EA046194B1 - ANTIVIRAL PRODRUGS AND THEIR NANO COMPOUNDS - Google Patents

Description

This invention claims priority under 35 U.S.C. §119(e). (U.S.C.) Provisional U.S. Patent Application No. 62/748,798, filed October 22, 2018. The above application is incorporated herein by reference.

The present invention was carried out with government support under grant numbers R01 МН104147, Р01 DA028555, R01 NS036126, Р01 NS031492, R01 NS034239, Р01 МН064570, P30 МН062261, P30 AI078498, R01 AG043540 and R56 AI138613 from the National Institutes of Health. The Government has certain rights to the present invention.

Field of technology to which the present invention relates

The present invention generally relates to the delivery of therapeutic agents. More specifically, the present invention relates to compositions and methods for delivering therapeutic agents to a patient for the treatment of a disease or disorder.

BACKGROUND OF THE INVENTION

Significant progress has been made in developing effective diagnostics and therapeutic agents against human immunodeficiency virus type 1 (HIV-1). Antiretroviral therapy (ART) has significantly reduced the incidence of disease-associated clinical manifestations and mortality, resulting in an almost normal quality of life in infected people (Vittinghoff, et al. (1999), J. Infect Dis., 179(3):717-720; Lewden, et al (2007), J. Acquir. Immune Defic. Syndr., 46(l):72-77). However, ART requires lifelong treatment to suppress viral replication and prevent the onset of AIDS. Moreover, the effectiveness of ART may be hampered by HIV-1 resistance, drug toxicity, and poor patient compliance (Wensing, et al. (2014), Top. Antivir. Med., 22(3):642-650; Siliciano, et al. (2013), Cur Opin. Virol., 3(5):487-494; Prosperi, et al. (2012), BMC Infect. Dis., 12:296-296; Van den Berk, et al. (2016), abstract number 948, 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, comorbid psychiatric symptoms, and/or substance abuse may result in failure to adhere to critical ART regimens (Tucker, et al. (2017), EBioMedicine, 17:163-171).

Long-acting antiretroviral drugs (LAP) are characterized by improved adherence to treatment (Spreen, et al. (2013), Curr. Opin. HIV AIDS, 8(6):565-571). Reducing the treatment regimen from daily to monthly or even less frequent administration allows for greater patient privacy and satisfaction, and improves 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):18071813) . However, only a few antiretroviral drugs have been successfully reformulated into LAPs.

Cabotegravir (CAB) is an integrase inhibitor or integrase strand transfer inhibitor (INSTI) with low aqueous solubility, high melting point, high activity, 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):239245). These properties allow the CAB to be formulated into a 200 mg/mL suspension (LAP CAB) for monthly or even less frequent intramuscular administration (Margolis, et al. (2017), Lancet, 390(10101):1499-1510; Spreen, W.W. (2014), J. Acquir. Immune Defic. Syndr., 67(5):481-486). Notably, CAB plus rilpivirine (RPV) represents the first long-acting ART combination regimen in which monthly or bimonthly LAP formulations of CAB and RPV demonstrated antiretroviral activity comparable to daily orally administered three-drug combinations in the maintenance setting. therapy (Margolis, et al. (2017), Lancet, 390(10101):1499-1510).

CAB is primarily metabolized by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and has 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). LAP CAB provides high protection against rectal, vaginal and intravenous transmission of simian/human immunodeficiency virus (SHIV) 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 LAP CAB dosing regimen has limitations. In particular, split injections are required, which are performed in volumes of 2 ml, which leads to discontinuation of treatment due to intolerable reactions at the injection site (Margolis, et al. (2017), Lancet 390(10101):1499-510; Markowitz, et al. (2017), Lancet HIV, 4(8):331-340). Moreover, the maximum interval between dosing is only 8 weeks. Administration of LAP CAB every 12 weeks was recently studied to maintain plasma CAB concentrations above 4 times the concentration providing 90% inhibition.

- 1046194 and adjusted for protein binding (4xPA-1C ₉₀ , 660 ng/ml), a concentration that has been demonstrated to provide protection against emerging infections 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 participants had faster than expected drug absorption, resulting in plasma drug concentrations that were below the target effective concentration of 4xPA-1C ₉₀ at 12 weeks. Thus, there is a significant need for methods to extend dosing intervals beyond 8 weeks and reduce injection volumes to improve adherence (Boyd, et al. (2017), Lancet, 390(10101):1468-1470).

Summary of the present invention

In accordance with the present invention, prodrugs of integrase inhibitors are provided. In a specific embodiment, the prodrug comprises an integrase inhibitor modified with an ester containing an aliphatic or alkyl group (eg, an aliphatic or alkyl group containing from about 3 to about 30 carbons). In a particular embodiment, the aliphatic or alkyl group is a fatty acid alkyl chain or a saturated linear aliphatic chain, optionally substituted with at least one heteroatom. 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). Also included in the present invention are compositions containing at least one prodrug of the present invention and at least one pharmaceutically acceptable carrier.

In accordance with yet another aspect of the present invention, nanoparticles are provided comprising at least one prodrug of the present invention and at least one polymer or surfactant. In accordance with a particular embodiment, the prodrug is crystalline. In a specific embodiment, the polymer or surfactant is an amphiphilic block copolymer, such as an amphiphilic block copolymer containing at least one poly(oxyethylene) block and at least one poly(oxypropylene) block (e.g., poloxamer 407 ). The nanoparticle may contain a polymer or surfactant bound to at least one targeting ligand. A single nanoparticle may contain targeting and non-targeting surfactants. In accordance with a specific embodiment, the nanoparticles have a diameter ranging from approximately 100 nm to 1 μm. Also included in the present invention are compositions comprising at least one nanoparticle of the present invention and at least one pharmaceutically acceptable carrier.

In accordance with another aspect of the present invention, methods are provided for treating, suppressing and/or preventing a disease or disorder in a subject in need thereof. The methods involve administering to a subject at least one prodrug or nanoparticle of the present invention, optionally in a composition containing a pharmaceutically acceptable carrier. In a particular embodiment, the disease or disorder is a viral infection (eg, a retroviral infection). In accordance with a particular embodiment, the method further comprises administering at least one additional therapeutic agent or therapy for the disease or disorder, such as at least one additional anti-HIV compound.

Brief description of drawings

In fig. Figure 1A shows images of NM2CAB (top) and NCAB (bottom) nanoparticles, which were determined using scanning electron microscopy.

In fig. Figure 1B shows graphs of the stability of NM2CAB nanoparticles at 25°C for the indicated periods of time. Specifically, the zeta potential (top), polydispersity coefficient (middle), and particle diameter (bottom) for the nanoparticles were determined using dynamic light scattering.

In fig. 2A is a graph (top) of drug uptake as measured by UPLC-UV/Vis by human monocyte-derived macrophages (MDM) and a graph (bottom) of drug retention MDMs collected at the indicated time points to analyze intracellular drug levels.

In fig. 2B is a graph (top) of HIV-1 reverse transcriptase activity at the indicated drug concentration and a graph (bottom) of HIV-1 reverse transcriptase activity in MDMs treated with drug, challenged with HIV-1ADA, and measured at the indicated time points after treatment.

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In fig. Figure 2C shows images of p24-stained MDM cells after challenge.

In fig. 3A is a graph of plasma CAB levels following a single intramuscular (IM) dose of NCAB, NMCAB, or NM2CAB in female NSG (NOD scid gamma mouse) mice. The dose administered was 45 mg CAB equivalents (eq)/kg. The upper dashed bold line indicates the plasma 4xPA-IC ₉₀ CAB value of 664 ng/ml, and the lower dashed line indicates the plasma 1xPA-IC ₉₀ CAB value of 166 ng/ml.

In fig. 3B is a graph of plasma CAB levels following a single intramuscular (IM) dose of NM3CAB in female NSG (NOD scid gamma mouse) mice. The dose administered was 45 mg CAB equivalents (eq)/kg. The upper dashed bold line indicates the plasma 4xPA-IC ₉₀ CAB value of 664 ng/ml, and the lower dashed line indicates the plasma 1xPA-IC ₉₀ CAB value of 166 ng/ml.

In fig. Figure 4 shows images of the antiretroviral dose-dependent effect of NM2CAB at a concentration of 10, 50 or 100 μM, obtained using immunocytochemical staining for the HIV-1 p24 antigen.

In fig. Figure 5 shows graphs of prodrug levels. The biodistribution of prodrugs (MCAB or M2CAB) in tissues and blood was assessed 14, 28, 42 and 364 days after a single IM injection of NMCAB or NM2CAB. Prodrug levels were measured in the spleen, liver, intestine, lung, brain, rectal tissue, kidney, anatomically associated lymph node tissue, and blood. Prodrug levels were determined using LC-MS/MS (liquid chromatography tandem mass spectrometry). Data are expressed as mean ± SEM. For the day 14, 28 and 42 groups, the numbers of animals in each group were N=5, and for the day 364 group, the numbers of animals were N=3 (NMCAB), N=4 (NM2CAB). One-way ANOVA followed by Tukey's post hoc test was used to compare tissue drug levels between the three treatments (*P<0.05, **P<0.01, ***P<0.001).

In fig. 6A and 6B are plots of the pharmacokinetic parameters and biodistribution of NM2CAB in rhesus monkeys. Four rhesus monkeys were dosed with 45 mg/kg CAB equivalent of NM2CAB via a single IM injection. Blood plasma samples were collected and analyzed for CAB (upper lines) and M2CAB (lower lines) levels up to day 393 (Fig. 6A). Biopsies of rectal, lymph node, and adipose tissue were collected on day 204 postdrug administration and analyzed for both CAB (top) and M2CAB (bottom) concentrations (Fig. 6B). Drug concentrations in both plasma and tissues were determined using LCMS/MS.

Detailed Description of the Present Invention

Maximum limitation of the viral load in the tissues of infected areas may facilitate viral eradication strategies. This can be achieved through the formation of active lipophilic and hydrophobic antiretroviral prodrug nanocrystals stabilized by surfactants. Hydrophobicity, drug hydrolysis rate, and antiretroviral activities must be balanced for optimal therapeutic effect. This paper demonstrates that manipulation of the hydrophobic and lipophilic carbon chain length of a prodrug can optimize therapeutic efficacy, particularly in the case of slow-release, long-acting antiretroviral therapy (LASER ART). LASER ART refers to a long-acting antiretroviral drug formed from prodrug nanocrystals with a lipid tail. A myristoylated CAB prodrug nanocrystal provides steady-state plasma concentrations of CAB in PA-IC90 for 4 months in rhesus monkeys after a single dose of 45 mg/kg CAB-equivalent administered via intramuscular injection. We show here that additional chemical modifications, unexpectedly, serve to enhance the lipophilicity and hydrophobicity of CAB, improve drug potency, and delay prodrug hydrolysis, thereby significantly extending the half-life of the parent drug. New prodrugs of cabotegravir (M2CAB) enhance drug encapsulation with appropriate excipients and stabilizers such as poloxamer 407 (P407).

M2CAB nanoformulations (NM2CAB) provide sustained drug release and site-specific delivery of antiretroviral drug. Prodrugs contain the native drug conjugated to hydrophobic moieties through hydrolyzable covalent bonds. NM2CAB nanoformulations were rapidly taken up by human monocyte-derived macrophages (MDMs) with sustained drug retention for 30 days in vitro; while nanoformulated parent drug (NCAB) or first-generation myristoylated cabotegravir (NMCAB) demonstrated HIV-1 breakthrough in MDM within one or 20 days of treatment, respectively. Notably, MDMs treated with NM2CAB exhibited robust antiretroviral activities after challenge

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HIV-1 for a period of up to 30 days after a single treatment with the drug. HIV-1 p24 was undetectable in the NM2CAB-treated group when measured on day 5 and at subsequent time points in 5-day increments over a period of up to 30 days or longer. Additionally, a single intramuscular (IM) injection of NM2CAB at a dose of 45 mg CAB-eq/kg in female NSG (NOD scid gamma mouse) mice demonstrated zero-order controlled release kinetics for active CAB and provided drug levels approximately equal to or greater than 4- multiples of PA-IC ₉₀ , for more than 5 months. The NM2CAB nanoformulations presented herein provide improvements to current combination ART regimens that require multiple daily dosings by reducing the number of drug dosage units a patient takes, reducing the risk of viral load rebound, limiting toxicities, and/or allowing drug entry into viral reservoirs. . Importantly, NM2CAB also provides a dosing interval of once every six months (or less frequently) to maximize the effectiveness of PrEP or treatment regimens.

Long-acting, sustained-release ART (LASER ART) formulations can prolong 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., Antimicrobial Agents Chemother (2018), 62:e01316-17). Also provided herein are novel integrase inhibitor prodrugs, sustained-release, sustained-release formulations thereof, and methods for their synthesis and use. Integrase inhibitors (integrase strand transfer inhibitors (INSTIs)) are a class of antiretroviral drugs designed to block the action of integrase (eg, HIV integrase), a viral enzyme that integrates the viral genome into the host cell's DNA. 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, USA). Hydrophobic and lipophilic prodrugs and their sustained release formulations exhibit increased potency and efficacy, increased cell and tissue penetration, and extended half-lives compared to the parent integrase inhibitor. The prodrugs and their compositions according to the present invention, as well as combinations thereof, can be used in the control of viral (eg, retroviral) infections.

Agents for the treatment of viral infections, particularly HIV infections, that are currently available include viral entry inhibitors, nucleoside reverse transcriptase inhibitors, nucleotide reverse transcriptase inhibitors, integrase and protease inhibitors. Resistance is associated with the short half-life of the drug, the life cycle of the virus, and rapid mutations resulting in high genetic variability. Combination therapies, such as antiretroviral therapy (ART), which are considered drug cocktail therapies, have received particular attention. Beneficial effects include reduced viral persistence, limited toxicity, improved compliance with therapeutic regimens, and sustained antiretroviral efficacy. Combination therapies minimize potential drug resistance by inhibiting the replication of a virus (eg, HIV), thereby reducing the number of spontaneously arising resistant mutants. Treatment failure is due, in part, to the short half-life of the drug. Moreover, ineffectiveness may also be due, in part, to limited access of the drug to tissue and cellular viral reservoirs, thereby hindering attempts to eradicate the virus. In this regard, the development of platforms based on cell- and tissue-targeted prodrugs (nanoparticles) contained in nanoformulations is of significant interest for the control of viral (for example, HIV) infections. Pre-exposure prophylaxis (PrEP) is another strategy used in controlling the transmission of a virus (eg, HIV). For example, TRUVADA® (tenofovir/emtricitabine) has been approved for pre-exposure prophylaxis for HIV infection. In addition, the combination of lamivudine and zidovudine (COMBIVIR®) has been used as pre-exposure prophylaxis and post-exposure prophylaxis.

The prodrugs and nanoformulated prodrugs (nanoparticles) presented herein unexpectedly prolong drug half-life, increase hydrophobicity and lipophilicity, and improve antiretroviral efficacy. This will have a beneficial effect on people who must receive high doses daily or even multiple doses per day, since a lower dosage with less frequency of dosing will not only reduce side effects, but will also be convenient for patients. The prodrugs and nanoformulated prodrugs (nanoparticles) provided herein may also be used as post-exposure treatment and/or pre-exposure prophylaxis (eg, for people who are at high risk of HIV-1 infection). In other words, prodrugs and nanoparticles according to the present

- 4 046194 the invention, as well as a combination thereof, can be used to prevent a viral infection (eg, HIV infection) and/or treat or suppress an acute or chronic viral infection (eg, HIV infection). Although the prodrugs and nanoformulations of the present invention are generally described as anti-HIV agents, the prodrugs and nanoformulations of the present invention are also effective against other viral infections, including, but not limited to: retroviruses (e.g., lentiviruses), hepatitis B (HBV), hepatitis C virus (HCV) and human T-cell leukemia viruses (HTLV), especially retroviruses.

The present invention describes new, broad-spectrum active prodrugs with improved biological activity compared to the parent drugs. Methods are also provided for encapsulating prodrugs into effective, sustained release formulations for efficient intracellular and tissue delivery and extended drug half-lives. The long-acting, sustained-release (LASER) compositions described herein demonstrate enhanced activity and can be used as effective therapeutic or prophylactic interventions against viral infections (eg, retroviral infections).

The prodrugs of the present invention enable effective intracellular delivery of integrase inhibitors. Presented herein are prodrugs, which are derivatives of integrase inhibitors in which a chemical moiety, particularly an oxygen-containing moiety such as a hydroxyl group, has been replaced by an ester moiety containing a hydrophobic and lipophilic cleavable moiety (eg, therapeutic fatty alcohols). The hydrophobic and lipophilic cleavable moiety (eg, therapeutic fatty alcohols) may exhibit antiviral activity against enveloped viruses (Katz, et al., Ann. NY Acad. Sci. (1994), 724:472-88). Notably, synergistic interactions between therapeutic fatty alcohols and nucleoside analogues can significantly enhance the antiviral activity of nucleosides (Marcelletti, et al., Antiviral Res. (2002), 56:153-66).

As described herein, prodrugs may contain labile therapeutic fatty alcohols to improve drug activity, accelerate cell and tissue penetration, protein binding, and bioavailability. The hydrophobic nature of the synthesized prodrugs facilitates drug encapsulation into nanocrystals for sustained release and long-lasting action with improved biopharmaceutical properties. The nanoformulations of the present invention may consist of prodrug particles dispersed in sterile aqueous suspensions and stabilized by polymeric excipients, lipids and/or surfactants or polymers. Without being bound by theory, the drug release mechanism involves dissolution of the prodrug from the nanoparticle followed by efficient cleavage to form two bioactive agents, i.e. integrase inhibitor and broad-spectrum antiviral fatty alcohols.

The advantages of the system described herein include, but are not limited to, improved drug potency, bioavailability, and extended half-life for patient convenience. Indeed, the nanoformulations described in the present invention demonstrated a significant increase in drug uptake by monocyte-derived macrophages (MDM). The modified drug and nanoparticles also showed increased activity due to increased and long-lasting suppression of viral replication. Thus, the nanoformulations according to the present invention provide the ability to enhance antiviral activity and accelerate drug delivery to anatomical reservoirs of infection.

In accordance with the present invention, prodrugs of integrase inhibitors are provided. In accordance with a specific embodiment, the prodrug contains an integrase inhibitor in which a chemical moiety, such as a hydroxyl group, is replaced by an ester containing an optionally substituted aliphatic or alkyl group. In a particular embodiment, the ester contains a hydrocarbon chain, in particular a hydrocarbon chain of 16-20 carbon atoms in length or 18 carbon atoms in length (the numbering in this case includes the carbon in the C=O of the ester).

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:

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ralegravir (RAL).

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

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wherein R represents an optionally substituted aliphatic or alkyl group.

The aliphatic or alkyl group may be unsaturated or saturated and may be substituted with at least one heteroatom (eg, O, N, or S). In accordance with a particular embodiment, R may contain an aromatic moiety, which may be substituted with at least one heteroatom (eg, O, N or S).

In accordance with a specific embodiment, the alkyl or aliphatic group is hydrophobic. In accordance with a particular embodiment, R represents an optionally substituted hydrocarbon chain, in particular a saturated one. In accordance with a particular embodiment, R is a saturated linear aliphatic chain. In a particular embodiment, the alkyl or aliphatic group contains from about 3 to about 30 carbons (eg, in the backbone of the alkyl or aliphatic group), which may be substituted by at least one heteroatom (eg, O, N, or S). In accordance with a specific embodiment, R represents a C ₁₄ -C ₂₁ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₄ -C ₁₉ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₄ -C ₁₇ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₅ -C ₂₁ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₅ -C ₁₉ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₅ -C ₁₇ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₆ -C ₂₁ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₆ -C ₁₉ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₇ -C ₂₁ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In accordance with a specific embodiment, R represents a C ₁₇ -C ₁₉ unsaturated or saturated alkyl or aliphatic group, which may be substituted with at least one heteroatom (eg, O, N or S). In a particular embodiment, R is a C17 unsaturated or saturated alkyl or aliphatic group that may be substituted with at least one heteroatom (eg, O, N, or S).

In a particular embodiment, R is an alkyl chain

- 7 046194 fatty acid (saturated or unsaturated), in particular C16-C22 fatty acid, C16-C20 fatty acid, _C16 - _C18 fatty acid, C18-C22 fatty acid, _C18 - _C20 fatty acid or _C18 fatty acid (numbering in this case includes the carbon in the C=O ester).

In a specific embodiment, R is a saturated linear aliphatic chain or hydrocarbon chain of at least 14 carbons in length (e.g., 14-21 carbons in chain length, 14-19 carbons in chain length, 14-17 carbons in chain length, 15-21 carbons per chain, 15-19 carbons per chain long, 15-17 carbons per chain long, or 17 carbons per chain long). In a specific embodiment, R is a saturated linear aliphatic chain or hydrocarbon chain of 14, 15, 16, 17, 18, 19, 20 or 21 carbons in length, in particular 14, 15, 16, 17, 18 or 19 carbons in length, 15, 16 or 17 carbons long or 17 carbons long. In a particular embodiment, R is a saturated linear aliphatic chain or a 17 carbon hydrocarbon chain.

According to a specific embodiment, the prodrug of the present invention is:

(M2CAB) or a pharmaceutically acceptable salt thereof.

Also included in the present invention are nanoparticles (sometimes referred to herein as nanoformulations) containing a prodrug of the present invention. Nanoparticles can be used to deliver compounds into a cell or host (eg, in vitro or in vivo). In accordance with a specific embodiment, the nanoparticle is used to deliver an antiretroviral therapeutic agent to a subject. The nanoparticles of the present invention contain at least one prodrug and at least one surfactant or polymer. In a specific embodiment, the nanoparticles are characterized by a spectroscopy-determined surfactant/drug polymer ratio that supports optimal targeting of the drug-loaded nanoparticle for persistence in the macrophage depot. These nanoparticle components, along with other optional components, are described herein below.

Methods for synthesizing nanoparticles according to the present invention are known in the art. In accordance with a particular embodiment, the methods form nanoparticles containing a prodrug (eg, crystalline or amorphous) coated (either partially or completely) with a polymer and/or surfactant. Examples of synthesis methods include, but are not limited to, milling (eg, wet milling), homogenization (eg, high pressure homogenization), particle replication in non-wettable matrices (PRINT) technology, and/or ultrasonication techniques. For example, US Patent Application Publication No. 2013/0236553, incorporated herein by reference, discloses methods suitable for synthesizing nanoparticles of the present invention. In a particular embodiment, the polymers or surfactants are first chemically modified with targeting ligands and then applied directly or mixed with non-targeting polymers or surfactants in specific molar ratios to coat the surface of the prodrug suspensions. for example, through the use of a nanoparticle synthesis process (eg, a crystalline nanoparticle synthesis process) such as milling (eg, wet milling), homogenization (eg, high pressure homogenization), particle replication in non-wettable matrices (PRINT) technology, and/or ultrasonication techniques , thus obtaining targeted nanoformulations. Nanoparticles can be used with or without additional purification, although avoiding additional purification is desirable for faster production of nanoparticles. In accordance with a specific embodiment, the nanoparticles are synthesized using milling and/or homogenization. Targeted nanoparticles (eg, using ligands (optionally high molecular weight)) can be developed through either physical or chemical coating and/or surface binding of polymers or surfactants and/or drug nanosuspensions.

In a specific embodiment, nanoparticles of the present invention are synthesized by adding a prodrug (eg, crystals) to a polymer or surfactant solution and then forming the nanoparticles (eg, by wet milling or high pressure homogenization). The prodrug and polymer or surfactant solution may be shaken prior to wet milling or high pressure homogenization.

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Nanoparticles of the present invention can be used to deliver at least one prodrug of the present invention into a cell or subject (including non-human animals). The nanoparticles according to the present invention may further contain at least one other agent or compound, in particular a biologically active agent, in particular a therapeutic agent (eg an antiviral compound), or a diagnostic agent, in particular at least one antiviral or antiretroviral agent. In accordance with a specific embodiment, the nanoparticles according to the present invention contain at least two therapeutic agents, in particular, at least one is a prodrug according to the present invention. For example, the nanoparticle may contain a prodrug of the integrase inhibitor of the present invention and at least one other therapeutic agent (eg, an anti-HIV agent).

In accordance with a specific embodiment, the nanoparticles of the present invention are a submicron colloidal dispersion of nanosized prodrug crystals stabilized by polymers or surfactants (eg, surfactant-coated drug crystals; nanoformulation). In a specific embodiment, the prodrug may be crystalline (solids having the characteristics of crystals), amorphous, or it is a solid-state prodrug nanoparticle that is formed into a crystal that combines the drug and the polymer or surfactant. In accordance with a particular embodiment, the prodrug is crystalline. As used herein, the term crystalline refers to an ordered state (ie, other than amorphous) and/or a substance exhibiting long-range order in three dimensions. In accordance with a particular embodiment, the majority (eg, at least 50, 60, 70, 80, 90, 95% or more) of the prodrug and, optionally, the hydrophobic portion of the surfactant are crystalline.

In a specific embodiment, the nanoparticle of the present invention has a diameter of up to about 2 or 3 μm (e.g., z-average diameter), or its largest linear dimension, particularly up to about 1 μm (e.g., from about 100 nm to approximately 1 µm). For example, the diameter or largest linear dimension of a nanoparticle can be from about 50 to about 800 nm. In a specific embodiment, the diameter or largest linear dimension of the nanoparticle is from about 50 to about 750 nm, from about 50 to about 500 nm, from about 200 to about 500 nm, from about 200 to about 400 nm, or from about 250 to about 350 nm. For example, nanoparticles can be rod-shaped, elongated rod-shaped, irregular, or round. The nanoparticles of the present invention may be neutral or charged. Nanoparticles can be positively or negatively charged.

As stated above herein, the nanoparticles of the present invention contain at least one polymer or surfactant. Surfactant refers to a surfactant including substances commonly called wetting agents, detergents, dispersing agents or emulsifying agents. Surfactants are usually organic compounds that are amphiphilic.

Examples of polymers or surfactants include, but are not limited to, synthetic or natural phospholipids, PEGylated lipids (eg, PEGylated phospholipid), lipid derivatives, polysorbates, amphiphilic copolymers, amphiphilic block copolymers, poly(ethylene glycol) copolymers, and lactide-glycolide copolymers ( PEG-PLGA), derivatives thereof, ligand-conjugated derivatives and combinations thereof. The present invention can also use other polymers or surfactants and combinations thereof that can form stable nanosuspensions and/or can chemically/physically bind to targeting ligands for HIV-susceptible/HIV-infected CD4+ T cells, macrophages and dendritic cells. 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 lactide-glycolide copolymer (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); glyceryl esters, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, sorbitan esters, glycerol monostearate, polyethylene glycols, polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, arylalkyl polyether alcohols, polyoxyethylene-polyoxyethylene copolymers propylene, poloxamines, cellulose, methylcellulose, hydroxylmethylcellulose, hydroxypropylcellulose, guide

- 9 046194 roxypropyl methylcellulose, polysaccharides, starch and their derivatives, hydroxyethyl starch, polyvinyl alcohol (PVA), polyvinylpyrrolidone and a combination thereof); and 2) ionic surfactants (eg, phospholipids, amphiphilic lipids, 1,2-dialkylglycero-3-alkylphosphocholines, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn -glycero-3-phosphoethanolamine-Ncarboxy(polyethylene glycol) (DSPE-PEG), dimethylaminoethanecarbamoyl-cholesterol (DC-Chol), Na[1-(2,3-dioleoyloxy)propyl]-N, Natrimethylammonium (DOTAP), halides alkylpyridinium, quaternary ammonium compounds, lauryldimethylbenzylammonium, acylcarnitine hydrochlorides, dimethyldioctadecylammonium (DDAB), n-octylamines, oleylamines, benzalkonium, cetyltrimethylammonium, chitosan, chitosan salts, poly(ethylenimine) (PEI), poly(Ni-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, alginic acid salts, hyaluronic acid, hyaluronic acid salts, gelatins, dioctyl sodium sulfosuccinate, sodium carboxymethylcellulose, cellulose sulfate, dextran sulfate and carboxymethylcellulose, 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, their derivatives and combinations thereof).

The polymer or surfactant of the present invention may be charged or neutral. In accordance with a particular embodiment, the polymer or surfactant is neutral or negatively charged (eg, poloxamers, polysorbates, phospholipids and derivatives thereof).

In accordance with a specific embodiment, the polymer or surfactant is an amphiphilic block copolymer or a lipid derivative. According to a specific embodiment, the at least one polymer or surfactant in the nanoparticle is an amphiphilic block copolymer, in particular a copolymer comprising at least one poly(oxyethylene) block and at least one poly(oxypropylene) block . In accordance with a specific embodiment, the polymer or surfactant is an amphiphilic triblock copolymer. In a specific embodiment, the polymer or surfactant is an amphiphilic triblock copolymer comprising a central hydrophobic propylene glycol block flanked by two hydrophilic polyethylene glycol blocks. In accordance with a specific embodiment, the surfactant is poloxamer 407.

According to a specific embodiment, the amphiphilic block copolymer is a copolymer comprising at least one poly(oxyethylene) block and at least one poly(oxypropylene) block. In accordance with a specific embodiment, the amphiphilic block copolymer is a poloxamer. Examples of poloxamers include, but are not limited to, Pluronic® L31, L35, F38, L42, L43, L44, L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88 , L92, F98, L101, P103, P104, P105, F108, L121, L122, L123, F127, 10R5, 10R8, 12R3, 17R1, 17R2, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25 R5, 25R8, 31R1 , 31R2 and 31R4. In a specific embodiment, the poloxamer is poloxamer 407 (Pluronic® F127).

In accordance with a specific embodiment of the present invention, the polymer or surfactant is present in the nanoparticle and/or nanoparticle synthesis solution (as described herein) at a concentration ranging from about 0.0001 to about 10 or 15% by weight . In accordance with a specific embodiment, the concentration of polymer or surfactant is in the range of from about 0.01 to about 15%, from about 0.01 to about 10%, from about 0.1 to about 10%, or from about 0. 1 to approximately 6% by weight. In accordance with a specific embodiment, the nanoparticle contains at least about 50, 75, 80, 85, 90, 95, 97, 98, 99% or more by weight of the therapeutic agent (prodrug). In accordance with a specific embodiment, the nanoparticles are characterized by a specific 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, from about 20:6 to about 500:6, from about 50:6 to approximately 200:6 or approximately 100:6.

As discussed above herein, the polymer or surfactant of the present invention may be associated with a targeting ligand. Targeting nanoparticles (e.g., to a macrophage) may provide better targeting, reduced excretion rates, reduced toxicity, and extended half-life compared to unbound drug or untargeted nanoparticles. The targeting ligand is

- 10 046194 combat compound that specifically binds to a specific tissue type or cell type (eg, at a desired target:cell ratio). For example, a targeting ligand can be used to capture or bind to a surface marker or receptor of a target cell (eg, a macrophage), which may facilitate its uptake into the cell (eg, into a protected subcellular organelle that is free from metabolic degradation). In accordance with a specific embodiment, the targeting ligand is a ligand for a cell surface marker/receptor. The targeting ligand may be an antibody or antigen-binding fragment thereof that is immunologically specific for a cell surface marker (eg, protein or carbohydrate) predominantly or exclusively expressed on the target tissue or cell type. The targeting ligand can be linked to the polymer or surfactant directly or through a linker. Typically, a linker is a chemical moiety containing a covalent bond or chain of atoms that covalently attaches a ligand to a polymer or surfactant. The linker can be linked to any synthetically feasible position in the ligand and the polymer or surfactant. Exemplary linkers may contain at least one optionally substituted; saturated or unsaturated; a linear, branched or cyclic aliphatic group, an alkyl group or an optionally substituted aryl group. The linker may be a lower alkyl or an aliphatic group. The linker may also be a polypeptide (eg, a polypeptide of about 1 to about 10 amino acids, in particular about 1 to about 5). In accordance with a particular embodiment, the targeting moiety is coupled to one or both ends of the polymer or surfactant. The linker may be non-degradable and may be a covalent bond or any other chemical structure that cannot undergo significant or complete degradation in physiological environments or under physiological conditions.

The nanoparticles/nanoformulations of the present invention may contain targeting and/or non-targeting polymers or surfactants. In a specific embodiment, the molar ratio of targeting and non-targeting polymers or surfactants in the nanoparticles/nanoformulations of the present invention is from about 0.001 to 100%, from about 1 to about 99%, from about 5 to about 95% , from about 10 to about 90%, from about 25 to about 75%, from about 30 to about 60%, or about 40%. In a particular embodiment, the nanoparticle contains only targeting polymers or surfactants. In a specific embodiment, the nanoparticles/nanoformulations of the present invention comprise a folate-targeting polymer or surfactant and a non-targeting version of the polymer or surfactant. In accordance with a specific embodiment, the nanoparticles/nanoformulations of the present invention comprise folate-poloxamer 407 (FA-P407) and/or poloxamer 407.

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 accordance with a specific embodiment, the targeting ligand is a ligand for targeting macrophages. The targeted nanoformulations of the present invention may contain a targeting ligand to target the nanoparticles to druggable tissues and cells/tissue and cellular reservoirs of HIV (e.g., central nervous system, gut-associated lymphoid tissue (GALT), CD4+ T cells, macrophages, dendritic cells, etc.). Ligands for targeting macrophages 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 (eg, mannose), formyl peptide receptor (FPR) ligands (eg, 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 for CD4, CCR5, CXCR4, CD7, CD111, CD204, CD49a, CD29, CD19, CD20, CD22, CD171, CD33, Leis-Y, WT-1, R0R1, MUC16, MUC1, MUC4, estrogen receptor, transferrin receptors, EGF receptors (eg, HER2), folate receptor, VEGF receptor, FGF receptor, androgen receptor, NGR, integrins and GD2. In accordance with a specific embodiment, the targeting ligand is folic acid.

As discussed above herein, the nanoparticles of the present invention may contain an additional therapeutic agent. The present invention also includes therapeutic methods in which the prodrug and/or nanoparticles of the present invention are co-administered with another therapeutic agent. In accordance with a specific implementation option

- 11 046194 The therapeutic agent is hydrophobic, is a water-insoluble compound or a slightly water-soluble compound, in particular when included in a nanoparticle. For example, 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 aqueous environment in the pH range 0-14, preferably pH from 4 to 10, in particular at 20°C.

In accordance with a specific embodiment, the therapeutic agent is an antiviral or antiretroviral agent. An antiretroviral agent may be effective against or specific for lentiviruses. Lentiviruses 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 infectious anemia virus in horses (EIA). In accordance with a specific embodiment, the therapeutic agent is an anti-HIV agent. An anti-HIV compound, or anti-HIV agent, is a compound that inhibits HIV (eg, inhibits HIV replication and/or infection). Examples of anti-HIV agents include, but are not limited to:

(I) Nucleoside analogue reverse transcriptase inhibitors (NRTIs). NRTIs refer to nucleosides and nucleotides and their analogs that inhibit the activity of reverse transcriptase, in particular HIV-1 reverse transcriptase. NRTIs contain a sugar and a base. Examples of nucleoside analogue reverse transcriptase inhibitors include, but are not limited to, adefovir dipivoxil, adefovir, lamivudine, telbivudine, entecavir, tenofovir, stavudine, abacavir, didanosine, emtricitabine, zalcitabine, and zidovudine.

(II) Non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIs are allosteric inhibitors that bind reversibly at a site other than the substrate binding site on reverse transcriptase, particularly 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; Rescriptor®), efavirenz (EFV, DMP-266, SUSTIVA®), nevirapine (NVP, Viramune®), PNU-142721, capravirine (S -1153, AG-1549), emivirine (+)-calanolide A (NSC-675451) and B, etavirine (ETR, TMC-125, Intelence®), rilpivirine (RPV, TMC278, Edurant™), DAPY (TMC120), doravirine (Pifeltro™), BILR-355 BS, PHI-236 and PHI-443 (TMC-278).

(III) Protease inhibitors (PI). Protease inhibitors are inhibitors of viral protease, in particular HIV-1 protease. Examples of protease inhibitors include, but are not limited to, darunavir, amprenavir (141W94, AGENERASE®), tipranivir (PNU-140690, APTIVUS®), indinavir (MK-639; CRDQVAN®), saquinavir (INVIRASE®, FORTOVASE®), fosamprenavir (LEXIVA®), lopinavir (AVT-378), ritonavir (AVT-538, NORVIR®), atazanavir (REYATAZ®), nelfinavir (AG-1343, VIRACEPT®), lasinavir (BMS-234475/CGP-61755), BMS -2322623, GW-640385X (VX-385), AG-001859 and SM-309515.

(IV) Fusion or entry inhibitors. Fusion or entry inhibitors are compounds, such as peptides, that block HIV from entering a cell (eg, by binding to the HIV envelope protein and blocking the structural changes required for fusion of the virus with the host cell). Examples of fusion inhibitors include, but are not limited to, CCR5 receptor antagonists (eg, maraviroc (Selzentry®, Celsentri)), enfuvirtide (INN, FUZEON®), T-20 (DP-178, FUZEON®) and T-1249.

(V) Integrase inhibitors (other than the prodrug of the present invention). Integrase inhibitors are a class of antiretroviral drugs designed to block the action of integrase (eg, HIV integrase), a viral enzyme that integrates the viral genome into the host cell's DNA. Examples of integrase inhibitors include, but are not limited to, raltegravir, elvitegravir, GSK1265744 (cabotegravir), GSK1349572 (dolutegravir), GS-9883 (bictegravir) and MK-2048.

Anti-HIV compounds also include maturation inhibitors (eg, bevirimat). Maturation inhibitors are generally compounds that bind to HIV gag and disrupt its processing during viral maturation. Anti-HIV compounds also include HIV vaccines such as, but not limited to, ALVAC® HIV (vCP1521), AIDSVAX®B/E (gp120) and combinations thereof. Anti-HIV compounds also include anti-HIV antibodies (eg, anti-gp120 or anti-gp41 antibodies), particularly broad-spectrum neutralizing antibodies.

More than one anti-HIV agent can be used, particularly when the agents have different mechanisms of action (as outlined above). For example, anti-HIV agents that are not NNRTIs can be combined with NNRTI prodrugs according to the present invention. In a specific embodiment, the anti-HIV therapy is highly active antiretroviral therapy (HAART).

- 12 046194

The present invention includes compositions (eg, pharmaceutical compositions) containing at least one prodrug and/or nanoparticle according to the present invention and at least one pharmaceutically acceptable carrier. As stated above herein, a nanoparticle may contain more than one therapeutic agent. In accordance with 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 and second prodrugs are different. In a specific embodiment, the first prodrug is a prodrug of the present invention and the second prodrug is a non-nucleoside reverse transcriptase inhibitor (NNRTI) prodrug, particularly rilpivirine (RPV). The compositions (eg, pharmaceutical compositions) of the present invention may further contain other therapeutic agents (eg, other anti-HIV compounds (eg, those described herein)).

The present invention also includes methods for preventing, suppressing and/or treating a disease or disorder. The methods involve administering a prodrug and/or nanoparticle according to the present invention (optionally in a composition) to a subject in need thereof. In a particular embodiment, the disease or disorder is a viral infection (eg, a retroviral infection). Examples of viral infections include, but are not limited to, HIV, hepatitis B, hepatitis C and HTLV. In accordance with a specific embodiment, the viral infection is a retroviral or lentiviral infection, in particular an HIV infection (eg, HIV-1). The prodrugs and/or nanoparticles of the present invention (optionally in a composition) can be administered to an animal, particularly a mammal, more particularly a human, for the purpose of treating/suppressing/preventing a disease or disorder (eg, a retroviral infection such as HIV infection). The pharmaceutical compositions of the present invention may also contain at least one other therapeutic agent, such as an antiviral agent, in particular at least one anti-HIV compound/anti-HIV agent. An additional anti-HIV compound may also be administered in a separate pharmaceutical composition to the prodrug or compositions of the present invention. The pharmaceutical compositions can be administered simultaneously or at different points in time (eg, sequentially).

The dosage ranges for administering the prodrugs, nanoparticles, and/or compositions of the present invention are large enough to provide the desired effect (e.g., cure, ameliorate, treat, and/or prevent a disease or disorder (e.g., HIV infection), its symptoms (e.g., AIDS, ARC (AIDS-associated complex)) or predisposition to them). In accordance with a specific embodiment, the pharmaceutical composition of the present invention is administered to a subject in an amount of from about 5 μg/kg to about 500 mg/kg. In accordance with a specific embodiment, the pharmaceutical composition of the present invention is 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 accordance with a specific embodiment, the pharmaceutical composition of the present invention is administered to a subject in an amount of from about 0.5 to about 100 mg/kg, from about 10 to about 100 mg/kg, or from about 15 to about 50 mg/kg. The dosage should not be so large as to cause significant adverse side effects such as unwanted cross-reactions, anaphylactic reactions, etc. Typically, the dosage will vary with the age, condition, sex and severity of the disease of the patient, and can be determined by one skilled in the art. The dosage can be adjusted by a specific doctor in case of any contraindications.

The prodrugs and nanoparticles described herein will typically be administered to a patient in the form of a pharmaceutical composition. As used herein, the term patient refers to human subjects or animal subjects. These prodrugs and nanoparticles can be used for therapeutic purposes under the guidance of a physician.

Pharmaceutical compositions containing prodrugs and/or nanoparticles of the present invention can be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). For example, the complexes can be formulated with a suitable vehicle such as water, buffered saline, ethanol, polyol (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents, or the like. suitable mixtures, in particular an aqueous solution. The concentration of prodrugs and/or nanoparticles in the selected medium can be varied, and the medium can be selected based on the desired route of administration of the pharmaceutical composition. Unless any conventional medium or agent is incompatible with the nanoparticles to be administered, they are intended to be used in a pharmaceutical composition.

Dose and dosage schedule of prodrugs and/or nanoparticles according to the present invention, which

- 13 046194 which are suitable for administration to a particular patient can be determined by the physician based on the age, gender, weight, general health of the patient and the specific condition for which the nanoparticles are being administered and its severity. The physician may also take into account the route of administration, pharmaceutical carrier, and biological activity of the nanoparticle.

The selection of a suitable pharmaceutical composition will also depend on the chosen route of administration. For example, the nanoparticles of the present invention can be administered by direct injection or intravenously. In this case, the pharmaceutical composition contains a prodrug and/or a nanoparticle dispersed in a medium that is compatible with the injection site.

The prodrugs and/or nanoparticles of the present invention can be administered by any route. For example, the prodrugs and/or nanoparticles of the present invention can be administered, without limitation, parenterally, subcutaneously, orally, topically, pulmonaryly, rectally, vaginally, intravenously, intraperitoneally, intrathecally, intracerebrally, epidurally, intramuscularly, intradermally, or intracarotidly. In accordance with a specific embodiment, the prodrug and/or nanoparticle is administered parenterally. In a specific embodiment, the prodrug and/or nanoparticle is administered orally, intramuscularly, subcutaneously, or into the bloodstream (eg, intravenously). In accordance with a specific embodiment, the prodrug and/or nanoparticle is administered intramuscularly or subcutaneously. Pharmaceutical compositions for injection are known in the art. If injection is chosen as the route of administration of the prodrug and/or nanoparticle, steps must be taken to ensure that sufficient quantities of the molecules or cells reach their target cells to exhibit a biological effect. Oral dosage forms include, but are not limited to, tablets (e.g., coated and uncoated, chewable), gelatin capsules (e.g., soft or hard), lozenges, dragees, solutions, emulsions, suspensions, syrups, elixirs, powders/granules (eg resuspended or dispersible), chewing gums and effervescent tablets. Dosage forms for parenteral administration include, but are not limited to, solutions, emulsions, suspensions, dispersions, and powders/granules for resuspension. Topical dosage forms include, but are not limited to, creams, gels, ointments, balms, patches, and transdermal delivery systems.

Pharmaceutical compositions containing a prodrug and/or nanoparticle according to the present invention as an active ingredient in a uniform mixture with a pharmaceutically acceptable carrier can be prepared in accordance with conventional techniques for the preparation of pharmaceutical compositions. The carrier can take a wide range of forms depending on the form of the pharmaceutical composition desired for administration, for example, intravenous, oral, direct injection, intracranial and intravitreal.

The pharmaceutical composition of the present invention can be formulated in unit dosage form for ease of administration and uniformity of dosage. A unit dosage form as used herein refers to a physically discrete unit of a pharmaceutical composition suitable for a patient being treated. Each dose must contain an amount of active ingredient calculated to produce the desired effect, in combination with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. In accordance with a specific embodiment, the prodrugs and/or nanoparticles of the present invention, due to their long-acting therapeutic effect, can be administered once every 1-12 months or less frequently. For example, the nanoformulations of the present invention can be administered once every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, 24 months or less frequently. In accordance with a specific embodiment, the prodrugs and/or nanoparticles of the present invention are administered less than once every two months. According to a specific embodiment, the prodrugs and/or prodrug nanoformulations are administered once a month, once every two months, in particular 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 often.

Dosage units can be proportionally increased or decreased based on the patient's weight. Suitable concentrations for alleviating a particular pathological condition can be determined by calculations based on a concentration-dose curve, as is known in the art.

In accordance with the present invention, a suitable dosage unit for administering nanoparticles can be determined by assessing the toxicity of the molecules or cells in animal models. Various concentrations of nanoparticles in the pharmaceutical composition can be administered to mice, and minimum and maximum dosages can be determined based on the beneficial results and side effects observed as a result of treatment. The appropriate dosage unit can also be determined by assessing the effectiveness of nanoparticle treatments in combination with other standard drugs. Dosage units of nanoparticles can be determined alone or in combination with each therapeutic agent according to the observed effect.

- 14 046194

The pharmaceutical composition containing 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 usually depend on the patient's condition.

The present invention includes methods of treating a disease/disorder comprising administering to a subject in need thereof a pharmaceutical composition containing 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 a subject is treated through ex vivo therapy. Specifically, the method involves removing cells from a subject, exposing the cells/contacting the cells in vitro to nanoparticles of the present invention, and returning the cells to the subject. In accordance with a specific embodiment, the cells contain macrophages. Other methods of treating a disease or disorder can be combined with the methods of the present invention and may be co-administered with the pharmaceutical compositions of the present invention.

The present invention also includes delivery of a nanoparticle according to the present invention to a cell in vitro (eg, in culture). The nanoparticle can be delivered into the cell in at least one carrier.

Definitions

The following definitions are presented to facilitate understanding of the present invention.

Singular forms include corresponding plural forms unless the context clearly requires otherwise.

The term pharmaceutically acceptable indicates approval by a US federal or state government regulatory agency or presence in the US Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans.

The term 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 agent, bulking agent (eg lactose, mannitol), excipient, excipient or medium with which the active agent according to the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous solutions of dextrose and glycerol are preferably used as carriers, in particular for injection solutions. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A.R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and in Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

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, as compared to a drug, is chemically modified in such a way that the modification makes it less active, substantially inactive, or inactive compared to the drug. However, the chemical modification is such that the corresponding drug is formed due to metabolic or other biological processes, usually after administration of the prodrug.

As used herein, the term treatment refers to any type of treatment that has a beneficial effect on a patient affected by a disease, including improving the patient's condition (eg, one or more symptoms), delaying the progression of the condition, etc. In accordance with a particular embodiment, treatment of a retroviral infection results in at least suppression/reduction in the number of infected cells and/or detectable levels of virus.

As used herein, the term prevention refers to prophylactic treatment of a subject who is at risk of developing a condition (eg, HIV infection), resulting in a reduction in the likelihood that the subject will develop the condition.

A therapeutically effective amount of a compound or pharmaceutical composition refers to an amount effective to prevent, suppress, treat, or reduce the symptoms of a particular disorder or disease. Treating a microbial infection (eg, HIV infection) as used herein may refer to curing, reducing, and/or preventing a microbial infection, symptom(s) thereof, or predisposition thereto.

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, which can be used to treat a condition, disease

- 15 046194 or impairment or reduction of symptoms of a condition, disease or impairment.

As used herein, the term small molecule refers to a substance or compound that has a relatively low molecular weight (eg, less than 4000, less than 2000, in particular less than 1 kDa or 800 Da). Typically, small molecules are organic and are not proteins, polypeptides or nucleic acids, although they may be amino acids or dipeptides.

As used herein, the term antimicrobial means a substance that kills microorganisms or inhibits the growth of microorganisms such as bacteria, fungi, viruses or protozoa.

As used herein, the term antiviral agent refers to a substance that destroys a virus and/or inhibits the replication of a virus. For example, the antiviral agent may inhibit and/or prevent the production of viral particles, maturation of viral particles, virus attachment, virus uptake into cells, virus assembly, virus egress/budding, virus integration, etc.

As used herein, the term highly active antiretroviral therapy (HAART) refers to anti-HIV therapy with various combinations of therapeutic agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, HIV protease inhibitors and fusion inhibitors.

As used herein, the term amphiphilic refers to the ability to dissolve in both water and lipids/non-polar media. Typically, an amphiphilic compound contains a hydrophilic portion and a hydrophobic portion. The term hydrophobic denotes a preference for non-polar media (for example, a hydrophobic substance or moiety is more easily dissolved or wetted by non-polar solvents such as hydrocarbons than by water). Hydrophobic compounds are mostly insoluble in water. As used herein, the term hydrophilic means the ability to dissolve in water.

As used herein, the term polymer refers to molecules formed by the chemical combination of two or more repeating units or monomers. The term block copolymer, in its simplest sense, refers to conjugates of at least two distinct polymer segments, each polymer segment containing two or more adjacent units of the same species.

An antibody or antibody molecule is any immunoglobulin, including antibodies and fragments thereof (eg, scFv), that bind to a specific antigen. As used herein, an antibody or antibody molecule includes intact immunoglobulin molecules, immunologically active portions of immunoglobulin molecules, and fusion proteins with immunologically active portions of immunoglobulin molecules.

As used herein, the term immunologically specific refers to proteins/polypeptides, in particular antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not recognize or bind significantly to other molecules in a sample containing mixed population of antigenic biological molecules.

As used herein, the term targeting ligand refers to any compound that specifically binds to a particular tissue type or cell type, in particular, without significant binding to other tissue types 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.

The term aliphatic refers to a non-aromatic hydrocarbon moiety. Aliphatic compounds may be acyclic (eg, linear or branched) or cyclic moieties (eg, cycloalkyl) and may be saturated or unsaturated (eg, alkyl, alkenyl, and alkynyl). Aliphatic compounds may contain a predominantly carbon backbone (eg, from 1 to about 30 carbons) and may contain heteroatoms and/or substituents (see below).

The term alkyl as used herein includes saturated or unsaturated straight or branched chain hydrocarbons containing from 1 to about 30 carbons in the straight/main chain. The hydrocarbon chain in alkyl groups may be interrupted by one or more heteroatoms (for example, oxygen, nitrogen, or sulfur). The alkyl (or aliphatic) group may optionally be substituted (eg, less than about 8, less than about 6, or 1 to about 4 substituents).

The term lower alkyl or lower aliphatic group refers to an alkyl or aliphatic group, respectively, that contains from 1 to 3 carbons in the hydrocarbon chain. Alkyl or aliphatic substituents include, but are not limited to, alkyl (eg, lower alkyl), alkenyl, halogen (such as F, Cl, Br, I), haloalkyl (eg, CCl ₃ or CF ₃ ), alkoxy, alkylthio, hydroxy , methoxy, carboxyl, oxo, epoxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl

- 16 046194 (for example, 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 alkyl groups having at least about 5 carbons in the backbone are generally hydrophobic and lack large substitutions with hydrophilic substituents.

The term aryl, as used herein, refers to monocyclic and bicyclic aromatic groups containing 6-10 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. Aryl groups may optionally include one to three additional rings fused to a cycloalkyl ring or heterocyclic ring. Aryl groups may optionally be substituted on available carbon atoms, for example, with 1, 2 or 3 groups selected from hydrogen, halogen, alkyl, polyhaloalkyl, alkoxy, alkenyl, trifluoromethyl, trifluoromethoxy, alkynyl, aryl, heterocyclo, aralkyl, aryloxy, aryloxyalkyl, aralkoxy, arylthio, arylazo, heterocyclooxy, hydroxy, nitro, cyano, sulfonyl anion, amino or substituted amino. The aryl group may be heteroaryl.

The term heteroaryl refers to an optionally substituted mono-, di-, tri- or other polycyclic aromatic ring system that includes at least one and preferably from 1 to about 4 sulfur, oxygen or nitrogen heteroatoms as ring members. For example, heteroaryl groups can have from about 3 to about 50 carbon atoms (and all combinations and subcombinations of ranges and specific amounts of carbon atoms therein), with about 4 to about 10 carbon atoms being preferred.

The following examples provide illustrative methods for practicing the present embodiment and are not intended to limit the scope of the present invention in any way.

Examples

Example 1.

Maximum reduction of residual HIV-1 levels in drug-resistant tissue sites, which include the brain, lymph nodes, bone marrow, gut-associated lymphoid tissue, and genital tract, may be achieved through the development of reservoir-targeted long-acting drugs. actions. In addition to the beneficial effects of long dosing intervals, long-acting injectable drug formulations can be formulated using receptor-mediated processes to achieve improved cell targeting, extended drug half-life, and improved tissue biodistribution. CAB is a highly active viral integrase inhibitor and has been formulated as LAP (LAP CAB), which exhibits sustained drug plasma levels in humans following a single intramuscular dose. Long-acting injectable nanoformulations for rilpivirine and LAP CAB already provide once-monthly injection options 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 requirement for high doses and large injection volumes. For this purpose, slow effective release and sustained release antiretroviral therapy (LASER ART) agents have been developed by synthesizing lipophilic and hydrophobic prodrug nanocrystals that allow rapid drug penetration across physiological barriers. LASER ART maximizes the drug content with limited excipient use, while maintaining the possibility of proportional dose adjustment and long-term storage. Myristoylated prodrugs have been formulated with poloxamer surfactants. Improved potency, bioavailability, and tissue distribution of CAB were demonstrated by increasing drug lipophilicity, which maintained plasma CAB concentrations at PA-IC _90i levels for 4 months in rhesus monkeys following a single intramuscular injection of 45 mg/kg CAB-equivalent. . Here, improved prodrugs and nanoformulations have been synthesized that reduce dosing frequency while improving viral reservoir targeting and drug activity.

Disclosed herein is an active ester prodrug CAB that has physicochemical properties that permit use of the LASER ART formulation for infrequent administration, such as at dosing intervals of six to twelve months or less. Criteria evaluated in selecting the optimal CAB prodrug candidate included drug activity, lipophilicity profile, efficient in vivo conversion to CAB with minimal systemic circulation of the prodrug in the bloodstream, and sustained CAB concentrations four times the PA-IC90 over periods of six months or longer after

- 17 046194 single intramuscular injection of the prodrug composition. Surprisingly, the present invention demonstrates that changing the hydrocarbon chain length of a fatty acid ester prodrug significantly improves active drug release and retention. This culminated in the identification of M2CAB, a prodrug of CAB as an 18-carbon fatty acid ester, with unexpectedly superior controlled release kinetics of CAB compared to MCAB or other fatty acid hydrocarbon chain lengths.

Prodrugs of the present invention are derivatives of an integrase inhibitor, such as CAB, conjugated to hydrophobic cleavable moieties. Thus, the hydrophobic parent compound is converted into a more hydrophobic ester derivative. This is achieved through the attachment of a fatty acid moiety, which can improve drug protein binding and bioavailability. The ester linkage between the integrase inhibitor (eg, CAB) and the derivatizing moieties is prone to enzymatic or hydrolytic degradation. The mechanism of drug release from the particle involves dissolution of the prodrug from the excipient followed by efficient cleavage of the ester to form the active parent compound. The developed NM2CAB significantly improved drug absorption by MDM with sustained drug retention over the 30-day observation period; whereas NCAB or NMCAB formulations were released from macrophages after one day or 20 days of treatment, respectively. Similarly, MDM treated with NM2CAB exhibited increased and sustained antiretroviral activities compared with NCAB or NMCAB when challenged with HIV-1 for up to 30 days after a single drug treatment. HIV-1 p24 was not detected in the NM2CAB-treated group at any of these time points. Advantages of this system include unexpectedly improved bioavailability and extended half-life of the drug. NM2CAB's prolonged plasma and tissue concentrations of CAB demonstrate that an effective dosing interval of every six months can be achieved.

Synthesis of M2CAB.

The synthesis of M2CAB prodrugs was carried out as described:

Briefly, the preparation of the M2CAB prodrug was accomplished by 1) deprotonation of the phenolic functionality using a suitable base such as ^^diisopropylethylamine; and 2) reactions with either a carboxylic acid chloride or an activated carboxylic acid in an alkyl fatty acid.

Both stages 1) and 2) were carried out in one vessel. In particular, the hydroxyl group was deprotonated using an appropriate reagent. The alcohol anion then combines with a fatty acid chloride or activated carboxylic acid to form prodrugs. Examples of coupling reagents that can be used to activate a carboxylic acid include, but are not limited to, uranium salts, carbodiimide reagents, and phosphonium salts. An example of a base that can be used in the coupling reaction is, but is not limited to, diisopropylethylamine (DIEA). Examples of polar aprotic solvents that can be used include, but are not limited to, dimethylformamide (DMF), tetrahydrofuran (THF), and acetonitrile. The reagents were mixed at 0°C and gradually warmed to over 12-24 hours. The final compounds were purified by silica column chromatography and characterized by nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-tandem mass spectrometry.

Deprotonation of the hydroxyl group and binding to the fatty acid.

A solution of CAB (2 g, 4.93 mol, 1.0 eq.) in anhydrous dimethylformamide (20 ml) was cooled to 0° C. under argon. H^Diisopropylethylamine (1.7 mL, 9.86 mol, 2.0 eq.) was then added dropwise to the pre-cooled drug solution. Stearoyl chloride (3.3 mL, 9.86 mmol, 2.0 eq.) was then added to the deprotonated phenol solution. The mixture was gradually warmed to room temperature with stirring over 16 hours, concentrated and purified by flash chromatography eluting with an 80 to 90% EtOAc/Hex gradient to give the prodrug in 90% yield. ^{The 1} H-NMR spectrum of M2CAB shows the presence of a broad peak at 1.20-1.50 ppm and peaks corresponding to aliphatic protons on the fatty acid moiety.

Synthesis of the composition.

M2CAB nanocrystals were coated with poloxamer 407 (P407), 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene

- 18 046194 glycol)-2000 (DSPE-PEG) and/or polyvinyl alcohol (PVA). Nanocrystals can also be stabilized using surfactants in the form of polysorbate and polyethylene glycol. Based on proton NMR spectroscopy data, a drug to surfactant ratio of 100:6 by weight was used to produce nanoformulated M2CAB, MCAB, and CAB. Briefly, 1-5% (w/v) M2CAB, MCAB or CAB and 0.06-0.3% (w/v) P407 were mixed in endotoxin-free water. Premixed slurry formulations were prepared by wet grinding or high pressure homogenization at 20,000 psi. inch until the desired size and polydispersity index (PDI) have been achieved. The nanoformulations were characterized for particle size, polydispersity index (PDI), and zeta potential using dynamic light scattering (Figure 1). It was performed using the Malvern Zetasizer, Nano Series Nano-ZS instrument (Malvern Instruments Inc, Westboro, MA, USA). The morphology of the nanoparticles was determined using scanning electron microscopy (SEM). UPLC MS/MS (Ultra Performance Liquid Chromatography Tandem Mass Spectrometry) was used for drug quantification.

Uptake and retention in macrophages.

Human monocytes were obtained by leukapheresis from HIV-1/2 and hepatitis B seronegative donors and then purified by countercurrent elutriation centrifugation (Balkundi et al., Intl. J. Nanomed. (2011), 6:3393-3404; Nowacek et al., Nanomed (2009), 4(8):903-917). Human monocytes were seeded in a 12-well plate at a density of 1.0 x 10 ⁶ cells/well using DMEM supplemented with 10% heat-inactivated pooled human serum, 1% glutamine, 10 μg/ml ciprofloxacin and 50 μg/ml gentamicin. Cells were maintained at 37°C in a 5% CO2 incubator. After 7-10 days of differentiation in the presence of 1000 U/ml recombinant human macrophage colony-stimulating factor (MCSF), MDMs were treated with a number of nanoformulations and native drugs. Drug absorption was assessed by measuring intracellular drug concentrations at various time points after treatment. For drug retention studies, cells were treated for 8 days, then washed with PBS and maintained by replacing half the volume of medium every other day until harvested at the indicated time points. For both studies, attached MDMs were washed with PBS (3x1 ml), then scraped into 1 ml of fresh PBS and counted at the indicated time points using a Countess™ automated cell counter (Invitrogen, Carlsbad, CA, USA). Cells were pelleted by centrifugation at 950xg for 8 min at 4°C. The cell pellet was resuspended in 200 μl of HPLC grade methanol and probe sonicated followed by centrifugation at 20,000xg for 20 min. The supernatant was analyzed for drug content using HPLC.

As can be seen in FIG. 2A, NM2CAB was taken up by MDM at higher levels than NMCAB, with statistically significant differences. Moreover, MDMs retained NM2CAB at higher levels and for longer periods of time than NMCAB, with statistically significant differences (Figure 2A).

Antiretroviral activities.

Antiretroviral efficacy was determined by measurements of HIV reverse transcriptase (RT) activity (Fig. 2B and 2C). To assess antiretroviral efficacy, MDMs were treated with 100 μM of either CAB-LAP, NMCAB, or NM2CAB for 8 h. After treatment, cells were washed with PBS to remove excess unbound drug and nanoparticles and cells were cultured with fresh medium, replacing half the volume of medium every two times. days. MDM were challenged with HIV-TADA at an MOI of 0.01 infectious viral particles/cell for up to 30 days. The production of progeny virions was measured using RT activity in the culture medium (Kalter, et al. (1992), J. Clin. Microbiol., 30(4):993-995). The expression of HIV-1 p24 protein antigens was assessed (Guo, et al. (2014), J. Virol., 88(17):9504-9513). MDMs were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were blocked using 10% BSA containing 1% Triton X-100 in PBS for 30 min at room temperature. After blocking, cells were incubated with mouse monoclonal antibody to HIV-1 p24 (1:50; Dako, Carpinteria, CA, USA) overnight at 4°C, followed by incubation for 1 h at room temperature. HRP polymer-labeled anti-mouse secondary antibody (Dako EnVision® System) was added (one drop/well). Hematoxylin was added to counterstain the nuclei, and images were obtained using a Nikon TE300 microscope with a 20× objective. As can be seen in FIG. 2B and 2C, unexpectedly better antiretroviral efficacy was observed for nanoformulated M2CAB compared to nanoformulated CAB or MCAB.

- 19 046194

Pharmacokinetic/biodistribution study.

Female NSG mice were administered a single IM dose of 45 mg/kg CAB equivalent of NM2CAB, NMCAB, or NCAB. Plasma and tissue drug levels were analyzed by UPLC-MS/MS. Plasma drug levels were monitored weekly. A single IM injection of NM2CAB exhibits zero-order controlled release kinetics for active CAB, which is maintained at a concentration four times higher than PA-IC _90i compared to NMCAB or NCAB (Figure 3). At day 364 post-injection, plasma CAB levels were 345.2 ng/mL for NM2CAB, 8.5 ng/mL for NMCAB, and undetectable for NCAB (detection limit 0.5 ng/mL).

Mice were also given a single IM dose of 45 mg/kg CAB equivalent of NM3CAB (C22). Plasma drug levels were monitored weekly. At day 28 post-injection, plasma CAB levels were 233.2 ng/mL (Fig. 3B). Accordingly, it is clear that NM2CAB (C18) performed better in maintaining long-term effective drug release compared to the shorter NMCAB (C14) and the longer NM3CAB (C22) with statistical significance of the differences.

The hydrophobicity of CAB was significantly improved after derivatization into the prodrug M2CAB. The improved hydrophobicity of M2CAB facilitated the preparation of stable formulations with high drug capacity. Moreover, conversion of CAB to the more hydrophobic M2CAB and formation of nanoparticles significantly improved intracellular drug accumulation compared to nanoformulated CAB or MCAB. Significant improvements in MDM retention and antiretroviral efficacy were also observed for nanoformulated M2CAB compared to nanoformulated CAB or MCAB. A single IM injection of NM2CAB at a dose of 45 mg CAB-equivalents/kg into female NSG mice also unexpectedly demonstrated plasma CAB concentrations four times higher than PA-IC90 for more than five months, significantly longer than nanoformulated CAB or MCAB. .

Example 2.

Current antiretroviral (ARV) therapeutic regimens that are both active and well-tolerated provide 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 treatment adherence, which in turn is influenced by comorbidities, social stigma, behavior, concurrent illicit drug use, and costs (Fauci, et al., JAMA (2019) , 321:844-845). However, even strict adherence to daily drug doses often leads to drug toxicity, drug-drug interactions, and the emergence of viral drug resistance. All drug regimens do not eliminate the virus (Dash, et al., Nat. Commun. (2019), 10:2753). This highlights the fact that all therapies require lifelong adherence to sustained HIV-1 suppression and disease attenuation. This has led scientists to develop long-acting (LA) therapeutic approaches. They all focus on improving treatment adherence and drug potency (Gendelman, et al., Trends Microbiol. (2019), 27:593606). The two most active and approaching clinical approval by the US Food and Drug Administration are injectable LA ARV formulations, while implantable drug devices are still in development (Margolis, et al. , Lancet (2017), 390:1499-1510; Taylor, et al., Topics Antiviral Med. (2019), 27:50-68).

Studies to date have shown promise for an injectable LA formulation for widespread use in humans (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). As a result, injectable LA nanoformulations of cabotegravir (CAB) and rilpivirine (RPV) as a two-drug combination will receive approval for monthly dosing, likely by the end of 2019 (Margolis, et al., Lancet (2017), 390:1499- 1510; Taylor, et al., Topics Antiviral Med. (2019), 27:5068; Kerrigan, et al., PloS One (2018), 13:e0190487). Both Antiretroviral Therapy as Long-Acting Suppression (ATLAS) and First Long-Acting Injectable Regimen (FLAIR) strategies have demonstrated promising safety, efficacy, and tolerability (Taylor, et al. al., Topics Antiviral Med. (2019), 27:50-68). Combination treatment demonstrated noninferiority when compared with standard three oral drug regimens (Taylor, et al., Topics Antiviral Med. (2019), 27:50-68). Likewise, 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 many limitations, including in situ reactions.

- 20 046194 injections, large volumes injected, dose frequency, limited penetration of 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). Moreover, these newer therapeutic approaches require more frequent use of a healthcare provider, either to provide the injections themselves or to perform implant insertion and removal procedures. Results from measuring tissue and plasma drug levels correlate with LA ARV efficacy, which includes drug penetration into mucosal tissues, lymphoid tissues, and the central nervous system, as well as long-term safety. Based on the extent of the influence of these unknown factors, there is an urgent need for additional improvements in LA ARV regimens. Any future LA ARVs will need to be administered in reduced volumes without systemic toxicity. If this is achieved, ARV regimens may also provide properties similar to ARV vaccine mimetics. Success will prevent new infections and reduce new transmission, and such properties may provide a functional cure for HIV-1.

To this end, LA ARV libraries have been generated across the antiretroviral spectrum (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), 33:585588; Sillman, et al., Nat. Commun. (2018), 9:443; Smith, et al., Biomaterials (2019), 223:119476; Soni, et al., Biomaterials (2019), 222:119441). Herein, CAB prodrugs were designed to prolong the apparent half-life and antiretroviral activities while providing tight control of hydrolysis. The synthesis and comprehensive physicochemical characterization of three CAB prodrugs with 14-, 18-, and 22-carbon linker chains (MCAB, M2CAB, and M3CAB, respectively) are presented, with full evaluation of the corresponding nanoformulations with them (NMCAB, NM2CAB, and NM3CAB). These analyzes continued previous research on 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 monocyte-macrophages and demonstrated long-term protection against HIV-1 challenge when administered monthly. NM2CAB provided plasma CAB concentrations above the 90% protein binding inhibition concentration (PA-1C _eo ) of 166 ng/mL for 52 weeks. This correlated with significant levels of biodistribution in lymphoid, mucosal and intestinal tissues after a single parenteral injection. Systemic adverse events were not recorded. Concurrently measured plasma drug concentrations in drug-injected normal and immunodeficient mice and rhesus monkeys confirmed sustained drug release over long periods of time in prophylactic and therapeutic regimens. Taken together, the results indicate that prodrugs can be used for the same purpose as a prophylactic HIV-1 vaccine.

Materials and methods.

Reagents.

CAB was purchased from BOC sciences (Shirley, New York, USA). Pyridine, dimethylformamide (DMF), ^^diisopropylethylamine (DIEA), myristoyl chloride, stearoyl 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). Diethyl ether, ethyl acetate, hexanes, acetonitrile (ACN), methanol, LC-MS grade water, cell culture grade water (endotoxin free), gentamicin, monobasic potassium phosphate (KH2PO4), bovine serum albumin (BSA), Triton™ X-100, and TRIzol® reagent were purchased from Fisher Scientific (Hampton, NH, USA). Mouse monoclonal antibody to human HIV-1 p24 protein (clone Kal-1) and polymer-based HRP-conjugated secondary antibody EnVision™ ⁺ were purchased from Dako (Carpinteria, CA, USA). Heat-inactivated pooled human serum was purchased from Innovative Biologies (Herndon, VA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Corning Life Sciences (Tewksbury, MA, USA).

Synthesis and characterization of CAB prodrugs.

A series of three prodrugs were synthesized by esterification at the 10-hydroxyl group on CAB, yielding lipophilic prodrugs with 14-, 18-, and 22-carbon linkers, named MCAB, M2CAB, and M3CAB. CAB was first dried from anhydrous pyridine and then suspended in anhydrous DMF. The mixture was cooled to 0°C in an argon atmosphere. DIEA (2 equivalents) was used to deprotonate the 10-hydroxyl group of CAB, which was further reacted with 2 equiv. myristoyl chloride or stearoyl chloride for 24 hours to obtain MCAB or M2CAB, respectively. The synthesis of M3CAB was a two-step process. At the first stage, behenylchlo

- 21 046194 read was synthesized by chlorination of the carboxyl group of behenic acid in anhydrous chloroform using 4 eq. thionyl chloride. CAB, dried from anhydrous pyridine and resuspended in anhydrous DMF in the presence of 4 eq. triethylamine was further added to behenyl chloride in DMF at 0°C under argon followed by heating the reaction mixture to 50°C for 24 hours. All resulting prodrugs were purified by silica gel column chromatography using the original mobile phase in in the form of a mixture of ethyl acetate: hexanes in a ratio of 4:1 for the initial fractions, and then using a mixture of ethyl acetate hexanes in a ratio of 9:1 for the rest. Finally, the prodrugs were precipitated and washed in diethyl ether, dried in vacuum to obtain a white powder with an average yield of 85-95%. Successful synthesis of prodrugs was confirmed by nuclear magnetic resonance spectra of protons and carbon ( ^1H and ^13C NMR), which were recorded using a Bruker Avance-III HD instrument (Billerica, MA, USA), operating at 500 MHz, magnetic field strength 11.7 Tl. Fourier transform infrared (FT-IR) analysis was performed using a universal attenuated total internal reflection (UATR) spectroscopy method on a Spectrum Two (PerkinElmer, Waltham, MA, USA). Comparative crystallographic analyzes of CAB and prodrugs by powder X-ray diffraction (XRD) were carried out in the 2θ angle range of 2-70° at a rate of 1°/s using a PANalytical Empyrean diffractometer (PANalytical Inc., Westboro, MA, USA) using Cu-Κα radiation (1.5418 A) with voltage settings of 40 kV, current 45 mA. Molecular weight was determined by direct infusion into a Waters TQD mass spectrometer.

Quantification of CAB, MCAB, M2CAB and M3CAB by UPLC-UV/Vis.

A Waters ACQUITY H-Class ultra-performance liquid chromatography (UPLC) system with TUV detector and Empower 3 software (Milford, MA, USA) was used to measure drug concentrations. Samples CAB, MCAB, M2CAB and M3CAB were separated on a Phenomenex Kinetex 5 µm C18 column (150 x 4.6 mm) (Torrance, CA, USA). CAB was detected at 254 nm using a mobile phase consisting of 65% 50 mM potassium phosphate monobasic (KH ₂ PO ₄ ), pH3.2, and 35% acetonitrile (ACN) and a flow rate of 1.0 ml /min. MCAB, M2CAB and M3CAB were detected at a wavelength of 230 nm using a mobile phase consisting of 90% ACN and 10% water, 95% ACN and 5% water, 98% ACN and 2% water, respectively, and at a flow rate of 1.0 ml/min. Drug content was determined relative to the peak areas of drug standards (0.05-50 μg/ml) in methanol.

Measuring the solubility of a drug formulation in water.

The 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 obtain a saturated aqueous solution. The mixture was stirred for 24 hours at room temperature. Subsequently, the solution was centrifuged at 14,000 rpm for 10 min to precipitate the undissolved powder. The supernatant was collected, lyophilized, and dissolved in methanol for drug content analysis by UPLC UV/Vis.

Chemical stability.

The stability of MCAB, M2CAB and M3CAB was determined under acidic (pH 1), basic (pH 11) and neutral (pH 7) conditions at room temperature and elevated temperature (37°C). A stock solution of each prodrug was prepared in DMSO at a concentration of 1 mg/mL. For assays under acidic, basic, and neutral conditions, 100 μL of each prodrug stock solution 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 at 40°C with shaking (innova® 42 shaker, 150 rpm). Samples were removed after 0, 2, 4, 8 and 24 hours and stored at -80°C. The samples were then analyzed for drug content using UPLC-UV/Vis.

Kinetic characteristics of hydrolytic cleavage of a drug in blood plasma.

The hydrolysis kinetics of MCAB, M2CAB and M3CAB and the corresponding active drug release were determined in plasma from different species (mouse, rat, rabbit, monkey, dog and human). MCAB, M2CAB, or M3CAB (1 μM) were incubated in 100 μl of blood plasma at 37°C. At various time points, 1 mL of acidified methanol (0.1% formic acid and 25 mM ammonium formate to avoid additional hydrolysis of the prodrug) was added to each sample and vortexed for 3 min to stop the reaction. At time 0 min, a stock solution of the prodrug was added to 100 μl of ice-cold blood plasma, and 1 ml of ice-cold acidified methanol was immediately added. After addition of methanol, samples were centrifuged at 15,000xg for 10 min and the collected supernatant was analyzed for drug content using UPLC-MS/MS (Waters Xevo TQ-XS).

- 22 046194

Synthesis and characterization of nanoparticles.

Nanoformulations of parent CAB (NCAB) and its prodrugs (NMCAB, NM2CAB, and NM3CAB) were prepared by high-pressure homogenization using the surfactant poloxamer 407 (P407). Drug/prodrug and P407 were premixed (10:1 w/w) in endotoxin-free water for 24 h at 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. inch to obtain uniform nanocrystals with the desired particle size. Nanoparticles were characterized for particle size (Deff), polydispersity index (PDI) and zeta potential by dynamic light scattering (DLS) using a Malvern Nano-ZS instrument (Worcestershire, UK). The stability of the nanoformulations was monitored at temperatures of 4, 25 and 37°C for 3 months. The drug/prodrug content of the nanoformulation was measured by dissolving the nanoformulation in methanol (dilution range: 1000-10000) and analyzed by UPLC UV/Vis. The following equation was used to calculate encapsulation efficiency:

Encapsulation efficiency (%) = (weight of drug in the composition/initial weight of added drug) x 100.

The morphology of the nanoparticles was assessed using scanning electron microscopy (SEM). Nanoparticles were fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.2) at 4°C for 24 h and processed for visualization. Briefly, nanosuspensions were air-dried on a coverslip mounted on an SEM sample stage and sputter-coated with approximately 50 nm of gold/palladium alloy. Samples were analyzed using an FEI Quanta 200 scanning electron microscope (Hillsboro, OR, USA) operated at 5.0 kV (Sillman, et al., Nat. Commun. (2018), 9:443).

Human monocyte-derived macrophages (MDM).

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

Cytotoxicity assay.

Cell viability after treatment with nanoparticles was assessed by performing the MTT assay. Human MDMs seeded in 96-well plates at a density of 0.08 x 10 ⁶ cells per well were treated with 10, 25, 50, 100, 200, or 400 μM NCAB, NMCAB, NM2CAB, or NM3CAB for 24 h. Untreated cells were used as controls. For each group, samples were present in three parallels. Cells were washed with PBS and incubated with MTT solution (5 mg/ml) in an amount of 100 μl/well for 45 min at 37°C. After incubation, the MTT solution was removed and the cells were washed with PBS. 200 μL of DMSO was then added to each well and absorbance was measured at 490 nm on a Molecular Devices SpectraMax® M3 plate reader using SoftMax Pro 6.2 software (Sunnyvale, CA, USA).

Studies on the uptake, retention and release of drug particles from MDM.

MDM uptake and retention studies were carried out in clear flat-bottomed 12-well plates at a density of 1.0 x 10 ⁶ cells/well, with treatments in each group carried out in triplicate. For cellular uptake studies, MDMs were treated with 100 μM NCAB, NMCAB, NM2CAB, or NM3CAB. MDMs were collected at 2, 4, 8, and 24 h post-treatment to measure intracellular drug and prodrug levels. For retention studies, MDMs were treated with 100 μM NCAB, NMCAB, NM2CAB, or NM3CAB for 8 h and then washed twice with phosphate-buffered saline (PBS). Fresh culture medium was added and half the volume of medium was replaced every two days. MDMs were collected after 1, 5, 10, 15, 20, 25, and 30 days to analyze intracellular drug and prodrug concentrations. For both studies, at the initial time points, attached MDMs were washed twice with PBS. Cells were then scraped into PBS and counted using an Invitrogen Countess™ automated cell counter (Carlsbad, CA, USA). The cell suspension in PBS was centrifuged at 3000 rpm for 8 min at 4°C. The resulting cell pellets were sonicated in 200 μl of methanol using a sonication probe with ce

- 23 046194 pour extraction of intracellular drugs. The resulting samples were centrifuged at 20,000xg for 10 min at 4°C to separate cellular debris from the drug-containing supernatant. The samples were further analyzed for drug and prodrug content using UPLC-UV/Vis. For release studies, culture media at time points similar to those used in the retention study were collected to quantify drug and prodrug released by MDMs. The culture medium was mixed with methanol to precipitate insoluble components in the culture medium and to extract the drug and prodrug. The mixture was centrifuged at 17,000xg for 10 min at 4°C to separate the insoluble sediment. The supernatant was transferred to new tubes for drying in a centrifugal vacuum concentrator. The dried contents were suspended in methanol for further analysis by UPLC-UV/Vis.

Particle characterization using transmission electron microscopy (TEM).

MDMs were treated with NCAB, NMCAB, NM2CAB, or NM3CAB at 100 μM for 8 h and then washed twice with PBS. Fresh culture medium was added and half the volume of medium was replaced every two days. Supernatant fluids from the MDM culture were collected 0, 15, and 30 days after drug particle treatment and analyzed by TEM to visualize intracellular nanoparticles. On day 0, cells were harvested immediately after the 8 h treatment. At the indicated time points, cells were washed, scraped in PBS, pelleted at 3000 rpm for 8 min at room temperature, and fixed in 2% glutaraldehyde, 2% paraformaldehyde in 0 .1 M Sorensen's phosphate buffer (pH 6.2). A drop of the fixed cell suspension was placed on a 200 mesh formvar/silica copper grid and allowed to settle for 2 min. Excess solution was removed with a swab and the sample was allowed to dry. A drop of NanoVan vanadium negative stain was placed on the grid for 1 min, then swabbed away and the sample was allowed to dry. Grids were evaluated on a FEI Tecnai G2 Spirit TWIN transmission electron microscope (Hillsboro, OR, USA) operated at 80 kV. Images were acquired digitally using the AMT Digital Imaging System (Wooburn, MA, USA) (Sillman, et al., Nat. Commun. (2018), 9:443).

HIV-1 infection and measurement of reverse transcriptase (RT) activity in fluids from infected cells.

MDMs were seeded in clear flat-bottomed 24-well plates at a density of 0.8 x 106 cells/well. MDMs were treated with 100 μM NCAB, NMCAB, NM2CAB, or NM3CAB for 8 h. After treatment, cells were washed with PBS and cultured in fresh culture medium, replacing half the volume of medium every two days. At 1, 5, 10, 15, 20, 25, and 30 days after treatment, cells were infected with HIV-IADA (a virus strain exhibiting tropism for macrophages) at a multiplicity of infection (MOI) of 0.1 infectious particles per cell for 16 h. After the infection period, MDMs were washed with PBS and replenished with fresh medium. The cells were cultured for the next ten days, replacing half the volume of medium every two days and completely replacing the medium on the 8th day. Culture medium was collected on day 10 postinfection to measure HIV-1 RT activity (Kalter, et al., J. Immunol. (1991), 146:3396-3404; Nowacek, et al., J. Neuroimmune Pharm. ( 2010), 5:592-601). The infection rate was expressed as a percentage of RT activity in infected MDMs that were not treated. Cells were fixed in 2% PFA and HIV-1 p24 antigen expression was determined by immunocytochemical staining (Nowacek, et al., J. Neuroimmune Pharm. (2010), 5:592-601).

Analysis of half-maximal effective concentration (EC ₅₀ ).

MDMs were seeded in clear flat-bottomed 96-well plates (0.08 x 106 cells/well). Cells were treated with drug concentrations ranging from 0.01–1000 nM CAB, MCAB, M2CAB, M3CAB, NCAB, NMCAB, NM2CAB, or NM3CAB for 1 h before infection with HIV-Iada (MOI of 0.1 infectious particles per cell) at for 4 hours. After 4 hours of control infection with the virus, the cells were washed and fresh medium containing the drug (0.1-1000 nM) was added. Subsequently, cell supernatants were collected after 10 days and analyzed for HIV-1 RT activity as described above.

Nanoparticle uptake into CD4+ T cells using CEM-ss cells as standards.

CEM-ss CD4+ T cells were suspended in RPMI supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cell suspension (1 ml/well) was added to transparent flat-bottomed 12-well plates, pre-coated with a solution of poly-L-lysine (500 μg/ml in distilled water) for 1 hour. After attachment to the surface of the wells, the cells were treated with 25 μM NCAB , NMCAB, NM2CAB or NM3CAB. After 2, 4, and 8 h, cells were washed and scraped in PBS. The cell suspension was centrifuged at 200xg for 5 min and intracellular drug and prodrug concentrations were quantified using a Waters TQD mass spectrometer. Uptake of nanoparticles into the CEM-ss CD4+ T cell line was confirmed

- 24 046194 was expected by TEM imaging after 8 hours of treatment at a concentration of 25 µM. Sample processing for TEM imaging is described above.

Pharmacokinetic (PK) and biodistribution (BD) studies in mice.

NSG mice (female, 6–8 weeks, Jackson Labs, Bar Harbor, ME, USA) were treated with 45 mg/kg CAB equivalents of NCAB, NMCAB, NM2CAB, or NM3CAB via a single intramuscular (IM, hamstring) injection at a dose of 40 µl/25 g mouse. Following injection, blood samples were collected in heparinized tubes on day 1 post-injection and then weekly until day 364 via buccal puncture (from the submandibular vein, MEDIpoint, Inc., Mineola, NY, USA). Collected blood (25 μl) was immediately diluted in 1 ml ACN and stored at -80°C until drug concentration measurements. The remaining blood samples were centrifuged at 2000xg for 8 min to collect blood plasma. Blood plasma was collected and stored at -80°C for drug content analysis. On days 14, 28, 42, and 364 post-administration, animals were humanely euthanized by isoflurane inhalation followed by cervical dislocation. Spleen, lymph nodes, liver, lung, intestine, kidney, brain, vaginal, and rectal tissue were collected to quantify CAB and prodrug concentrations. CAB, MCAB, M2CAB and M3CAB were quantified in mouse plasma, blood and tissue by UPLC-MS/MS using a Waters ACQUITY H-class UPLC instrument (Waters, Milford, MA, USA) coupled to a Xevo compact mass spectrometer TQ-S. All solvents for sample processing and UPLC-MS/MS analysis were of LCMS grade (Fisher). For plasma and blood samples, 25 μl of sample was added to 1 ml of acetonitrile (ACN), to which 10 μl of internal standard (IS) was added. d3-dolutegravir (d3-DTG), myristoylated dolutegravir (MDTG), and stearoylated darunavir (SDRV) at final concentrations of 200, 20, and 20 ng/mL, respectively, were used as ISs for the CAB, MCAB, and M2CAB/M3CAB assays, respectively. The samples were mixed on a vortex mixer and centrifuged at 17000xg for 10 min at 4°C. Supernatants were collected and dried using SpeedVac® and resuspended in 100 μl of 80% methanol; 10 μL was injected for analysis of MCAB, M2CAB, and M3CAB by UPLC-MS/MS. Standard curves were prepared from mouse plasma/blood blank samples over a concentration range of 0.2–2000 ng/mL for CAB, MCAB, M2CAB, and M3CAB. To quantify the drug in tissues, 3-200 mg of sample was homogenized in 4-29 volumes of 0.1% v/v. formic acid and 2.5 mM ammonium formate containing 90% methanol. To 100 μl of tissue homogenate, 280 μl of methanol containing 0.1% formic acid and 2.5 mM ammonium formate, 80% methanol (10 μl) and IS (10 μl) was added, followed by vortex mixing for 3 min and centrifugation at 17000xg for 15 min. For the MCAB, M2CAB, and M3CAB assays, 85 μL of supernatant was mixed with 15 μL of water. For the CAB assay, 20 μL of supernatant was mixed with 80 μL of 50% ACN. These aliquots were vortexed, centrifuged at 17,000xg for 10 min, and 10 μL of the supernatant was used for LC-MS/MS analysis. Standards were prepared in a similar manner using tissue homogenate blanks with 10 μl of spike solution (CAB/MSAB/M2CAB/M3CAB, 5-20,000 ng/ml in 50% ACN). For CAB quantitation, chromatographic separation of a 10 µL CAB sample was performed on a Waters ACQUITY UPLC BEN Shield RP18 column (1.7 µm, 2.1 x 100 mm) using a 10 min 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. During the first 3.5 min, the mobile phase contained 35% B, and then its content was increased to 95% B in 0.5 min and kept constant for 1.5 min. Mobile phase B was then returned to 35% in 0.5 min and the column was equilibrated for 1 min before the next injection. In the case of quantitation of MCAB and M2CAB, chromatographic separation was performed on a shorter 30 mm column (PN) using an 8 min gradient method with a flow rate of 0.28 ml/min. The initial mobile phase contained 80% B for the first 2 min, and then the content was increased to 95% B in 4 min, held constant for 0.75 min, returned to 80% in 0.25 min, and the column was equilibrated for 1 min before the next injection.In the case of M3CAB quantitation, chromatographic separation was also carried out at more short 30 mm column using an 8-minute gradient method with a flow rate of 0.35 ml/min. The initial mobile phase contained 88% B for the first 5 minutes, and then its content was increased to 95% B in 0.25 minutes, maintained constant for 1.5 min, returned 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 at cone voltage values of 10, 24, 2 and 20 V, respectively, and collision energies of 24, 18, 24 and 26 V, respectively, in the positive ion detection mode. The multiple reaction monitoring (MRM) transitions used 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 spectra were analyzed and quantified using a pro

- 25 046194 MassLynx software version 4.1. All quantifications were determined using ratios of the peak area for the analyte to the peak area for the internal standard. After analyzing PK and BD in NSG mice, NM2CAB, the best among all formulations, was again evaluated in another mouse strain, BALB/cJ. (males, 6-8 weeks, Jackson Labs). NCAB was used as a control. Mice injected with NM2CAB were humanely euthanized on day 280 post-administration. Quantification of drug and prodrug in collected plasma and tissue was similar to that described above. PK analysis using a non-compartmental model for CAB in plasma was performed using Phoenix WinNonlin-8.0 (Certara, Princeton, NJ, USA) for studies in NSG mice.

PK and BD in rhesus monkeys (RM).

Four male RMs (4.4–6.7 kg; PrimeGen) were anesthetized with ketamine (10 mg/kg) and then administered 45 mg/kg SAV-equivalent NM2CAB and the laboratory-derived prodrug RPV via IM injection (quadriceps muscle). thighs, 0.5 ml/kg). Blood samples were collected in potassium EDTA-coated tubes for clinical blood count (CBC) and metabolic profiling. Blood plasma was separated for drug concentration measurements. Biopsy tissues from lymph nodes, adipose tissue, and rectal tissue were collected on day 204 post-injection for drug quantitation. Quantification methods for drug and prodrug in plasma and tissues were similar to those described above in the mouse studies.

Statistical analyses.

Statistical analysis was performed using GraphPad Prism 7.0 software (La Jolla, CA, USA). Data from in vitro studies were expressed as the mean ±SEM of a minimum of 3 biological replicates, whereas results from in vivo studies were expressed as the mean ±SEM of a minimum of 3 biological replicates. Student's t test (two-tailed) was used for comparisons between two groups. One-way ANOVA followed by Tukey's test was used to compare three or more groups. Levels of statistical significance of differences were determined as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Research coordination.

All animal studies were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC) in accordance with the standards included in the Guide of the Care and Use of Laboratory Animals of Laboratory Animals (National Research Council of the National Academies, 2011) Human monocytes were isolated by leukapheresis from HIV-1/2 and hepatitis B seronegative donors according to a protocol approved by the UNMC institutional review board.

Results.

Synthesis and characterization of the prodrug.

CAB was chemically modified by attaching fatty acid chains with variable chain lengths of 14, 18, and 22 carbon atoms, yielding MCAB esters, M2CAB, and M3CAB, respectively. The prodrugs were further characterized by nuclear magnetic resonance (NMR) to confirm the synthesis. The 1H NMR spectra for all three prodrugs showed triplets in the range of 0.86-0.89, 1.77-1.78 and 2.66-2.70 ppm and a broad singlet in the range of 1.24-1.25 ppm, which corresponds to the terminal methyl and repeating methylene protons in the alkyl chain of a fatty acid. The disappearance of the phenolic proton peak at 11.5 ppm confirmed the replacement of the CAB hydroxyl proton by fatty acid moieties. ^{The 13} C NMR spectrum for each prodrug confirmed carbon atoms in the conjugated aliphatic chain. Electrospray ionization mass spectrometry (ESI-MS) confirmed the molecular masses of all prodrugs. ESI-MS infusion for MCAB generated a strong signal at 616.28 m/z, ESI-MS infusion for M2CAB generated a strong signal at 672.34 m/z, and ESI-MS infusion for M3CAB generated a strong signal at 728.47 m/z. Characterization of the prodrug by Fourier transform infrared spectroscopy (FTIR) produced bands not observed for CAB in the ranges 2908-2935 and 1748-1775 cm ^-1 . They correspond to the C-H stretches in aliphatic methylene groups and the C=O stretch corresponding to the carboxyl group in the ester bond, respectively. XRD spectra in the range 2θ=2-70° demonstrated the crystalline nature of the prodrugs MCAB, M2CAB and M3CAB. M2CAB nanoparticles retained the crystalline properties of the prodrug. A significant consistent decrease in water solubility was observed for MCAB (2.83 ± 1.37 μg/ml), M2CAB (1.91 ± 0.23 μg/ml) and M3CAB (1.51 ± 0.7 μg/ml) according to compared with CAB (12.12±0.41 μg/ml). Moreover, compared with CAB (0.09±0.002 mg/ml), a parallel significant increase in solubility in 1-octanol was observed for MCAB (2.31±28.15 mg/ml), M2CAB (2.64±0.02 mg/ml) and M3CAB. These results confirmed the increased hydrophobicity and lipophilicity of the prodrugs compared to CAB as a result.

- 26 046194 for chemical modifications with fatty acid conjugates.

Prodrugs are pharmacologically inactive compounds that require enzymatic or hydrolytic activation for biological conversion to active drugs under physiological conditions. Therefore, the kinetic characteristics of MCAB, M2CAB and M3CAB hydrolysis and subsequent CAB formation were assessed in plasma from different species (mouse, rat, rabbit, monkey, dog and human). When incubated in blood plasma from all species tested, MCAB demonstrated more than 85% degradation within 30 min, M2CAB demonstrated an average of 75% degradation after 2 hours and 80% degradation after 6 hours. M3CAB demonstrated the lowest rate of degradation, with about 50% prodrug remained after 24 hours of incubation in blood plasma. These studies showed that MCAB was rapidly converted to CAB, while M3CAB showed less conversion to CAB. CAB formation from M2CAB hydrolysis averaged 71.1% after 6 h, indicating that M2CAB is an optimal prodrug in terms of stability and active drug formation under physiological conditions. There were some differences in the rates of prodrug hydrolysis in plasma between different animal species. Variations in plasma CAB concentrations among study species may result from differences in body fat distribution, muscle mass, physical activity, and differences in plasma cholinesterase enzymes involved in 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). Moreover, the chemical stability of M2CAB was determined at room temperature and at elevated temperature (37°C) under acidic (pH 1), neutral (pH 7) and basic conditions (pH 11) for 24 h to determine the long-term stability of the prodrug nanoformulations under different conditions. storage conditions. At room temperature after 24 h, 19% hydrolysis under acidic conditions and 24% hydrolysis under neutral conditions were determined. However, under basal conditions, M2CAB was completely hydrolyzed and was not detected. At 37°C after 24 h, approximately >95% of the prodrugs were hydrolyzed under acidic conditions and 28% of the prodrugs were hydrolyzed under neutral conditions. However, under basal conditions, M2CAB was completely hydrolyzed within 2 hours. The half-maximal effective concentration (EC ₅₀ ) values for the prodrugs, MCAB, M2CAB and M3CAB, were compared with CAB to determine changes in antiretroviral activity due to chemical modification, if present. Measurements of HIV-1 RT activity in the culture medium of HIV-IADA-infected MDMs demonstrated that the EC50 values for CAB (28.39 nM), MCAB (34.19 nM), M2CAB (44.71 nM) and M3CAB (51. 15 nM) were comparable, indicating no significant effect on drug activity.

Preparation and characterization of nanocomposition.

Nanoformulations CAB (NCAB), MCAB (NMCAB), M2CAB (NM2CAB) and M3CAB (NM3CAB) were prepared by top-down synthesis using high-pressure homogenization. The encapsulation efficiency of the drug or any of the prodrugs was >85%. The surfactant poloxamer 407 (P407) provided surface stabilization of the particle. Nanoparticle size, polydispersity index (PDI) and zeta potential were determined by DLS at room temperature (RT), 4 and 37 °C. All formulations remained stable for up to 98 days, meaning that these formulations can maintain their physical integrity under a variety of storage conditions. Moreover, temperature fluctuations did not affect the physicochemical properties of either formulation during the study period. On the day of receipt, the mean particle size, PDI, and zeta potential for NCAB were 294.5 ± 1.8 nm, 0.23 ± 0.01, -28.3 ± 0.21 mV; for NMCAB they were 302.1±10.8 nm, 0.23±0.01, -31.1±1.1 mV; for NM2CAB they were 359.7±3.63 nm, 0.23±0.02, -31.3±0.1 mV and for NM3CAB they were 268.47±6.9 nm, 0.23±0. 03, -31.1±0.06 mV. At day 98 after receipt, physicochemical parameters for NCAB were 327.5±4.9 nm, 0.21±0.04, -18.2±0.25 mV; for NMCAB they were 388.7±6.4 nm, 0.22±0.03, -24.8±2.87 mV; for NM2CAB they were 369.5±2.5 nm, 0.19±0.03, -19.6±0.53 mV and for NM3CAB they were 245.6±2.6 nm, 0.27±0. 06, -25.7±0.56 mV. The SEM images demonstrated that the NCAB, NMCAB, NM2CAB, and NM3CAB nanoparticles had a uniform rod-shaped morphology. To confirm reproducibility, the NM2CAB formulation was prepared in 11 separate batches (see table). The nanoparticle sizes ranged from 243.00±2.48 to 378.00±1.90 nm with a narrow PDI (from 0.18±0.03 to 0.33±0.03).

- 27 046194

Reproducibility of NM2CAB synthesis

Production batch no. Size±8EM (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 AND 243.00±2.48 0.22±0.01

The effect of nanoformulations on the antiretroviral activity of prodrugs (EC ₅₀ ) was determined. The antiviral activity of NCAB, NMCAB, NM2CAB, or NM3CAB was determined in MDM over a concentration range (0.01-1000 nM) and measured using HIV-1 reverse transcriptase activity after challenge with HIV-IADA virus at an MOI of 0.1. EC50 values were increased compared to non-nanoformulated drug or non-nanoformulated prodrugs, likely due to the dissolution of the nanoparticles required before degradation of the prodrugs. EC50 values were comparable for NCAB (39.83 nM), NMCAB (89.67 nM), NM2CAB (37.02 nM). However, the EC50 value of NM3CAB was significantly increased (~1.78E+06 nM). This may be a consequence of the slower degradation of the prodrug and subsequent formation of active CAB, as well as the stability of the nanoformulations inside cells. To further characterize prodrug nanoformulations and determine treatment-providing concentrations for in vitro studies, cell viability was assessed in MDM and CEM-ss CD4+ T cells using the MTT assay. In MDM, no toxicity was observed in the concentration range tested (10–400 μM) for all nanoformulations. In CEM-ss CD4+ T cells, cytotoxicity was detected at concentrations of 50 μM and above. Thus, treatment concentrations for all nanoformulations were 100 μM for MDM assays and 25 μM for CEM-ss CD4+ T cell assays.

In vitro screening in MDM u CEM-ss CD4 ⁺ T cells.

Can be successfully used as cellular drug depots and carriers. Due to their phagocytic nature 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), MDMs can serve as drug delivery systems in virus reservoirs. Therefore, MDM has been used as an in vitro system for the evaluation of 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).

Uptake analysis was performed in MDM by measuring drug and prodrug levels after treatment with 100 µM NCAB, NMCAB, NM2CAB or NM3CAB for up to 24 hours. Intracellular prodrug levels measured for NMCAB, NM2CAB and NM3CAB were 61.69±0.78 , 84.07±5.82 and 73.34±13.59 nmol/10 ⁶ cells, respectively, after 24 hours; and intracellular CAB levels were 0.58±0.11, 12.31±0.46, 17.79±2.92 and 7.97±1.76 nmol/10 ⁶ cells for NCAB, NMCAB, NM2CAB or NM3CAB, respectively . Thereafter, the ability of macrophages to retain drug and prodrug within cells was assessed over a period of 30 days after a single treatment. MDMs were treated with 100 μM NCAB, NMCAB, NM2CAB, or NM3CAB for 8 hours. Intracellular prodrug levels were sustained for up to 30 days after a single treatment. Specifically, the amount of intracellular prodrug measured at day 30 for NMCAB, NM2CAB, and NM3CAB was 0.41 ± 0.09, 14.21 ± 2.28, and 26.70 ± 3.29 nmol/10 ⁶ cells. Likewise, intracellular levels of CAB generated from prodrug cleavage were measured over a period of up to 30 days. Intracellular CAB levels measured at day 30 for NM2CAB and NM3CAB were 1.71 ± 0.35 and 2.05 ± 0.10 nmol/10 ⁶ cells. In contrast, intracellular CAB levels fell below the limit of quantification within 24 h of NCAB treatment; and intracellular CAB concentration after NMCAB treatment was measured until day 25 (0.09 ± 0.04 nmol/10 ⁶ cells), and it was not detected on day 30. In parallel

- 28 046194 retention assay, the amount of CAB released into the culture fluid was measured over 30 days. NM2CAB exhibited the most sustained release of CAB, with drug levels of 1.0 ± 0.10 nmol/10 ⁶ cells on day 30. CAB was not detected with NCAB and NM3CAB treatment. No prodrugs were detected in the culture medium for any of the treatments, indicating biological conversion of the prodrug.

TEM images of MDMs were then obtained on days 0, 15, 30 after treatment with nanoformulations for 8 h to assess the presence of nanoparticles in the cytoplasmic vesicles. TEM images confirmed the presence of prodrug nanoformulations (NM2CAB and NM3CAB) for up to day 30 in MDM, demonstrating the drug depot properties of MDM; and confirmed the results of uptake, retention and release assays. Uptake of NCAB, NMCAB, NM2CAB, or NM3CAB was determined in CD4' T cells after treatment at 25 μM, reflecting the pattern observed in MDM. TEM imaging performed on CEM-ss CD4+ T cells after a single treatment with NCAB, NMCAB, NM2CAB, or NM3CAB for 8 h confirmed the presence of nanoparticles in the cytoplasmic compartments.

To assess whether sustained drug retention in MDM would provide protection against HIV-1 infection, cells were challenged with HIV-IADA for up to 30 days after 8-hour treatment with 100 μM NCAB, NMCAB, or NM2CAB and quantitated in regarding HIV-1 RT activity, as well as qualitative determination of HIV-1 p24 antigen expression. NM3CAB was not selected for this study because it did not demonstrate significant protection with the desired EC ₅₀ value. Treatment with NM2CAB suppressed HIV1 RT activity until day 30, and this was confirmed by the absence of HIV-1 p24 expression. In contrast, complete viral breakthrough occurred on day 1 after NCAB treatment and on day 20 after NMCAB treatment. Thus, the improved MDM drug retention exhibited by NM2CAB provided better protection against HIV-1 challenge compared with NACB and NMCAB. In addition, the dose-dependent antiretroviral activity of NM2CAB was determined (Fig. 4). MDMs were treated with 10, 50, or 100 μM NM2CAB for 8 h and challenged with HIV-Iada. Antiretroviral activity was determined in a similar manner over a period of up to 30 days. Complete viral suppression was observed with 50 and 100 μM NM2CAB treatments, while 57% protection was observed with 10 μM treatment after 30 days, and this was confirmed by HIV-1 p24 expression (Fig. 4).

Pharmacokinetic parameters (PK) and biodistribution.

To assess PK and biodistribution profiles, female NSG mice were administered by IM injection a single dose of 45 mg/kg CAB equivalents of NCAB, or NMCAB, or NM2CAB. Female immunodeficient NSG mice were used for evaluation to simulate disease in the form of immunocompensatory pathology and to assess biodistribution in the genitourinary tract. NCAB is an effective equivalent formulation to CAB-LA, as both formulations produced similar output levels of CAB in plasma up to day 49 after administration of BALB/cJ to mice.

At day 1 post-injection, NCAB treatment provided higher plasma concentrations of CAB compared to both NMCAB and NM2CAB and exhibited faster degradation kinetics over the study period compared to NM2CAB. In the case of NCAB treatment, plasma concentrations of CAB were maintained at levels greater than four times the concentration providing 90% inhibition based on protein binding (4xPA-IC ₉₀ ; 664 ng/ml) for the period up to day 35 (792.7 ng/mL), then rapidly fell below the PA-IC90 (166 ng/mL) by day 49 (75 ng/mL) before falling below the limit of quantitation (0.5 ng/mL) by day 126. NMCAB treatment exhibited slower degradation and maintained plasma CAB levels above 4xPA-IC ₉₀ during the period up to day 91 (673.8 ng/ml) and above PA-IC _9O during the period up to day 168 (186.7 ng/ml) . At day 364 after NMCAB treatment, CAB levels were quantified as 8.5 ng/ml. In stark contrast, NM2CAB treatment produced slower plasma breakdown kinetics compared to both NCAB and NMCAB during the period up to day 364, maintaining a steady-state plasma concentration of CAB above 4xPA-IC ₉₀ during the period up to day 231 (702 ng/ml) and higher PA-IC _9O during the period up to day 364 (354.2 ng/ml). Plasma pharmacokinetic parameters for CAB were determined using non-compartmental model analysis for all treatment groups. Quantification of PK parameters demonstrated that the apparent half-life of CAB after NM2CAB treatment (131.56 days) was 17- and 3-fold longer than that of NCAB (7.80 days) and NMCAB (44.40 days), respectively. Similarly, the CAB mean residence time (MRT) of NM2CAB was 21 times longer than that of NCAB (201.94 days vs. 9.79 days, respectively) and 7 times longer than that of NMCAB (201.94 days vs. 30.76 days, respectively).

NM2CAB also resulted in significantly higher levels of CAB in tissue over a period of up to one year. Tissue biodistribution of CAB was assessed on days 14, 28, 42 and 364 after a single IM injection in vaginal tissue, rectal tissue, spleen, liver, intestine, brain,

- 29 046194 kidney, lung and tissues anatomically associated with lymph nodes. Lymph node drug levels were determined in anatomical regions associated with lymph nodes only on days 28 and 364 due to their immature state in immunodeficient NSG mice. Notably, MCAB levels were lower than M2CAB on days 14, 28 and 42, and were undetectable on day 364. For NM2CAB on day 364, prodrug levels were 3414.8 ng/g (spleen), 909.8 ng/g ( liver), 52.7 ng/g (lung), 50.3 ng/g (brain), 3.9 ng/g (kidney) and 18710.1 ng/g (lymph nodes). At day 28, CAB levels in all tissues were comparable between NCAB and NM2CAB. However, by day 42, CAB levels in all tissues tested following NCAB treatment were significantly lower compared to levels following NM2CAB treatment (vaginal tissue, spleen, intestine, brain, kidney and lung, rectal tissue, and brain). In contrast, tissue CAB levels up to day 42 after NMCAB treatment were significantly higher compared to NCAB and NM2CAB treatments. At day 364 following NM2CAB treatment, CAB concentrations were significantly higher in all tissues tested compared to other formulations, and CAB levels were measured at 27 ng/g (vaginal tissue), 19.7 ng/g (rectum), 41.1 ng/g (spleen), 67.62 ng/g (tissues anatomically associated with lymph nodes), 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 (lung). In contrast, CAB levels were significantly below or below the limit of quantitation (0.5 ng/g) in all tissues at day 364 after treatment with NCAB or NMCAB.

Concentrations of the prodrug (MCAB or M2CAB) in blood and all tissues were also examined (Fig. 5). Blood concentrations of both prodrugs were lower on day 1 postinjection, 22 ng/mL for MCAB and 31.3 ng/mL for M2CAB, and rapidly fell below the limit of quantitation, suggesting biological conversion of the prodrugs to the active parent drug. All tissues screened represented significant depots of M2CAB and had sustained levels of M2CAB during the study period. A single IM injection of NM3CAB (45 mg/kg CAB equivalents) in NSG females produced low plasma CAB levels (2248 ng/ml) on day 1 post-administration compared to NCAB (41237.6 ng/ml), NMCAB (30148 .9 ng/ml) and NM2CAB (7076.1 ng/ml). Plasma CAB levels reached values around PA- _1C9O (233.2 ng/ml) within 28 days after treatment. Plasma and tissue levels of CAB and M3CAB were measured on day 28. To confirm slower hydrolysis of M3CAB, PK assessment was repeated in BALB/cJ mice after a single IM injection of NM3CAB at the same dose. Similar to the PK results in NSG mice, NM3CAB produced low plasma CAB levels on day 1 post-administration (777.8 ng/ml); and plasma CAB levels fell below a single PA-IC90 value within 28 days (98.9 ng/ml). These data supported the slower hydrolysis of the M3CAB prodrug and subsequent biological conversion to CAB.

The improved PK and BD profiles of NM2CAB across all formulations were confirmed in another strain of BALB/cJ (male) mice. Here, wild-type mice were used to confirm the results from immunodeficient NSG mice. Plasma and tissue PK and BD measurements for NM2CAB were performed in parallel with measurements in NSG mice. Specifically, plasma CAB concentrations were higher than PA- _1C9O at day 231 (170.8 ng/mL) for NM2CAB, confirming an improvement in the apparent drug half-life. NCAB was used as a control in which CAB levels fell below PA- _1C9O by day 28 (12.28 ng/ml).

To confirm the results observed in rodents, rhesus monkeys were administered by IM injection a single dose of 45 mg/kg CAB equivalents of NM2CAB. Plasma levels of CAB and prodrug (M2CAB) were measured up to day 393. Similar to the results in mice, NM2CAB treatment produced slower plasma breakdown kinetics, maintaining a steady-state plasma concentration of CAB through day 393. On day 393 CAB levels measured averaged 56.1 ng/mL. As expected, plasma concentrations of M2CAB were lower during the study compared to CAB levels, suggesting biological conversion of the prodrug to its active parent drug (Fig. 6A). Biopsies of rectal tissue, lymph nodes, and adipose tissue were collected on day 204 after NM2CAB administration and analyzed for CAB and M2CAB levels. CAB concentrations in rectal, 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 node and adipose tissue (33.3 and 233.2 ng/g, respectively), with lower levels observed in rectal tissue (1.7 ng/g) (Fig. 6C).

Toxicity assessment.

Following administration of NM2CAB, toxicological analyzes were performed in both mice and rhesus monkeys. To assess toxicity in NSG mice, animal weights were recorded weekly for a year; and at study completion (day 364), plasma and tissues were collected for metabolic profiles and histopathological analyses, respectively. Age-matched, untreated mice were used as controls. No differences in weight were observed between animals from all groups. A comprehensive set of serum chemistry parameters were quantified using a disk to determine a comprehensive diagnostic profile

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VetScan and the VetScan VS-2 instrument. The following parameters were assessed: 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). There were no significant differences between controls and NM2CAB treatment groups, indicating that NM2CAB did not adversely affect systemic organ function. Histological evaluation of tissue sections (liver, lung, intestine, spleen, kidney and brain) stained with H&E by a board-certified pathologist revealed no abnormal pathology in mice treated with NM2CAB. Moreover, the formulation was well tolerated by both strains of mice (NSG and BALB/cJ), and no injection site reactions or changes in behavior or locomotor activity were observed.

For the rhesus monkey assessment, weights were recorded from before formulation administration, and plasma samples and peripheral blood mononuclear cells (PBMCs) were collected for CBC and metabolic profiles up to day 393 after NM2CAB administration. Systemic toxicity was assessed by measuring both hematological (neutrophils, lymphocytes, monocytes) and metabolic (ALT, alkaline phosphatase, BUN/creatinine) profiles. No changes in weight were recorded in any of the animals after injection. The initial mild redness observed at the injection site subsided by day 3 in all animals. No inflammation or lump formation was noted 3 days after injection. Neutrophils, lymphocytes, and monocytes were counted before and after NM2CAB administration; and the counted blood cell numbers were consistent. On day 1 after injection, an increase in the counted number of neutrophils was recorded, and it became normal within 2 weeks in all animals. This change may be related to the injection and may not be related to the drug. Metabolic profiles for the liver and kidneys remained unchanged in all animals after treatment. Overall, no adverse events were observed following administration of NM2CAB.

Interdrug interactions.

Drug-drug interactions between two prodrug nanoformulations (NM2CAB and NM3PRV) of different drug classes: CAB (INSTI) and rilpivirine (RPV; NNRTI) were assessed. The choice of RPV as a drug along with CAB is due to the ongoing clinical trials of the combination of CAB and long-acting RPV nanoformulations. NM3RPV is a long-acting RPV formulation (Hilaire, et al., J. Control Release (2019), 311312:201-211). BALB/cJ mice received IM treatment with a single dose of NM2CAB alone (45 mg/kg CAB equivalents), NM3RPV alone (45 mg/kg RPV equivalents), or by coadministration of nanoformulations of both prodrugs (NM2CAB and NM3RPV, 45 mg/kg drug equivalents). funds). Plasma levels of CAB and RPV were measured and no differences in active drug levels were observed between animals treated with the formulations alone or in combination, suggesting the use of a combination of multiple prodrug nanoformulations for treatment or prevention.

Due to the failure of protective vaccines and pre-exposure prophylaxis (PrEP) to eliminate HIV infection in individuals at risk, long-term ART is the only approach available to prevent disease and stop new infections and transmission of the virus. This is emphasized in the concept of treatment as prevention for people at risk of infection. The development of injectable LA ARVs is focused on creating drugs with long half-lives. PrEP requires LA ARV coverage to exclude infections following HIV exposure. The development of viral infection should cease during periods of time when protective levels of ARV are detected in the blood plasma. Drugs must also be administered without unwanted side effects, which include gastrointestinal toxicity and any significant drug-drug interactions. Currently, LA agents are attracting enthusiasm among potential users due to the potential benefits of eliminating the social stigma of viral infections and not requiring dosing intervals of less than 24 hours, some of which can be dosed less frequently than every 2-3 months. LA ARVs administered via the subcutaneous or intramuscular route are highly effective and have already demonstrated improved quality of life and longevity (May, et al., AIDS (2014), 28:1193-1202; Antiretroviral Therapy Cohort, Lancet (2017), HIV 4: e349-e356). They help circumvent the problem of poor adherence, which remains a key challenge in treatment (Shubber, et al., PLoS Med. (2016), 13:e1002183; Osterberg, et al., New Eng. J. Med. (2005), 353:487-497). Indeed, any poor adherence to ARV treatment regimens resulting in treatment failure, emergence of drug resistance mutations, and new viral transmissions can be eliminated using improved adherence. In an attempt to improve existing LA ARV platforms, slow-release, long-acting antiretroviral therapy (LASER ART) prodrugs have been developed. These new formulations serve to reduce the frequency of injections while maintaining stable therapeutic ARV levels for longer.

- 31 046194 body period (Edagwa, et al., Exp. Opinion Drug Del. (2017), 14:1281-1291). LASER ART involves prodrug synthesis through chemical modifications of existing ARVs to provide slow dissolution of native drugs, poor aqueous solubility, enhanced penetration across biological cell membranes as well as tissue reservoirs, and limited systemic off-target toxicities. Prodrug synthesis enables the formation of ARV nanocrystals stabilized by polymeric excipients. The PK and potency (PD) of LASER ART formulations have been established for a number 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., Antimicrob. Agents Chemother. (2018), 62:e01316-17 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).

CAB is an HIV-1 integrase strand transfer inhibitor (INSTI) and is currently in development as both an oral formulation and an injectable LA formulation (McPherson, et al., Expert Opin. Investig. Drugs (2018), 27:413-420). Its unique inherent properties such as hydrophobic nature, long systemic half-life (approximately 40 hours after oral administration), high potency, sustained profile, low daily oral dosage requirement (>30 mg/day) and limited drug-drug interactions make it an attractive candidate for development into an injectable LA formulation (Trezza, et al., Curr. Opin. HIV AIDS (2015), 10:239-245). Herein, we demonstrate that a single injection of NM2CAB provided unexpectedly superior improvements in drug duration of action, as reflected by sustained plasma drug concentrations and tissue biodistribution, compared with either NCAB, NMCAB, or NM3CAB. NM2CAB provided sustained plasma degradation while maintaining drug levels above PA- _1C9O 166 ng/mL for 364 days after a single injection.

LA CAB, now approaching approval for clinical use, has been extensively studied in rhesus monkeys, supporting its potential as an injectable ARV for pre-exposure prophylaxis (PrEP) studies (Edagwa, et al., Exp. Opin. Drug Del. (2017), 14:1281-1291; Stellbrink, et al., Curr. Opin. HIV AIDS (2018), 13:334-340). These studies demonstrated that LA CAB provided a high degree of protection against vaginal, rectal, intravenous, and penial challenge with SHIV strains, justifying its future use as PrEP in those people at high risk of exposure to HIV and in intravenous drug users. Plasma levels above 3xPA-1C ₉₀ provided 100% protection, and concentrations above PA-IC _9i0 provided 97% protection against virus challenge (Edagwa, et al., Exp. Opin. Drug Del. (2017), 14:1281 -1291; Stellbrink, et al., Curr. Opin. HIV AIDS (2018), 13:334-340). Here, administration of NM2CAB provided plasma CAB concentrations above PA-IC _9i0 for more than six months, suggesting its superiority and its clinical effectiveness as PrEP.

Overall, the development of effective treatment and prevention interventions in HIV-1-infected patients with antiretroviral drugs (ARVs) has changed what was once an imminent death to a manageable lifelong chronic disease. The need to control the HIV-1 pandemic continues as an estimated 37.9 million people worldwide are infected. The advent of LA ARV will certainly increase opportunities to overcome the problem of suboptimal medication adherence and reduce the burden of HIV infection. To date, HIV chemoprevention with antiretroviral agents has been best demonstrated using compounds containing tenofovir diisopropyl fumarate (TDF). However, the required levels of adherence to daily or near-daily oral tablets have proven to be a challenge. LA drugs offer greater choice in achieving prevention with the understanding that safety, tolerability, and efficacy will continue to be part of therapeutic outcome assessments. LA ARVs that can be administered once a month or less frequently will improve patient compliance and expand PrEP options.

A number of publications and patent documents are cited in the above description for the purpose of describing the prior art to which the present invention relates. The full disclosure of each of these references cited is incorporated herein by reference.

Although certain of the preferred embodiments of the present invention have been described and specifically illustrated by the examples above, the present invention is not intended to be limited to such embodiments. Various modifications may be made thereto without departing from the scope and idea of the present invention, which are set out in the following claims.

Claims (11)

CLAIM 1. Compound of formula (I) o (I), or a pharmaceutically acceptable salt thereof, wherein R is a saturated linear aliphatic chain of 17 carbons in length. 2. A nanoparticle containing a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a polymer or surfactant, wherein the polymer or surfactant is an amphiphilic block copolymer containing a poly(oxyethylene) block and a poly(oxypropylene) block. 3. Nanoparticle according to claim 2, wherein the polymer or surfactant is poloxamer 407 (P407). 4. A pharmaceutical composition containing a compound according to claim 1 or a pharmaceutically acceptable salt or nanoparticle thereof according to claim 2 or 3 and a pharmaceutically acceptable carrier. 5. A method of treating HIV infection in a subject in need thereof, wherein said method comprises administering to said subject a therapeutically effective amount of a pharmaceutical composition according to claim 4. 6. The method according to claim 5, wherein the pharmaceutical composition is administered by injection. 7. The method according to claim 6, wherein the pharmaceutical composition is administered once over a 3-month period. 8. The method according to claim 6, wherein the pharmaceutical composition is administered once over a 6-month period. 9. The method according to claim 6, wherein the pharmaceutical composition is administered once over a 9-month period. 10. The method according to claim 6, wherein the pharmaceutical composition is administered once over a 12-month period. 11. The method according to claim 6, wherein the pharmaceutical composition is administered once over a 24-month period.