tissue, rectal tissue, spleen, liver, intestine, brain, kidney, lung and lymph node anatomical related tissues was evaluated at 14 days, 28 days, 42 days and 364 days after a single IM injection. Drug levels in lymph nodes were determined only in anatomical regions related to lymph nodes at 28 days and 364 days because of the immature state of lymph nodes in immunodeficient NSG mice. In particular, MCAB levels were lower than M2CAB at 14 days, 28 days and 42 days, and undetectable at 364 days. For NM2CAB, at 364 days, prodrug levels were 3414.8ng / g (spleen), 909.8ng / g (liver), 52.7ng / g (lung), 50.3ng / g (brain), 3.9ng / g (kidney) and 18710.1ng / g (lymph node). At day 28, CAB levels in all tissues were comparable between NCAB and NM2CAB. However, by day 42, CAB levels in all tissues tested were significantly lower after NCAB treatment compared to levels after NM2CAB treatment (vaginal tissue, spleen, intestine, brain, kidney and lung, rectal tissue, and brain). However, CAB levels in tissues were significantly higher after NMCAB treatment until day 42 compared to NCAB and NM2CAB treatment. At day 364 after treatment with NM2CAB, CAB concentrations in all tested tissues were significantly higher compared to the other formulations, and CAB levels were measured to be 27 ng / g (vaginal tissue), 19.7 ng / g (rectum), 41.1 ng / g (spleen), 67.62 ng / g (lymph node anatomical related tissue), 123.9 ng / g (liver), 10.3 ng / g (intestine), 7.5 ng / g (brain), 33.2 ng / g (kidney), and 35.5 ng / g (liver). In contrast, at day 364 after treatment with NCAB or NMCAB, CAB levels in all tissues were significantly low or below the limit of quantitation (0.5 ng / g).

[0182] Prodrug (MCAB or M2CAB) concentrations in blood and all tissues were also tested ( Figure 5). On the first day after injection, the concentrations of both prodrugs in the blood were low, 22 ng / mL for MCAB and 31.3 ng / mL for M2CAB, and quickly became below the limit of quantification, indicating that the prodrugs were bioconverted to the active parent drug. All screened tissues were important reservoirs of M2CAB, and the levels of M2CAB were maintained throughout the study. A single IM injection of NM3CAB (45 mg / kg CAB equivalent) in female NSG mice produced low levels of plasma CAB (2248 ng / mL) on the first day after administration, compared to NCAB (41237.6 ng / mL), NMCAB (30148.9 ng / mL), and NM2CAB (7076.1 ng / mL). Plasma CAB levels reached approximately PA-IC within 28 days after treatment. 90 (233.2 ng / mL). Plasma and tissue levels of CAB and M3CAB were measured on day 28. To verify the slower hydrolysis of M3CAB, the PK evaluation was repeated after a single IM injection of NM3CAB at the same dose in BALB / cJ mice. Similar to the PK results in NSG mice, NM3CAB produced low levels of plasma CAB (777.8 ng / mL) on day 1 after administration; and plasma CAB levels decreased to less than 1 times the PA-IC within 28 days. 90 (98.9 ng / mL). These data confirm the slower prodrug hydrolysis and subsequent bioconversion of the M3CAB prodrug to CAB.

[0183] The superior PK and BD profiles of NM2CAB in all formulations were confirmed in another mouse strain, BALB / cJ (male). Here, wild-type mice were used to validate the results from immunodeficient NSG mice. The PK and BD measurements of NM2CAB in plasma and tissues paralleled those in NSG mice. In particular, plasma CAB concentrations of NM2CAB were higher than those of PA-IC by day 231. 90 (170.8 ng / mL), demonstrating the improvement in the apparent half-life of the drug. NCAB was used as a control, where CAB levels became lower than PA-IC by day 28. 90 (12.28ng / mL).

[0184] To validate the results observed in rodents, rhesus monkeys were injected IM with a single dose of 45 mg / kg CAB equivalent NM2CAB. Plasma CAB and prodrug (M2CAB) levels were measured until day 393. Similar to the results in mice, NM2CAB treatment provided slower plasma decay kinetics, maintaining sustained plasma CAB concentrations until day 393. On day 393, CAB levels were measured as an average of 56.1 ng / mL. As expected, plasma M2CAB concentrations were lower throughout the study compared to CAB levels, indicating that the prodrug was bioconverted to its active parent drug ( Fig. 6A ). Rectal, lymph node, and adipose tissue biopsies were collected on day 204 after NM2CAB administration, and CAB levels and M2CAB levels were analyzed. CAB concentrations in rectum, lymph node, and adipose tissue were 10.12 ng / g, 22.7 ng / g, and 29.5 ng / g, respectively ( Figure 6B M2CAB was present at high levels in lymph nodes and adipose tissue (33.3 ng / g and 233.2 ng / g, respectively) and at lower levels in rectal tissue (1.7 ng / g) ( Figure 6B ).

[0185] Toxicity evaluation

[0186] After NM2CAB was used, toxicological determination was performed in both mice and rhesus monkeys. For the toxicity assessment in NSG mice, animal weight was recorded weekly for one year; and when the study concluded (day 364), plasma and tissue were collected for metabolic profile and histopathology respectively. Untreated mice of age matching were used as controls. No weight difference was observed in animals from all groups. Comprehensive serum chemistry parameters were quantitatively analyzed using VetScan comprehensive diagnostic profile disc and VetScan VS-2 instrument. The parameters examined were alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), amylase (AMY), total calcium (CA++), creatinine (CRE), globulin (GLOB), glucose (GLU), phosphorus (PHOS), potassium (K+), sodium (NA+), total bilirubin (TBIL), total protein (TP) and urea nitrogen (BUN). No significant differences were noted between the control and NM2CAB treated groups, indicating that NM2CAB did not adversely affect the function of systemic organs. Histological examination of tissue sections (liver, lung, intestine, spleen, kidney and brain) stained with H&E by a certified pathologist showed no abnormal pathology in NM2CAB treated animals. In addition, both strains of mice (NSG and BALB / cJ) tolerated the formulation well, and no injection site reactions and changes in behavior or movement were observed.

[0187] For the evaluation in rhesus monkeys, body weight was recorded before the formulation was applied, and plasma and peripheral blood mononuclear cells (PBMC) were collected for complete blood cell count and metabolic profile until the 393rd day after NM2CAB was applied. Systemic toxicity was evaluated by measuring hematological profiles (neutrophils, lymphocytes, monocytes) and metabolic profiles (ALT, alkaline phosphatase, BUN / creatinine). After injection, no weight change was recorded in any animal. The initial slight redness observed at the injection site in all animals was resolved before the 3rd day. No inflammation or bolus was noted 3 days after injection. Before and after NM2CAB was applied, neutrophils, lymphocytes and monocytes were counted; and blood cell counts were consistent. On the 1st day after injection, the increase in neutrophil counts was recorded, and neutrophil counts returned to normal in all animals within 2 weeks. Such changes may be related to injection, and may be unrelated to drugs. After treatment, there was no change in the metabolic profiles of liver and kidney in all animals. Overall, no adverse events were observed following NM2CAB administration.

[0188] Drug-drug interactions

[0189] Drug-drug interactions between two prodrug nanoformulations (NM2CAB and NM3PRV) of different classes of drugs (CAB (INSTI) and rilpivirine (RPV; NNRTI)) were evaluated. Due to the current combined clinical development of long-acting CAB and RPV nanoformulations, RPV is the drug of choice together with CAB. NM3RPV is a long-acting formulation of RPV (Hilaire et al., J. Control Release (2019) 311-312: 201-211). BALB / cJ mice were treated IM with a single dose of NM2CAB alone (45 mg / kg CAB equivalent), NM3RPV alone (45 mg / kg RPV equivalent), or co-administration of two prodrug nanoformulations (NM2CAB and NM3RPV, 45 mg / kg drug equivalent). Plasma levels of CAB and RPV were measured and no differences in active drug levels were observed between animals treated with either the individual or combined formulations, suggesting the utility of combinations of more than one prodrug nanoformulation for treatment or prevention.

[0190] In the absence of eradication of HIV infection, protective vaccines and pre-exposure prophylaxis (PrEP) and long-acting ART for people at risk are the only methods available for preventing disease and preventing new infections and viral transmission. For those at risk of infection, "treatment as prevention" highlights this point. The focus of developing LAARV injectables is to create drugs with long half-lives. For PrEP, LA ARVs need to prevent "coverage" of infection after HIV exposure. Viral infection needs to be stopped during the time period when protective ARV levels are detected in plasma. The drug must also be given without adverse side effects, including gastrointestinal toxicity and any significant drug-drug interactions. At present, LA agents have been welcomed by potential users due to the potential advantages of eliminating the stigma of viral infection and requiring less than daily dosing intervals (some are administered at a frequency of every 2 to 3 months). LA ARVs administered by subcutaneous or intramuscular routes are highly effective and have been shown to improve quality of life and life expectancy (May et al., AIDS (2014) 28: 1193-1202; Antiretroviral Therapy Cohort, Lancet (2017) HIV 4: e349-e356). They help avoid lack of compliance with medications, which remains a key treatment challenge (Shubber et al., PLoS Med. (2016) 13: e1002183; Osterberg et al., New Eng. J. Med. (2005) 353: 487-497). In fact, any poor compliance with ARV regimens that leads to treatment failure, drug-resistant mutations, and new viral transmission can be eliminated by improved compliance. In an attempt to improve existing LA ARV platforms, long-acting slow effective release antiretroviral therapy (LASER ART) prodrugs have been developed. These new formulations are used to reduce the frequency of injections while maintaining sustained therapeutic levels of ARVs over longer durations (Edagwa et al., Exp. Opinion Drug Del. (2017) 14: 1281-1291). LASER ART includes prodrug synthesis by chemical modification of existing ARVs to provide slow natural drug dissolution, poor water solubility, enhanced penetration through cell biofilms and tissue reservoirs, and limited systemic off-target toxicity. Prodrug synthesis allows the formation of ARV nanocrystals stabilized by polymer excipients.The PK and efficacy (PD) of LASER ART formulations have been demonstrated in a range of ARVs, including but not limited to dolutegravir (DTG), CAB, abacavir (ABC), lamivudine (3TC), and emtricitabine (FTC) (Zhou et al., Biomaterials (2018) 151:53-65; Hilaire et al., J. Control Release (2019) 311-312:201-211; Ibrahim et al., Int. J. Nanomed. (2019) 14:6231-6247; Lin et al., Chem. Commun. (2018) 54:8371-8374; McMillan et al., Antimicrob. Agents Chemother. (2018) 62: e01316-17; McMillan et al., AIDS (2019) 33: 585-588; Sillman et al., Nat. Commun. (2018) 9: 443; Smith et al., Biomaterials (2019) 223: 119476; Soni et al., Biomaterials (2019) 222: 119441).

[0191] CAB is an HIV-1 integrase strand transfer inhibitor (INSTI) and is currently being developed as both oral and LA injectables (McPherson et al., Expert Opin. Investig. Drugs (2018) 27: 413-420). Its unique intrinsic properties, such as hydrophobic nature, long systemic half-life (about 40 hours after oral administration), high potency, resistant profile, low daily oral dosing requirements (≤30 mg / day) and limited drug-drug interactions, make it an attractive candidate for development into LA injectables (Trezza et al., Curr. Opin. HIV AIDS (2015) 10: 239-245). Herein, it is demonstrated that a single injection of NM2CAB produces unexpectedly superior improvements in drug persistence, which is reflected by sustained drug plasma concentrations and tissue biodistribution compared to NCAB, NMCAB or NM3CAB. NM2CAB provided sustained plasma decay while maintaining drug levels above a PA-IC of 166 ng / mL for 364 days after a single injection 90 .

[0192] CAB LA, which is currently close to clinical approval, has been extensively studied in rhesus monkeys, confirming its ability to be used as an ARV injectable for pre-exposure prophylaxis (PrEP) research (Edagwa et al., Exp. Opin. Drug Del. (2017) 14: 1281-1291; Stellbrink et al., Curr. Opin. HIV AIDS (2018) 13: 334-340). These studies have demonstrated that CAB LA provides a high degree of protection against vaginal, rectal, intravenous and penile SHIV strain challenges, which supports its future use as PrEP for those at high risk of HIV exposure and intravenous drug users. Higher than 3×PA-IC 90 Plasma levels of 100% protection against viral challenge and were higher than those of PA-IC 90 Concentrations of 500 mg / kg provided 97% protection against viral challenge (Edagwa et al., Exp. Opin. Drug Del. (2017) 14: 1281-1291; Stellbrink et al., Curr. Opin. HIV AIDS (2018) 13: 334-340). Herein, NM2CAB administration produced a higher level of protection than PA-IC 90 Plasma CAB concentrations were sustained for more than 6 months, indicating its superiority and clinical efficacy as PrEP.

[0193] In summary, the development of effective antiretroviral (ARV) treatment and prevention measures for HIV-1 infected patients has transformed HIV infection from an inevitable death to a manageable lifelong chronic disease. The need to control the HIV-1 epidemic remains, as an estimated 37.9 million people are infected worldwide. The advent of LA ARVs will undoubtedly expand the options for overcoming the challenges of suboptimal medication adherence and reduce the burden of HIV infection. To date, chemoprevention of HIV antiretroviral agents has been best demonstrated using compounds containing tenofovir disoproxil fumarate (TDF). However, the level of compliance required for daily or near-daily oral tablets has proven to be challenging. LA products provide more options for achieving prevention, with the understanding that safety, tolerability, and efficacy will continue to be part of the evaluation of treatment outcomes. LA ARVs that can be administered monthly or less frequently will improve treatment adherence and extend the opportunity for PrEP.

[0194] In the previous specification, many publications and patent documents are cited in order to describe the prior art in the field to which the present invention belongs. The entire disclosure of each of these citations is incorporated herein by reference.

[0195] Although certain preferred embodiments of the present invention have been described and specifically illustrated above, the present invention is not intended to be limited to these embodiments. Various modifications may be made to these embodiments without departing from the scope and spirit of the present invention as set forth in the appended claims.

[0196] This article also provides the following items:

[0197] 1. A prodrug of an integrase inhibitor or a pharmaceutically acceptable salt thereof, wherein the prodrug comprises an ester containing an aliphatic group or a hydrocarbon group conjugated to the integrase inhibitor.

[0198] 2. The prodrug according to item 1, wherein the integrase inhibitor is selected from the group consisting of cabotegravir (CAB), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), bictegravir (BIC), BI 224436 and MK-2048.

[0199] 3. The prodrug according to item 1, wherein the integrase inhibitor is selected from the group consisting of cabotegravir (CAB), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG) and bictegravir (BIC).

[0200] 4. The prodrug or a pharmaceutically acceptable salt thereof according to item 1, wherein the prodrug is selected from the group consisting of:

[0201]

[0202]

[0203] wherein R is an optionally substituted aliphatic or hydrocarbyl group.

[0204] 5. The prodrug according to item 4, wherein R is a hydrocarbon chain of a fatty acid.

[0205] 6. The prodrug according to item 4, wherein R has a main chain of at least 15 carbons.

[0206] 7. The prodrug according to item 4, wherein R is a saturated linear aliphatic chain of 15 to 19 carbons in length.

[0207] 8. The prodrug according to item 4, wherein R is a saturated linear aliphatic chain of 15 to 17 carbons in length.

[0208] 9. The prodrug according to item 4, wherein R is a saturated linear aliphatic chain with a length of 17 carbons.

[0209] 10. The prodrug or a pharmaceutically acceptable salt thereof according to item 1, wherein the prodrug is

[0210]

[0211] 11. A nanoparticle comprising at least one prodrug according to any one of items 1 to 10 and at least one polymer or surfactant.

[0212] 12. The nanoparticle according to item 11, wherein the prodrug is crystalline.

[0213] 13. The nanoparticle according to item 11, wherein the polymer or surfactant is an amphiphilic block copolymer.

[0214] 14. The nanoparticle according to item 13, wherein the amphiphilic block copolymer comprises at least one poly(oxyethylene) block and at least one poly(oxypropylene) block.

[0215] 15. The nanoparticle according to item 11, wherein the polymer or surfactant is P407.

[0216] 16. The nanoparticle according to item 11, wherein the nanoparticle further comprises a polymer or surfactant attached to at least one targeting ligand.

[0217] 17. The nanoparticle according to item 11, wherein the diameter of the nanoparticle is about 100 nm to 1 μm.

[0218] 18. A composition comprising at least one nanoparticle according to item 11 and at least one pharmaceutically acceptable carrier.

[0219] 19. A composition comprising at least one prodrug according to any one of items 1 to 10 and at least one pharmaceutically acceptable carrier.

[0220] 20. A method for treating, inhibiting and / or preventing a disease or disorder in a subject in need thereof, the method comprising administering the prodrug of item 1 or the nanoparticle of item 11 to the subject.

[0221] 21. The method according to item 20, wherein the disease or disorder is a viral infection.

[0222] 22. The method of claim 21, wherein the disease or disorder is a retroviral infection.

[0223] 23. The method according to item 21, wherein the viral infection is HIV infection.

[0224] 24. The prodrug according to item 1 or the nanoparticle according to item 11 for use in treating a disease or disorder.

Claims

1. A compound as follows: or a pharmaceutically acceptable salt thereof, wherein R is a saturated linear aliphatic chain of 17 carbons in length.

2. A nanoparticle comprising the compound according to claim 1 and a polymer or a surfactant, wherein the polymer or the surfactant is P407.

3. The nanoparticle of claim 2, wherein the compound is crystalline. The nanoparticle according to claim 2 , wherein the diameter of the nanoparticle is 100 nm to 1 μm.

5. A composition comprising at least one nanoparticle according to any one of claims 2 to 4 and at least one pharmaceutically acceptable carrier.

6. A composition comprising the compound according to claim 1 and at least one pharmaceutically acceptable carrier.

7. Use of the compound according to claim 1, the nanoparticle according to any one of claims 2-4, or the composition according to claim 5 or 6 in the preparation of a medicament for treating HIV infection in a subject in need thereof.

8. The use according to claim 7, wherein when the medicament is used, the compound, the nanoparticle or the composition is administered to the subject via injection.

9. The use according to any one of claims 7-8, wherein when the medicament is used, the compound, the nanoparticle or the composition is administered to the subject once within a period of 3 months, 6 months, 9 months, 12 months, 18 months or 24 months.

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