Rosa I. Sanchez

CYTOCHROME P450 3A4 IS THE MAJOR ENZYME INVOLVED IN THE METABOLISM OF THE SUBSTANCE P RECEPTOR ANTAGONIST APREPITANT

The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas CYP3A4 catalyzed both N- and O-dealkylation reactions. The metabolism of aprepitant by human liver microsomes was inhibited completely by ketoconazole or troleandomycin. No inhibition was observed with other P450 isoform-selective inhibitors. Aprepitant was evaluated also as a P450 inhibitor in human liver microsomes. No significant inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP2E1 was observed in experiments with isoform-specific substrates (IC50 > 70 μM). Aprepitant was a moderate inhibitor of CYP3A4, with Ki values of ∼10 μM for the 1′- and 4-hydroxylation of midazolam, and the N-demethylation of diltiazem, respectively. Aprepitant was a very weak inhibitor of CYP2C9 and CYP2C19, with Ki values of 108 and 66 μM for the 7-hydroxylation of warfarin and the 4′-hydroxylation of S-mephenytoin, respectively. Collectively, these results indicated that aprepitant is both a substrate and a moderate inhibitor of CYP3A4.

THE INVOLVEMENT OF CYP3A4 AND CYP2C9 IN THE METABOLISM OF 17α-ETHINYLESTRADIOL

The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 μM). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (Vmax/Km) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoform-specific monoclonal antibodies, in which the inhibition of formation was ∼54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes.

Chemistry-enabled drug delivery (prodrugs): recent progress and challenges.

Prodrugs can be used to enhance the properties of an active pharmaceutical ingredient or drug, generally by improving solubility and/or permeability of the compound. However, discovery teams duly spend considerable time and resources on structure-activity optimization and formulation-enabled drug delivery technologies to achieve the target exposures and biopharmaceutical properties, when early implementation of prodrugs could lead to an improved drug molecule. The goal of this review is to provide an overview of the achievements during the past few years in developing prodrugs and highlighting the challenges of following this approach.

A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV challenges

OBJECTIVES Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission. METHODS Long-acting raltegravir was administered subcutaneously to BALB/c, NSG (NOD-scid-gamma) and humanized BLT (bone marrow-liver-thymus) mice and rhesus macaques. Raltegravir concentration in peripheral blood and tissue was analysed. Suppression of HIV replication was assessed in infected BLT mice. Two high-dose HIV vaginal challenges were used to evaluate protection from HIV transmission in BLT mice. RESULTS Two weeks after a single subcutaneous injection of long-acting raltegravir in BLT mice (7.5 mg) and rhesus macaques (160 mg), the plasma concentration of raltegravir was comparable to 400 mg orally, twice daily in humans. Serum collected from mice 3 weeks post-administration of long-acting raltegravir efficiently blocked HIV infection of TZM-bl indicator cells in vitro. Administration of long-acting raltegravir suppressed viral RNA in plasma and cervico-vaginal fluids of infected BLT mice, demonstrating penetration of active raltegravir into the female reproductive tract. Using transmitted/founder HIV we observed that BLT mice administered a single subcutaneous dose of long-acting raltegravir were protected from two high-dose HIV vaginal challenges 1 week and 4 weeks after drug administration. CONCLUSIONS These preclinical results demonstrated the efficacy of long-acting raltegravir in preventing vaginal HIV transmission.

In vivo analysis of the effect of panobinostat on cell-associated HIV RNA and DNA levels and latent HIV infection

BackgroundThe latent reservoir in resting CD4+ T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents.ResultsHere, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4+ T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4+ T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4+ T cells.ConclusionWe have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4+ T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.

Discovery of MK‐8970: An Acetal Carbonate Prodrug of Raltegravir with Enhanced Colonic Absorption

Developing new antiretroviral therapies for HIV‐1 infection with potential for less frequent dosing represents an important goal within drug discovery. Herein, we present the discovery of ethyl (1‐((4‐((4‐fluorobenzyl)carbamoyl)‐1‐methyl‐2‐(2‐(5‐methyl‐ 1,3,4‐oxadiazole‐2‐carboxamido)propan‐2‐yl)‐6‐oxo‐1,6‐dihydropyrimidin‐5‐yl)oxy)ethyl) carbonate (MK‐8970), a highly optimized prodrug of raltegravir (Isentress). Raltegravir is a small molecule HIV integrase strand‐transfer inhibitor approved for the treatment of HIV infection with twice‐daily administration. Two classes of prodrugs were designed to have enhanced colonic absorption, and derivatives were evaluated in pharmacokinetic studies, both in vitro and in vivo in different species, ultimately leading to the identification of MK‐8970 as a suitable candidate for development as an HIV therapeutic with the potential to require less frequent administration while maintaining the favorable efficacy, tolerability, and minimal drug–drug interaction profile of raltegravir.

Moderate Hepatic Impairment Does Not Affect Doravirine Pharmacokinetics

Doravirine is a novel, potent, nonnucleoside reverse‐transcriptase inhibitor currently in development for HIV‐1 infection treatment. As a substrate for CYP3A‐mediated metabolism, doravirine could potentially be affected by liver‐function changes. As a portion of the HIV‐1‐infected population has varying degrees of liver impairment, we investigated the effect of moderate hepatic impairment on the pharmacokinetic profile and tolerability of single‐dose doravirine 100 mg in otherwise healthy subjects. A total of 16 subjects aged 44–64 years took part in the open‐label, single‐dose trial: 8 with moderate hepatic impairment (Child‐Pugh score, 7–9; 6 men, 2 women) and 8 healthy individuals (mean age and height matched with the impairment group; 6 men, 2 women). Subjects with hepatic impairment were required to have chronic, stable hepatic impairment with features of cirrhosis of any etiology. Blood sampling revealed that doravirine exposure was similar in both groups. The observed geometric least‐squares mean ratio (90% confidence interval; moderately impaired/healthy subjects) was 0.99 (0.72–1.35) for AUC0–∞, 0.93 (0.74–1.18) for AUC0–24 h, 0.90 (0.66–1.24) for Cmax, and 0.99 (0.74–1.33) for C24 h. Geometric mean apparent terminal t½ was ∼18 hours for both groups, whereas median Tmax was 2 hours (range, 1–6 hours) and 2.5 hours (range, 1–3 hours) for impaired and healthy individuals, respectively. In addition, doravirine was generally well tolerated. The results demonstrate that moderate hepatic impairment does not have a clinically meaningful effect on doravirine pharmacokinetics. Therefore, dose adjustment should not be necessary in patients with both HIV‐1 and moderate hepatic impairment.

Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group

A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

Design of Prodrugs to Enhance Colonic Absorption by Increasing Lipophilicity and Blocking Ionization

Prodrugs are chemistry-enabled drug delivery modifications of active molecules designed to enhance their pharmacokinetic, pharmacodynamic and/or biopharmaceutical properties. Ideally, prodrugs are efficiently converted in vivo, through chemical or enzymatic transformations, to the active parent molecule. The goal of this work is to enhance the colonic absorption of a drug molecule with a short half-life via a prodrug approach to deliver sustained plasma exposure and enable once daily (QD) dosing. The compound has poor absorption in the colon and by the addition of a promoiety to block the ionization of the molecule as well as increase lipophilicity, the relative colonic absorption increased from 9% to 40% in the retrograde dog colonic model. A combination of acceptable solubility and stability in the gastrointestinal tract (GI) as well as permeability was used to select suitable prodrugs to optimize colonic absorption.

Results of a Doravirine-Atorvastatin Drug-Drug Interaction Study

ABSTRACT Doravirine is a novel, highly potent, nonnucleoside reverse transcriptase inhibitor that is administered once daily and that is in development for the treatment of HIV-1 infection. In vitro and clinical data suggest that doravirine is unlikely to cause significant drug-drug interactions via major drug-metabolizing enzymes or transporters. As a common HIV-1 infection comorbidity, hypercholesterolemia is often treated with statins, including the commonly prescribed atorvastatin. Atorvastatin is subject to drug-drug interactions with cytochrome P450 3A4 (CYP3A4) inhibitors. Increased exposure due to CYP3A4 inhibition may lead to serious adverse events (AEs), including rhabdomyolysis. Furthermore, atorvastatin is a substrate for breast cancer resistance protein (BCRP), of which doravirine may be a weak inhibitor; this may increase atorvastatin exposure. The potential of doravirine to affect atorvastatin pharmacokinetics was investigated in a two-period, fixed-sequence study in healthy individuals. In period 1, a single dose of atorvastatin at 20 mg was administered followed by a 72-h washout. In period 2, doravirine at 100 mg was administered once daily for 8 days, with a single dose of atorvastatin at 20 mg concomitantly being administered on day 5. Sixteen subjects were enrolled, and 14 completed the trial; 2 discontinued due to AEs unrelated to the treatment. The atorvastatin area under the curve from time zero to infinity was similar with and without doravirine (geometric mean ratio [GMR] for doravirine-atorvastatin/atorvastatin, 0.98; 90% confidence interval [CI], 0.90 to 1.06), while the maximum concentration decreased by 33% (GMR for doravirine-atorvastatin/atorvastatin, 0.67; 90% CI, 0.52 to 0.85). These changes were deemed not to be clinically meaningful. Both of the study drugs were generally well tolerated. Doravirine had no clinically relevant effect on atorvastatin pharmacokinetics in healthy subjects, providing support for the coadministration of doravirine and atorvastatin.