Quantification of clesrovimab, an investigational, half-life extended, anti-respiratory syncytial virus protein F human monoclonal antibody in the nasal epithelial lining fluid of healthy adults.

BACKGROUND: Clesrovimab (MK-1654) is an investigational, half-life extended human monoclonal antibody (mAb) against RSV F glycoprotein in clinical trials as a prophylactic agent against RSV infection for infants. METHODS: This adult study measured clesrovimab concentrations in the serum and nasal epithelial lining fluid (ELF) to establish the partitioning of the antibody after dosing. Clesrovimab concentrations in the nasal ELF were normalized for sampling dilution using urea concentrations from ELF and serum. Furthermore, in vitro RSV neutralization of human nasal ELF following dosing was also measured to examine the activity of clesrovimab in the nasal compartment. FINDINGS: mAbs with YTE mutations are reported in literature to partition ∼1-2 % of serum antibodies into nasal mucosa. Nasal: serum ratios of 1:69-1:30 were observed for clesrovimab in two separate adult human trials after urea normalization, translating to 1.4-3.3 % of serum concentrations. The nasal PK and estimates of peripheral volume of distribution correlated with higher extravascular distribution of clesrovimab. These higher concentration of the antibody in the nasal ELF corroborated with the nasal sample's ability to neutralize RSV ex vivo. An overall trend of decreased viral plaque AUC was also noted with increasing availability of clesrovimab in the nasal ELF from a human RSV challenge study. INTERPRETATION: Along with its extended half-life, the higher penetration of clesrovimab into the nasal epithelial lining fluid and the associated local increase in RSV neutralization activity could offer infants better protection against RSV infection.

An Investigation of Instability in Dried Blood Spot Samples for Pharmacokinetic Sampling in Phase 3 Trials of Verubecestat.

In-clinic dried blood spot (DBS) pharmacokinetic (PK) sampling was incorporated into two phase 3 studies of verubecestat for Alzheimer's disease (EPOCH [NCT01739348] and APECS [NCT01953601]), as a potential alternative to plasma PK sampling for improved logistical feasibility and decreased blood volume burden. However, an interim PK analysis revealed verubecestat concentrations in DBS samples declined with time to assay in both trials. An investigation revealed wide variation in implementation practices for DBS sample handling procedures resulting in insufficient desiccation which caused verubecestat instability. High-resolution mass spectrometry evaluations of stressed and aged verubecestat DBS samples revealed the presence of two hydrolysis degradants. To minimize instability, new DBS handling procedures were implemented that provided additional desiccant and minimized the time to analysis. Both verubecestat hydrolysis products were previously discovered and synthesized during active pharmaceutical ingredient stability characterization. A liquid chromatography-mass spectrometry assay to quantitate the dominant verubecestat degradant in DBS samples was developed and validated. The application of this method to stressed and aged verubecestat DBS samples confirmed that degradant concentrations accounted for the observed decreases in the verubecestat concentration. Furthermore, after increasing desiccant amounts, degradant concentrations accounted for approximately 7% of the verubecestat concentration in DBS clinical samples, indicating that issues with sample handling were minimized with new storage and shipping conditions. This case study illustrates the challenges with employing new sampling techniques in large, global trials, and the importance of anticipating and mitigating implementation risks.

Neutralization Activity of Anti-drug Antibodies Against a Biotherapeutic Can Be Predicted from a Comprehensive Pharmacokinetics, Pharmacodynamics, and Anti-drug Antibody Data Analysis.

Historically, a neutralization antibody (NAb) assay is considered critical in immunogenicity assessment of biologic therapeutics, even with low anti-drug antibody (ADA) positive rates. In 2019, FDA new guidelines issued on immunogenicity testing acknowledged the possibility of using "a highly sensitive PD marker or an appropriately designed PK assay or both that generate data that inform clinical activity" to replace a NAb assay. In the current manuscript, we present data for PK, PD, and ADA assays which collectively succeed to replace the standalone NAb assay. The data include a total LC/MS-based PK assay, a serum neutralization antibody (SNA) assay that essentially measures pharmacodynamically functional PK and can detect NAb activity in the presence of 1:1 ratio of drug, and a highly drug-tolerant ADA assay. In addition, a model-based meta-analysis (MBMA) demonstrated that the ability of SNA assay to detect NAb at 1:1 ratio of drug is sensitive enough to monitor clinically meaningful efficacy change, which is 50% reduction of SNA titer. Our strategy of preparing a holistic data package discussed here may provide a roadmap to the community for alternatives in assaying neutralizing activity of ADA.

A Phase 1 Randomized, Double-Blind, Placebo-Controlled Trial to Assess the Safety, Tolerability, and Pharmacokinetics of a Respiratory Syncytial Virus Neutralizing Monoclonal Antibody MK-1654 in Healthy Adults.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and related morbidity and mortality in infants. Passive immunization with an RSV-neutralizing antibody can provide rapid protection to this vulnerable population. Proof-of-concept for this approach has been demonstrated by palivizumab; however, the use of this antibody is generally restricted to the highest-risk infants due to monthly dosing requirements and its cost. To address the large unmet medical need for most infants, we are evaluating MK-1654, a fully human RSV-neutralizing antibody with half-life extending mutations targeting site IV of the fusion protein. In this 2-part, placebo-controlled, double-blind, first-in-human study, 152 healthy adults were randomized 3:1 to receive a single dose of MK-1654 or placebo in 5 cohorts (100 or 300 mg as an intramuscular dose or 300, 1000, or 3000 mg as an intravenous dose). Safety, pharmacokinetics, antidrug antibodies, and RSV serum-neutralizing antibody titers were evaluated through 1 year. MK-1654 serum concentrations increased proportionally with dose and resulted in corresponding elevations in RSV serum-neutralizing antibody titers. The antibody displayed a half-life of 73 to 88 days and an estimated bioavailability of 69% at the 300-mg dose. The overall safety profile of MK-1654 was similar to placebo, and treatment-emergent antidrug antibodies were low (2.6%) with no associated adverse events. These data support the continued development of MK-1654 for the prevention of RSV disease in infants.

Forward and reverse translational approaches to predict efficacy of neutralizing respiratory syncytial virus (RSV) antibody prophylaxis.

BACKGROUND: Neutralizing mAbs can prevent communicable viral diseases. MK-1654 is a respiratory syncytial virus (RSV) F glycoprotein neutralizing monoclonal antibody (mAb) under development to prevent RSV infection in infants. Development and validation of methods to predict efficacious doses of neutralizing antibodies across patient populations exposed to a time-varying force of infection (i.e., seasonal variation) are necessary. METHODS: Five decades of clinical trial literature were leveraged to build a model-based meta-analysis (MBMA) describing the relationship between RSV serum neutralizing activity (SNA) and clinical endpoints. The MBMA was validated by backward translation to animal challenge experiments and forward translation to predict results of a recent RSV mAb trial. MBMA predictions were evaluated against a human trial of 70 participants who received either placebo or one of four dose-levels of MK-1654 and were challenged with RSV [NCT04086472]. The MBMA was used to perform clinical trial simulations and predict efficacy of MK-1654 in the infant target population. FINDINGS: The MBMA established a quantitative relationship between RSV SNA and clinical endpoints. This relationship was quantitatively consistent with animal model challenge experiments and results of a recently published clinical trial. Additionally, SNA elicited by increasing doses of MK-1654 in humans reduced RSV symptomatic infection rates with a quantitative relationship that approximated the MBMA. The MBMA indicated a high probability that a single dose of ≥ 75 mg of MK-1654 will result in prophylactic efficacy (> 75% for 5 months) in infants. INTERPRETATION: An MBMA approach can predict efficacy of neutralizing antibodies against RSV and potentially other respiratory pathogens.

Clinical application of volumetric absorptive microsampling to the gefapixant development program.

In this paper we show the application of the Tasso OnDemand™, a novel automated sample collection device, in conjunction with volumetric absorptive microsampling (VAMS) for the development of gefapixant, a P2X3 receptor antagonist currently under clinical development for the treatment of refractory and unexplained chronic cough and endometriosis-related pain. A LC-MS/MS bioanalytical method was developed and validated using VAMS to support this development program. This method was utilized in a drug-drug interaction study to establish a mathematical bridging relationship with data obtained from a validated plasma assay used to support the program. The VAMS bioanalytical method and the predictability of the mathematical relationship is reported and discussed here.

Assessment of the CYP3A-mediated drug interaction potential of anacetrapib, a potent cholesteryl ester transfer protein (CETP) inhibitor, in healthy volunteers.

In this study, midazolam was used as a probe-sensitive CYP3A substrate to investigate the effect of anacetrapib on CYP3A activity, and ketoconazole was used as a probe-inhibitor to investigate the effect of potent CYP3A inhibition on the pharmacokinetics of anacetrapib, a novel cholesteryl ester transfer protein inhibitor in development for the treatment of dyslipidemia. Two partially blinded, randomized, 2-period, fixed-sequence studies were performed. Safety, tolerability, and midazolam and anacetrapib plasma concentrations were assessed. All treatments were generally well tolerated. The geometric mean ratios (90% confidence interval) of midazolam with anacetrapib/midazolam alone for AUC0-infinity and Cmax were 1.04 (0.94, 1.14) and 1.15 (0.97, 1.37), respectively. Exposure to anacetrapib was increased by ketoconazole--specifically, the geometric mean ratios (90% confidence interval) of anacetrapib with ketoconazole/anacetrapib alone for AUC0-infinity and Cmax were 4.58 (3.68, 5.71) and 2.37 (2.02, 2.78), respectively. The study showed that anacetrapib does not inhibit or induce CYP3A activity. Furthermore, anacetrapib appears to be a moderately sensitive substrate of CYP3A.

Determination of doravirine in human plasma using liquid-liquid extraction and HPLC-MS/MS.

Aim: A method to quantitate doravirine (MK-1439) in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics and efficacy of the compound. Methodology & results: The analyte was extracted using liquid-liquid extraction, separated on a reverse phase HPLC column, and detected on an API-4000 mass spectrometer using a Turbo-Ion spray source in positive ionization mode coupled with multiple reaction monitoring mode was used for quantification. The dynamic range for the assay was 0.02-10 ng/ml using 100 µl of human plasma. Conclusion: The assay was found to be sensitive, selective and reproducible and applied to support the doravirine clinical development program.

Use of minipig skin biopsy model as an innovative tool to design topical formulation to achieve desired pharmacokinetics in humans.

In vitro cadaver skin permeation studies are often conducted to characterize the permeation profile of compounds for dermal delivery. However, its utility could be limited in the case of topical products because of lack of reliable prediction of in vivo skin kinetics. In this paper, the use of in vivo skin biopsy data to guide topical formulation development is described. A formulation was developed by compounding MK-0873, a phosphodiesterase 4 (PDE4) inhibitor, into a commercially available cream base. The cream was characterized by skin pharmacokinetic studies in minipigs, which demonstrated that MK-0873 concentrations in the epidermis and dermis were substantially higher than the IC80 for human whole blood PDE4 inhibition of ∼200 nM, suggesting that cream should provide sufficient skin exposure to assess clinical efficacy. In toxicological studies, after 1 month repeat application in minipigs minor dermal irritation and minimal systemic exposure were observed. Based on these preclinical data, the cream formulation was chosen for single rising dose clinical studies, where plasma levels of MK-0873 were mostly below the LOQ, whereas skin biopsy concentrations ranged from 6.5 to 25.1 µM. These data suggested that minipig skin biopsy model can be a valuable tool to assess performance of topical formulations and guide formulation development.

Sensitive method for the quantitative determination of gemfibrozil in dog plasma by liquid-liquid cartridge extraction and liquid chromatography-tandem mass spectrometry.

A sensitive LC-MS/MS assay for the quantitative determination of gemfibrozil in dog plasma has been developed and validated and is described in this work. The assay involved the extraction of the analyte from 0.5-ml aliquots of dog plasma using Chem Elut cartridges and methyl tert.-butyl ether (MTBE). Chromatography was performed on a Metasil Basic column (50 x 2 mm I.D., 3 microm) using a mobile phase that consisted of 70:30 acetonitrile-ammonium acetate (1 mM, pH 5.0) with a flow-rate of 0.2 ml min(-1). The method showed excellent reproducibility with an inter- and intra-assay precision of <8.9% (%RSD), as well as excellent accuracy with an inter- and intra-assay accuracy between 99 and 101%. This method has a lower limit of quantitation (LLOQ) of 1.0 ng ml(-1) with a linear calibration range from 1.0 to 250 ng ml(-1). This new assay offers higher sensitivity and a much shorter run time over earlier methods.

Mechanistic studies on metabolic interactions between gemfibrozil and statins.

A series of studies were conducted to explore the mechanism of the pharmacokinetic interaction between simvastatin (SV) and gemfibrozil (GFZ) reported recently in human subjects. After administration of a single dose of SV (4 mg/kg p.o.) to dogs pretreated with GFZ (75 mg/kg p.o., twice daily for 5 days), there was an increase (approximately 4-fold) in systemic exposure to simvastatin hydroxy acid (SVA), but not to SV, similar to the observation in humans. GFZ pretreatment did not increase the ex vivo hydrolysis of SV to SVA in dog plasma. In dog and human liver microsomes, GFZ exerted a minimal inhibitory effect on CYP3A-mediated SVA oxidation, but did inhibit SVA glucuronidation. After i.v. administration of [(14)C]SVA to dogs, GFZ treatment significantly reduced (2-3-fold) the plasma clearance of SVA and the biliary excretion of SVA glucuronide (together with its cyclization product SV), but not the excretion of a major oxidative metabolite of SVA, consistent with the in vitro findings in dogs. Among six human UGT isozymes tested, UGT1A1 and 1A3 were capable of catalyzing the glucuronidation of both GFZ and SVA. Further studies conducted in human liver microsomes with atorvastatin (AVA) showed that, as with SVA, GFZ was a less potent inhibitor of the CYP3A4-mediated oxidation of this drug than its glucuronidation. However, with cerivastatin (CVA), the glucuronidation as well as the CYP2C8- and CYP3A4-mediated oxidation pathways were much more susceptible to inhibition by GFZ than was observed with SVA or AVA. Collectively, the results of these studies provide metabolic insight into the nature of drug-drug interaction between GFZ and statins, and a possible explanation for the enhanced susceptibility of CVA to interactions with GFZ.