Polyethylene Glycol 40-Modified Peptide with High Therapeutic Efficacy in Simian-Human Immunodeficiency Virus-Acutely Infected Rhesus Monkeys.

Anti-human immunodeficiency virus type 1 (anti-HIV-1) fusion peptides have been studied for nearly 2 decades, but few candidates have found useful clinical applications. One factor underlying the failure of such agents to reach the clinic is their poor pharmacokinetic properties, and many efforts have been made to overcome this problem. In this study, we modified C34, a peptide inhibitor of HIV-1 fusion, at its conserved glycosylation site using polyethylene glycols (PEGs) of different molecular weights. PEG40-NC, a conjugate of C34 and branched PEG 40 kDa (PEG40), which has been previously shown to improve the pharmacokinetic profiles of proteins, showed a significantly extended half-life (t1/2; 10.39 h in rats), which compensated for decreased in vitro activity (50% effective concentration [EC50] of 18.51 nM). PEG40-NC also showed a mechanism of action similar to that of C34. PEG40-NC monotherapy in acutely simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys significantly suppressed viral load compared with a control treatment. Efficacy was linked to the extended half-life and lymphatic exposure conferred by attached PEG40. These results highlight the potential of further clinical investigations of PEG40-NC in combination with antiretroviral therapy or other anti-HIV agents.IMPORTANCE Poor pharmacokinetics have severely hindered the clinical use of anti-HIV peptides. Different small molecules, such as lipid, cholesterol, and small PEG, were designed to modify peptides to improve their pharmacokinetics. In this study, we incorporated large branched PEG to anti-HIV peptide and obtained a conjugate with extended half-life and improved in vivo efficacy. The strategy we developed in this study can also be applicable for the development of other peptide candidates.

Pharmacokinetic and Chemical Synthesis Optimization of a Potent d-Peptide HIV Entry Inhibitor Suitable for Extended-Release Delivery.

Peptides often suffer from short in vivo half-lives due to proteolysis and renal clearance that limit their therapeutic potential in many indications, necessitating pharmacokinetic (PK) enhancement. d-Peptides, composed of mirror-image d-amino acids, overcome proteolytic degradation but are still vulnerable to renal filtration due to their small size. If renal filtration could be slowed, d-peptides would be promising therapeutic agents for infrequent dosing, such as in extended-release depots. Here, we tether a diverse set of PK-enhancing cargoes to our potent, protease-resistant d-peptide HIV entry inhibitor, PIE12-trimer. This inhibitor panel provides an opportunity to evaluate the PK impact of the cargoes independently of proteolysis. While all the PK-enhancing strategies (PEGylation, acylation, alkylation, and cholesterol conjugation) improved in vivo half-life, cholesterol conjugation of PIE12-trimer dramatically improves both antiviral potency and half-life in rats, making it our lead anti-HIV drug candidate. We designed its chemical synthesis for large-scale production (CPT31) and demonstrated that the PK profile in cynomolgous monkeys supports future development of monthly or less frequent depot dosing in humans. CPT31 could address an urgent need in both HIV prevention and treatment.

A minimally cytotoxic CD4 mimic as an HIV entry inhibitor.

Several CD4 mimics have been reported as HIV-1 entry inhibitors which can block the interaction between the viral envelope glycoprotein gp120 and the cell surface protein CD4. We previously found a lead compound 2 (YYA-021) with high anti-HIV activity and low cytotoxicity. Pharmacokinetic analysis however showed compound 2 to have wide tissue distribution and relatively high distribution volumes in rats and rhesus macaques. In the present study we searched for more hydrophilic CD4 mimics with a view to reducing tissue distribution. A new compound (5) with a 1,3-benzodioxolyl moiety was found to have relatively high anti-HIV activity and no significant cytotoxicity. Compound 5 is more hydrophilic than compound 2 and the pharmacokinetics of the intravenous administration of compound 5 in a rhesus macaque showed that compound 5 has lower tissue distribution than compound 2, suggesting that compound 5 possesses a better profile.

Pharmacokinetic interactions between BMS-626529, the active moiety of the HIV-1 attachment inhibitor prodrug BMS-663068, and ritonavir or ritonavir-boosted atazanavir in healthy subjects.

BMS-663068 is a prodrug of BMS-626529, a first-in-class attachment inhibitor that binds directly to HIV-1 gp120, preventing initial viral attachment and entry into host CD4(+) T cells. This open-label, multiple-dose, four-sequence, crossover study addressed potential two-way drug-drug interactions following coadministration of BMS-663068 (BMS-626529 is a CYP3A4 substrate), atazanavir (ATV), and ritonavir (RTV) (ATV and RTV are CYP3A4 inhibitors). Thirty-six healthy subjects were randomized 1:1:1:1 to receive one of four treatment sequences with three consecutive treatments: BMS-663068 at 600 mg twice daily (BID), BMS-663068 at 600 mg BID plus RTV at 100 mg once daily (QD), ATV at 300 mg QD plus RTV at 100 mg QD (RTV-boosted ATV [ATV/r]), or BMS-663068 at 600 mg BID plus ATV at 300 mg QD plus RTV at 100 mg QD. Compared with the results obtained by administration of BMS-663068 alone, coadministration of BMS-663068 with ATV/r increased the BMS-626529 maximum concentration in plasma (Cmax) and the area under the concentration-time curve in one dosing interval (AUCtau) by 68% and 54%, respectively. Similarly, coadministration of BMS-663068 with RTV increased the BMS-626529 Cmax and AUCtau by 53% and 45%, respectively. Compared with the results obtained by administration of ATV/r alone, ATV and RTV systemic exposures remained similar following coadministration of BMS-663068 with ATV/r. BMS-663068 was generally well tolerated, and there were no adverse events (AEs) leading to discontinuation, serious AEs, or deaths. Moderate increases in BMS-626529 systemic exposure were observed following coadministration of BMS-663068 with ATV/r or RTV. However, the addition of ATV to BMS-663068 plus RTV did not further increase BMS-626529 systemic exposure. ATV and RTV exposures remained similar following coadministration of BMS-663068 with either ATV/r or RTV. BMS-663068 was generally well tolerated alone or in combination with either RTV or ATV/r.

CYP3A5 genotype impacts maraviroc concentrations in healthy volunteers.

CYP3A5 plays a prominent role in the metabolism of maraviroc, an approved drug for human immunodeficiency virus (HIV)-1 treatment and a candidate for HIV-1 prevention. We studied the effect of the CYP3A5 genotype on pharmacokinetics of maraviroc and a primary CYP3A5-dependent metabolite of maraviroc denoted as metabolite 1 (M1). Volunteers were screened for health status and CYP3A5 genotype (wild-type allele *1 and dysfunctional alleles *2, *3, *6, and *7) to obtain 24 evaluable subjects in three groups (n = 8 each): homozygous dysfunctional (two dysfunctional alleles), heterozygous (one *1 allele and one dysfunctional allele), and homozygous wild-type (two *1 alleles). Subjects received 300 mg maraviroc orally followed by blood collection for 32 hours. The homozygous wild-type group exhibited lower mean plasma maraviroc concentrations at almost all sampling times. The median (interquartile range) maraviroc area under the plasma concentration-time curves from time 0 to infinity (AUC0-inf) were 2099 (1422-2568) ng⋅h/ml, 1761 (931-2640) ng⋅h/ml, and 1238 (1065-1407) ng⋅h/ml for the homozygous dysfunctional, heterozygous, and homozygous wild-type groups, respectively. The homozygous wild-type group had 41% lower maraviroc AUC0-inf and 66% higher apparent clearance compared with the homozygous dysfunctional group (P = 0.02). The AUC0-inf ratios of maraviroc to M1 in heterozygous and homozygous wild-type subjects were lower by 51 and 64% relative to the homozygous dysfunctional group, respectively (P < 0.001). In conclusion, the lower maraviroc concentrations in the homozygous wild-type group indicate that maraviroc may be underdosed in people homozygous for the CYP3A5*1 allele, including almost one-half of African Americans.

Pharmacokinetics and efficacy of a vaginally administered maraviroc gel in rhesus macaques.

OBJECTIVES: To investigate the pharmacokinetics (PK) of maraviroc, a CCR5-targeted HIV-1 entry inhibitor, in rhesus macaques following vaginal administration of various maraviroc-loaded aqueous hydroxyethylcellulose (HEC) gels, and to correlate the PK data with efficacy in a single high-dose vaginal SHIV-162P3 challenge model. METHODS: Maraviroc concentrations in vaginal fluid (Weck-Cel(®) sponge), vaginal tissue (punch biopsy) and plasma were assessed over 72 h following single-dose vaginal application of various maraviroc-loaded HEC gels. The range of maraviroc gel concentrations was sufficiently broad (0.003%-3.3% w/w) that test gels included both fully solubilized and predominantly dispersed formulations. The efficacy of the HEC gels against a single high-dose vaginal SHIV-162P3 challenge was also measured, and correlated with the PK concentrations. RESULTS: Maraviroc concentrations in vaginal fluid (range 10(4)-10(7) ng/mL), vaginal tissue (100-1200 ng/g) and plasma (<10(2) ng/mL) were highly dependent on maraviroc gel loading, irrespective of the form of the maraviroc component within the gel (solubilized versus dispersed). Fluid and plasma concentrations were generally highest 0.5 or 2 h after gel application, before declining steadily through to 72 h. Maraviroc concentrations in the various biological compartments correlated strongly with the extent of protection against vaginal SHIV-162P3 challenge. Complete protection was achieved with a 3.3% w/w maraviroc gel. CONCLUSIONS: A high degree of correlation between PK and efficacy was observed. Based on the data obtained with the 3.3% w/w maraviroc gel, maintenance of vaginal fluid and tissue levels in the order of 10(7) ng/mL and 10(3) ng/g, respectively, are required for complete protection with this compound.

Pharmacokinetic profile and safety of 150 mg of maraviroc dosed with 800/100 mg of darunavir/ritonavir all once daily, with and without nucleoside analogues, in HIV-infected subjects.

BACKGROUND: Once-daily nucleoside-sparing combination antiretroviral therapy regimens are attractive options for the treatment of HIV infection. However, the pharmacokinetic profiles of such regimens are often not established. METHODS: HIV-infected subjects receiving 245/200 mg of tenofovir/emtricitabine plus 800/100 mg of darunavir/ritonavir once daily with plasma HIV RNA <50 copies/mL were eligible. On day 1 (period 1), 150 mg of maraviroc daily was added and on day 11 (period 2), tenofovir/emtricitabine discontinued. At steady-state (days 10 and 20), intensive pharmacokinetic sampling was undertaken. We assessed (i) the number of subjects with trough (C(trough)) and average (C(avg)) maraviroc concentrations <25 and <75 ng/mL, respectively; (ii) geometric mean (GM) ratios for pharmacokinetic parameters for period 2 versus period 1; and (iii) factors associated with total maraviroc exposure. RESULTS: Eleven subjects completed the study procedures (mean age 49 years; range 35-59 years). In three subjects, maraviroc C(trough) and C(avg) were <25 and <75 ng/mL, respectively (C(avg), 68 ng/mL and C(trough), 14 and 21 ng/mL). Although not statistically significant, a trend was observed towards lower maraviroc, darunavir and ritonavir concentrations in period 2 versus period 1; total maraviroc exposure was 3579 ng·â€Šh/mL (95% CI: 2983-4294) and 2996 ng·â€Šh/mL (95% CI: 2374-3782) in periods 1 and 2, respectively, and the GM ratio was 0.84 (95% CI: 0.67-1.05). Only total ritonavir exposure was significantly associated with total maraviroc exposure (P=0.049; 95% CI: 0.01-0.91). No clinical safety concerns were observed. CONCLUSIONS: Within this novel nucleoside-sparing regimen, maraviroc exposure is dependent on ritonavir exposure, which was slightly reduced in the absence of tenofovir/emtricitabine.

A CD4 mimic as an HIV entry inhibitor: pharmacokinetics.

To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies.

Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic.

The pharmacologic utility of lengthy peptides can be hindered by loss of bioactive structure and rapid proteolysis, which limits bioavailability. For example, enfuvirtide (Fuzeon, T20, DP178), a 36-amino acid peptide that inhibits human immunodeficiency virus type 1 (HIV-1) infection by effectively targeting the viral fusion apparatus, has been relegated to a salvage treatment option mostly due to poor in vivo stability and lack of oral bioavailability. To overcome the proteolytic shortcomings of long peptides as therapeutics, we examined the biophysical, biological, and pharmacologic impact of inserting all-hydrocarbon staples into an HIV-1 fusion inhibitor. We find that peptide double-stapling confers striking protease resistance that translates into markedly improved pharmacokinetic properties, including oral absorption. We determined that the hydrocarbon staples create a proteolytic shield by combining reinforcement of overall alpha-helical structure, which slows the kinetics of proteolysis, with complete blockade of peptide cleavage at constrained sites in the immediate vicinity of the staple. Importantly, double-stapling also optimizes the antiviral activity of HIV-1 fusion peptides and the antiproteolytic feature extends to other therapeutic peptide templates, such as the diabetes drug exenatide (Byetta). Thus, hydrocarbon double-stapling may unlock the therapeutic potential of natural bioactive polypeptides by transforming them into structurally fortified agents with enhanced bioavailability.

Pharmacodynamics, safety, and pharmacokinetics of BMS-663068, an oral HIV-1 attachment inhibitor in HIV-1-infected subjects.

BACKGROUND: BMS-663068 is a prodrug of the small-molecule inhibitor BMS-626529, which inhibits human immunodeficiency virus type 1 (HIV-1) infection by binding to gp120 and interfering with the attachment of virus to CD4+ T-cells. METHODS: Fifty HIV-1-infected subjects were randomized to 1 of 5 regimen groups (600 mg BMS-663068 plus 100 mg ritonavir every 12 hours [Q12H], 1200 mg BMS-663068 plus 100 mg ritonavir every bedtime, 1200 mg BMS-663068 plus 100 mg ritonavir Q12H, 1200 mg BMS-663068 Q12H plus 100 mg ritonavir every morning, or 1200 mg BMS-663068 Q12H) for 8 days in this open-label, multiple-dose, parallel study. The study assessed the pharmacodynamics, pharmacokinetics, and safety of BMS-663068. RESULTS: The maximum median decrease in plasma HIV-1 RNA load from baseline ranged from 1.21 to 1.73 log(10) copies/mL. Plasma concentrations of BMS-626529 were not associated with an antiviral response, while low baseline inhibitory concentrations and the minimum and average steady-state BMS-626529 plasma concentrations, when adjusted by the baseline protein binding-adjusted 90% inhibitory concentration (inhibitory quotient), were linked with antiviral response. BMS-663068 was generally well tolerated. CONCLUSIONS: Administration of BMS-663068 for 8 days with or without ritonavir resulted in substantial declines in plasma HIV-1 RNA levels and was generally well tolerated. Longer-term clinical trials of BMS-663068 as part of combination antiretroviral therapy are warranted. Clinical Trials Registration.NCT01009814.

Pharmacokinetic effects of coadministration of lersivirine with raltegravir or maraviroc in healthy subjects.

Lersivirine (UK-453,061) is a new nonnucleoside reverse transcriptase inhibitor currently being developed as a treatment for human immunodeficiency virus type 1 infection. Lersivirine shows potent activity against wild-type and clinically relevant drug-resistant strains. Previous studies have demonstrated that lersivirine is metabolized by glucuronidation via UGT2B7 and by cytochrome P450 3A4 (CYP3A4). Lersivirine is also a weak inducer of the CYP3A4 enzyme. Therefore, coadministered lersivirine could potentially affect the pharmacokinetics of maraviroc, a CCR5 antagonist metabolized by CYP3A4, and raltegravir, an integrase inhibitor metabolized by glucuronidation. Two open-label studies assessed the pharmacokinetics of raltegravir and of maraviroc when they were coadministered with lersivirine and the pharmacokinetics of lersivirine when it was coadministered with raltegravir. Minor, clinically nonsignificant effects on the pharmacokinetics of raltegravir coadministered with lersivirine were observed at steady state for raltegravir, with estimated mean changes of -15%, -29%, and +25% in the area under the concentration-time profile from time zero to the end of the dosing interval (AUC(tau)), maximum plasma concentration (C(max)), and concentration observed 12 h postdose (C(12)), respectively. There were no clinically relevant effects of steady-state raltegravir on lersivirine AUC(tau), C(max), or concentration observed 24 h postdose (C(24)) (estimated mean changes of -2 to +5%). Coadministration of lersivirine at steady state with maraviroc resulted in no clinically relevant effects on maraviroc AUC(tau), C(max), or C(12) (estimated mean changes of +3.4 to +8.6%). Lersivirine appeared to be generally well tolerated in these studies and appears to be suitable for coadministration with raltegravir or maraviroc without the need for dose modification.

Correlation of the virological response to short-term maraviroc monotherapy with standard and deep-sequencing-based genotypic tropism prediction methods.

Genotypic tropism testing methods are emerging as the first step before prescription of the CCR5 antagonist maraviroc (MVC) to HIV-infected patients in Europe. Studies validating genotypic tests have included other active drugs that could have potentially convoluted the effects of MVC. The maraviroc clinical test (MCT) is an in vivo drug sensitivity test based on the virological response to a short-term exposure to MVC monotherapy. Thus, our aim was to compare the results of genotypic tropism testing methods with the short-term virological response to MVC monotherapy. A virological response in the MCT was defined as a ≥ 1-log(10) decrease in HIV RNA or undetectability after 8 days of drug exposure. Seventy-three patients undergoing the MCT were included in this study. We used both standard genotypic methods (n = 73) and deep sequencing (n = 27) on MCT samples at baseline. For the standard methods, the most widely used genotypic algorithms for analyzing the V3 loop sequence, geno2pheno and PSSM, were used. For deep sequencing, the geno2pheno algorithm was used with a false-positive rate cutoff of 3.5. The discordance rates between the standard genotypic methods and the virological response were approximately 20% (including mostly patients without a virological response). Interestingly, these discordance rates were similar to that obtained from deep sequencing (18.5%). The discordance rates between the genotypic methods (tropism assays predictive of the use of the CCR5 coreceptor) and the MCT (in vivo MVC sensitivity assay) indicate that the algorithms used by genotypic methods are still not sufficiently optimized.

Cytochrome P450 3A5 plays a prominent role in the oxidative metabolism of the anti-human immunodeficiency virus drug maraviroc.

Maraviroc is an anti-human immunodeficiency virus drug that acts by blocking viral entry into target cells. With use of ultra-performance liquid chromatography-mass spectrometry several monooxygenated, dioxygenated, and glucuronidated metabolites of maraviroc were identified both in vitro and in vivo. Characterization of the enzymes involved in the production of these metabolites determined that cytochrome P450 3A5 was the principal enzyme responsible for the formation of an abundant metabolite of maraviroc that resulted from oxygenation of the dichlorocyclohexane ring. For the formation of this metabolite, the V(max) values for CYP3A4 and CYP3A5 were 0.04 and 0.93 pmol · min⁻¹ · pmol P450⁻¹, and the K(m) values were 11.1 and 48.9 µM, respectively. Furthermore, human liver microsomes isolated from donors homozygous for the loss-of-function CYP3A5*3 allele exhibited a 79% decrease in formation of this metabolite compared with those homozygous for the wild-type CYP3A5*1 allele. To probe which divergent residues between CYP3A4 and CYP3A5 might play a role in the differential activities of these enzymes toward maraviroc, mutations were introduced into both enzymes and metabolism of maraviroc was measured. A CYP3A5 L57F mutant exhibited a 61% decrease in the formation of this metabolite, whereas formation by a CYP3A4 F57L mutant was increased by 337% compared with that of the wild type. Taken together, these data provide novel insights into the biotransformation of maraviroc as well as the potential role of CYP3A4 and CYP3A5 divergent residues in the enzymatic activities of these two highly homologous enzymes.

Enfuvirtide and cutaneous injection-site reactions.

Enfuvirtide belongs to a newer class of antiretroviral (ARV) agents called fusion inhibitors for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Enfuvirtide blocks attachment, binding, and entry of the viral capsid into the host CD4+ cell. Administration is only available subcutaneously in a twice-daily regimen particularly for those patients who have previously failed more than one ARV regimen. Common side effects of enfuvirtide administration include fatigue, insomnia, nausea, and diarrhea; however, injection-site reactions are the most common side effect and present in nearly all individuals undergoing treatment. The spectrum of cutaneous manifestations ranges from little to no reaction to cysts, nodules, induration, or sclerodermalike lesions. These reactions are mostly variants of iatrogenically induced hypersensitivity and are self-limited.

Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency.

Peptides derived from the heptad repeat 2 (HR2) region of the HIV fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, enfuvirtide, is used for the treatment of therapy-experienced AIDS patients. The mechanism of action of these peptides is binding to a critical intermediate along the virus-cell fusion pathway, and accordingly, increasing the affinity for the intermediate yields more potent inhibitors. We took a different approach, namely to increase the potency of the HR2 peptide inhibitor C34 by targeting it to the cell compartment where fusion occurs, and we show here that a simple, yet powerful way to accomplish this is attachment of a cholesterol group. C34 derivatized with cholesterol (C34-Chol) shows dramatically increased antiviral potency on a panel of primary isolates, with IC(90) values 15- to 300-fold lower than enfuvirtide and the second-generation inhibitor T1249, making C34-Chol the most potent HIV fusion inhibitor to date. Consistent with its anticipated mechanism of action, the antiviral activity of C34-Chol is unusually persistent: washing target cells after incubation with C34-Chol, but before triggering fusion, increases IC(50) only 7-fold, relative to a 400-fold increase observed for C34. Moreover, derivatization with cholesterol extends the half-life of the peptide in vivo. In the mouse, s.c. administration of 3.5 mg/kg C34-Chol yields a plasma concentration 24 h after injection >300-fold higher than the measured IC(90) values. Because the fusion machinery targeted by C34-Chol is similar in several other enveloped viruses, we believe that these findings may be of general utility.

Mechanism by which the lectin actinohivin blocks HIV infection of target cells.

Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man alpha(1-2)Man, Man alpha(1-2)Man alpha(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K(d) = 3.4 x 10(-8) M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high- and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the "cluster effect" of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.

Antiviral activity, pharmacokinetics, and safety of BMS-488043, a novel oral small-molecule HIV-1 attachment inhibitor, in HIV-1-infected subjects.

BMS-488043 is a novel and unique oral small-molecule inhibitor of the attachment of human immunodeficiency virus type 1 (HIV-1) to CD4(+) lymphocytes. The antiviral activity, pharmacokinetics, viral susceptibility, and safety of BMS-488043 were evaluated in an 8-day monotherapy trial. Thirty HIV-1-infected study subjects were randomly assigned to sequential, safety-guided dose panels of 800 and 1,800 mg BMS-488043 or a matched placebo in a 4:1 ratio, and the drug was administered every 12 h with a high-fat meal for 7 days and on the morning of day 8. Dose-related, albeit less-than-dose-proportional, increases in plasma BMS-488043 concentrations were observed. Mean plasma HIV-1 RNA decreases from the baseline for the BMS-488043 800- and 1,800-mg dose groups on day 8 were 0.72 and 0.96 log(10) copies/ml, respectively, compared with 0.02 log(10) copies/ml for the placebo group. A lower baseline BMS-488043 50% effective concentration (EC(50)) in the active-treatment groups was predictive of a greater antiviral response. Although absolute drug exposure was not associated with an antiviral response, the trough concentration (C(trough)), adjusted by the baseline EC(50) (C(trough)/EC(50)), was associated with antiviral activity. During dosing, four subjects experienced >10-fold reductions in viral susceptibility to BMS-488043, providing further support of the direct antiviral mechanism of BMS-488043. BMS-488043 was generally safe and well tolerated. These results suggest that further development of this novel class of oral HIV-1 attachment inhibitors is warranted.

Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding.

Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.

The use of the SAEM algorithm in MONOLIX software for estimation of population pharmacokinetic-pharmacodynamic-viral dynamics parameters of maraviroc in asymptomatic HIV subjects.

Using simulated viral load data for a given maraviroc monotherapy study design, the feasibility of different algorithms to perform parameter estimation for a pharmacokinetic-pharmacodynamic-viral dynamics (PKPD-VD) model was assessed. The assessed algorithms are the first-order conditional estimation method with interaction (FOCEI) implemented in NONMEM VI and the SAEM algorithm implemented in MONOLIX version 2.4. Simulated data were also used to test if an effect compartment and/or a lag time could be distinguished to describe an observed delay in onset of viral inhibition using SAEM. The preferred model was then used to describe the observed maraviroc monotherapy plasma concentration and viral load data using SAEM. In this last step, three modelling approaches were compared; (i) sequential PKPD-VD with fixed individual Empirical Bayesian Estimates (EBE) for PK, (ii) sequential PKPD-VD with fixed population PK parameters and including concentrations, and (iii) simultaneous PKPD-VD. Using FOCEI, many convergence problems (56%) were experienced with fitting the sequential PKPD-VD model to the simulated data. For the sequential modelling approach, SAEM (with default settings) took less time to generate population and individual estimates including diagnostics than with FOCEI without diagnostics. For the given maraviroc monotherapy sampling design, it was difficult to separate the viral dynamics system delay from a pharmacokinetic distributional delay or delay due to receptor binding and subsequent cellular signalling. The preferred model included a viral load lag time without inter-individual variability. Parameter estimates from the SAEM analysis of observed data were comparable among the three modelling approaches. For the sequential methods, computation time is approximately 25% less when fixing individual EBE of PK parameters with omission of the concentration data compared with fixed population PK parameters and retention of concentration data in the PD-VD estimation step. Computation times were similar for the sequential method with fixed population PK parameters and the simultaneous PKPD-VD modelling approach. The current analysis demonstrated that the SAEM algorithm in MONOLIX is useful for fitting complex mechanistic models requiring multiple differential equations. The SAEM algorithm allowed simultaneous estimation of PKPD and viral dynamics parameters, as well as investigation of different model sub-components during the model building process. This was not possible with the FOCEI method (NONMEM version VI or below). SAEM provides a more feasible alternative to FOCEI when facing lengthy computation times and convergence problems with complex models.

Hybrids of Small-Molecule CD4 Mimics with Polyethylene Glycol Units as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the interaction of gp120 with CD4. We have developed several CD4 mimics. Herein, hybrid molecules consisting of CD4 mimics with a long alkyl chain or a PEG unit attached through a self-cleavable linker were synthesized. In anti-HIV activity, modification with a PEG unit appeared to be more suitable than modification with a long alkyl chain. Thus, hybrid molecules of CD4 mimics, with PEG units attached through an uncleavable linker, were developed and showed high anti-HIV activity and low cytotoxicity. In investigation of pharmacokinetics in a rhesus macaque, a hybrid compound had a more effective PK profile than that of the parent compound, and intramuscular injection was a more useful administration route to maintain the high blood concentration of the CD4 mimic than intravenous injection. The presented hybrid molecules of CD4 mimics with a PEG unit would be practically useful when combined with a neutralizing antibody.