Efficacy, Pharmacokinetics, and Toxicity Profiles of a Broad Anti-SARS-CoV-2 Neutralizing Antibody.

We recently reported the isolation and characterization of an anti-SARS-CoV-2 antibody, called IgG-A7, that protects transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from an infection with SARS-CoV-2 Wuhan. We show here that IgG-A7 protected 100% of the transgenic mice infected with Delta (B.1.617.2) and Omicron (B.1.1.529) at doses of 0.5 and 5 mg/kg, respectively. In addition, we studied the pharmacokinetic (PK) profile and toxicology (Tox) of IgG-A7 in CD-1 mice at single doses of 100 and 200 mg/kg. The PK parameters at these high doses were proportional to the doses, with serum half-life of ~10.5 days. IgG-A7 was well tolerated with no signs of toxicity in urine and blood samples, nor in histopathology analyses. Tissue cross-reactivity (TCR) with a panel of mouse and human tissues showed no evidence of IgG-A7 interaction with the tissues of these species, supporting the PK/Tox results and suggesting that, while IgG-A7 has a broad efficacy profile, it is not toxic in humans. Thus, the information generated in the CD-1 mice as a PK/Tox model complemented with the mouse and human TCR, could be of relevance as an alternative to Non-Human Primates (NHPs) in rapidly emerging viral diseases and/or quickly evolving viruses such as SARS-CoV-2.

Development of an UPLC-MS/MS micromethod for quantitation of cinitapride in plasma and its application in a pharmacokinetic interaction trial.

AIM: Cinitapride (CIN) is a benzamide-derived molecule used for the treatment of gastroesophageal reflux and dyspepsia. Its pharmacokinetics are controversial due to the use of supratherapeutic doses and the lack of sensitive methodology. Therefore, a sensitive and accurate micromethod was developed for its quantitation in human plasma. RESULTS: CIN was extracted from 300 µl of heparinized plasma by liquid-liquid extraction using cisapride as internal standard, and analyzed with an ultra performance liquid chromatograph employing positive multiple-reaction monitoring-MS. CONCLUSION: The method proved to be rapid, accurate and stable within a range between 50 and 2000 pg/ml and was successfully validated and applied in a pharmacokinetic interaction trial, where it was demonstrated that oral co-administration of simethicone does not modify the bioavailability of CIN.

Development of a UPLC-MS/MS method for the quantification of sildenafil by DBS, and its use on pediatric pulmonary hypertension.

BACKGROUND: Sildenafil is used for the treatment of pediatric pulmonary hypertension. A dried blood spot (DBS)-based LC-MS method for sildenafil quantitation was developed and applied to a group of patients. RESULTS: DBS showed high portability and stability of samples, and the method was selective and linear for quantitation of sildenafil (5-3,000 ng/ml) and N-desmethyl-sildenafil (3-1,500 ng/ml). After a single oral dose of sildenafil (1 mg/kg), method evidenced poor metabolism in these patients. CONCLUSION: The method was successfully applied in peripheral blood and can be used for both pharmacokinetics and therapeutic drug monitoring. DBS proved to have advantages during sample translation and preservation of analytes. Data suggest that hazardous blood sildenafil levels may be reached in this population.

Small molecule specific run acceptance, specific assay operation, and chromatographic run quality assessment: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization teams.

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.

Development of a method by UPLC-MS/MS for the quantification of tizoxanide in human plasma and its pharmacokinetic application.

BACKGROUND: Nitazoxanide (NTZ) is used for the treatment of gastrointestinal tract colonization by anaerobic bacteria, viruses and other pathogens that represent a major cause of morbidity in Latin America. The aim of the present work was to develop and validate a UPLC-MS/MS method for the selective quantification of tizoxanide (TZN, the major metabolite of NTZ) in human plasma using niclosamide as internal standard; and examine its pharmacokinetic application in healthy volunteers. Nine male subjects received a single oral dose of a NTZ 500-mg tablet under fasting conditions. RESULTS: The method was linear between 0.1 and 10 µg/ml and capable of separating signals from free-TZN and those delivered by in-source collision-induced dissociation of TZN-glucuronide, quantifying it with accuracy and precision. Mean maximum plasma concentration was 6.79 µg/ml and was reached at 2.4 h post-dose. CONCLUSION: The method was validated, fulfilling regulatory guidelines. Results suggest low pharmacokinetic variability in the assayed population.

Comparison of fasting bioavailability among 100-mg commercial, 100-mg generic, and 50-mg chewable generic sildenafil tablets in healthy male Mexican volunteers: a single-dose, 3-period, crossover study.

BACKGROUND: Sildenafil citrate (SIL) was the first oral drug registered in Mexico for the treatment of erectile dysfunction. However, succinct pharmacokinetic data are available in the Mexican population. OBJECTIVE: The goals of the present work were: (1) to design a specific method to quantify SIL plasma levels by using UPLC-MS/MS; (2) to compare oral SIL bioavailability in Mexican men with pharmacokinetic data in other populations; (3) to fulfill local regulatory requests; and (4) to describe the relative tolerability of a new 50-mg chewable tablet. METHODS: This was a randomized, single-dose, 3-period, 6-sequence crossover study in healthy male volunteers. In each period, subjects received single oral doses of 100 mg of sildenafil (1 commercial [reference(⁎)], 1 generic [test 1(†)], or 2 chewable generic tablets [test 2(‡)]), with a 4-day washout period between each dose. Serial blood samples were collected for up to 24 hours. SIL was measured in heparinized plasma by using a validated UPLC-MS/MS method. Pharmacokinetic parameters included C(max), T(max), AUC(0-24), and AUC(0-∞). Bioequivalence was established if 90% CIs for mean test:reference ratios of log-transformed C(max) and AUC fell within the range of 0.80 to 1.25. Tolerability was assessed on the basis of a clinical interview with the subject and monitoring of vital signs. RESULTS: Demographic data showed a homogeneous population. Validation of analytical method proved to be linear within the range of 1 to 1000 ng/mL, with selectivity, accuracy, and precision. 90% CIs for test 1:reference ratios were 86.52 to 113.56, 94.75 to 108.84, and 94.97 to 108.82 for the logarithm parameters C(max), AUC(0-24), and AUC(0-∞), respectively. The 90% CIs for the test 2:reference ratios were 82.14 to 107.24, 98.26 to 112.56, and 99.19 to 113.34 for C(max), AUC(0-24), and AUC(0-∞). Regarding relative tolerability, slight cephalea was the most common adverse effect. CONCLUSIONS: The developed analytical method was validated in compliance with local requirements and was useful for sildenafil measurement. This single-dose study under fasting conditions suggests that both test products met the Mexican regulatory criteria for assuming bioequivalence in these healthy, male Mexican volunteers. The clinical data suggest that the chewable tablets were well tolerated by volunteers.

Pharmacokinetics of diphenylboroxazolidones of L-α-amino acids with activity on the CNS: quantification in rat DBS by UPLC-MS/MS.

BACKGROUND: A growing number of boron-containing compounds exhibit many important biological activities; of particular interest are the α-amino acid borinic derivatives with activity in the CNS. A validated, sensitive and specific UPLC-MS/MS technique for quantification of the diphenylboroxazolidones of glycine (DBPX-gly), L-aspartate (DPBX-L-asp) and L-glutamate (DPBX-L-glu) in dried blood spots (DBSs) is presented. RESULTS: The most intense signal corresponds to compounds with (11)B. The extraction procedure was liquid elution of 3.2-mm punched DBSs with acetonitrile:aqueous formic acid 0.1% (80:20 v/v). Assays proved to be linear, falling accurately and precisely within the range of 0.3-50 µg/ml for DPBX-L-asp and DPBX-L-glu and 0.1-5 µg/ml for DBPX-gly. Chromatograms exhibit clean 2.0-min running time peaks and S/N ratios for the LLOQ were approximately 15:1. The technique was used to evaluate the pharmacokinetics of the molecules and to correlate these with timecourse toxic effects. CONCLUSION: DBSs represent an advantage for the collection of small volumes of samples, and also in terms of processing and storage. UPLC-MS/MS allow us not only to identify the isotopic pattern of boron in DBPX, but also to quantify them with accuracy and specificity. Pharmacokinetics of these molecules exhibit a high apparent volume of distribution; it suggests a preference of DPBX-amino acids for fatty tissues such as the CNS.

A single-dose, three-period, six-sequence crossover study comparing the bioavailability of solution, suspension, and enteric-coated tablets of magnesium valproate in healthy Mexican volunteers under fasting conditions.

BACKGROUND: Valproic acid has been associated with a highly variable intersubject absorptive phase; therefore, magnesium salt (magnesium valproate [MgV]) was developed to diminish variation during enteric absorption. OBJECTIVES: The aims of this study were to assess the pharmacokinetics of single oral doses of MgV 500-mg solution, suspension, and enteric-coated tablets in a healthy Mexican population, and to compare formulation-related differences. METHODS: This was a randomized, single-dose, 3-period, 6-sequence crossover study in healthy Mexican volunteers aged 18 to 45 years. In each period, subjects received single oral doses of 500-mg MgV solution, suspension, and enteric-coated tablet formulations, with a 7-day washout period between each dosing period. Serial blood samples were collected at 0 hour (prior to MgV administration) and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 9, 12, 24, 48, and 72 hours after dosing. Valproate was measured by a new method of ultraperformance liquid chromatography coupled with mass spectrometry. Pharmacokinetic parameters of interest were C(max), T(max), AUC(0-72), AUC(0-infinity), t(1/2), V(d)/F, CL/F, and mean residence time (MRT). Formulation-related differences were assayed in accordance with the Mexican regulatory bioequivalence criteria. Log-transformed values of C(max) and AUC were used to construct a classic 90% CI. Bioequivalence was established if the 90% CI for the mean test:reference ratio of log-transformed C(max) and AUC were within the range of 0.80 to 1.25. Tolerability was assessed based on subject interview, vital sign monitoring, and clinical assessment. RESULTS: A total of 24 healthy volunteers (12 women and 12 men; mean [SD] age, 28.79 [6.5] years; height, 164 [9.8] cm; weight, 65.42 [8.95] kg; and body mass index, 24.28 [2.11] kg/m(2)) were included. For the MgV solution, the mean (SD) pharmacokinetic parameters of C(max), T(max), AUC(0-72), AUC(0-infinity), t(1/2), V(d)/F, CL/F, and MRT were 59.75 (8.24) microg/mL, 0.542 (0.14) hours, 1099.67 (241.70) microg h/mL, 1156.30 (264.01) microg h/mL, 16.19 (2.36) hours, 9633.68 (1892.70) mL, 418.35 (92.01) mL/h, and 18.36 (1.44) hours, respectively. For the MgV suspension, the mean (SD) pharmacokinetic parameters of C(max), T(max), AUC(0-72), AUC(0-infinity), t(1/2), V(d)/F, CL/F, and MRT were 55.04 (7.72) microg/mL, 0.773 (0.51) hour, 1057.76 (223.37) microg h/mL, 1111.09 (245.07) microg h/mL, 16.32 (2.20) hours, 1069.05 (1775.64) mL, 435.43 (99.59) mL/h, and 18.41 (1.43) hours, respectively. For the MgV enteric-coated tablets, the mean (SD) pharmacokinetic parameters of C(max), T(max), AUC(0-72), AUC(0-infinity), t(1/2), V(d)/F, CL/F, and MRT were 54.88 (6.73) microg/mL, 2.79 (0.89) hours, 1100.79 (216.70) microg h/mL, 1163.61 (238.36) microg h/mL, 16.48 (2.10) hours, 9675.15 (1659.36) mL, 412.36 (85.24) mL/h, and 19.95 (1.53) hours, respectively. The 90% CIs for the tablets:solution ratio were 82.15 to 95.44, 94.60 to 105.39, and 95.43 to 105.95 for C(max), AUC(0-72), and AUC(0-infinity), respectively. The 90% CIs for the suspension:solution ratio were 84.79 to 98.50, 88.89 to 99.02, and 89.15 to 98.97, respectively. The 90% CIs for the tablets:suspension ratio were 89.90 to 104.43, 100.84 to 112.34, and 101.60 to 112.80, respectively. CONCLUSION: This single-dose study found that the 3 formulations (solution, suspension, and enteric-coated tablets) of MgV met the regulatory criteria for bioequivalence in these healthy, fasting, Mexican volunteers.

Development of an ultra-performance liquid chromatography-tandem mass spectrometry micromethod for quantification of lamotrigine in human plasma and its use in a bioequivalence trial.

BACKGROUND: The aim of the present work was to develop a chromatographic technique coupled with mass spectrometry for the measurement of lamotrigine in plasma. Lamotrigine and guanabenz (internal standard) were measured by selected reaction monitoring. The method was validated and applied in a bioequivalence trial on 26 female volunteers. Lamotrigine chewable tablets (100 mg) were administered and monitored for up to 96 h. RESULTS: The method was linear between 0.05 and 5.0 µg/ml, with acceptable stability, accuracy and precision. Mean maximum plasma concentration was 1.37 µg/ml and was reached at 1.6 h postdose. Elimination half-life was 32.7 h. CONCLUSION: Lamotrigine tablets were bioequivalent. Ultra-performance liquid chromatography with tandem mass spectrometry represents a powerful tool in terms of sensitivity, specificity and high-throughput analysis.

Ultra-fast chromatographic micro-assay for quantification of diphenidol in plasma: application in an oral multi-dose switchability trial.

Pharmacokinetics of diphenidol (DPN) is limited due to the lack of analytical methodology. Here, a micro-assay for DPN quantification was developed, by coupling ultra-performance liquid chromatography with tandem mass spectrometry. The procedure involved plasma precipitation and injection of supernatant into UPLC with an Acquitytrade mark C18 column. Detection was in positive electrospray, following transitions of m/z 310.3 --> 292.3 and m/z 275.3 --> 230.2 for DPN and chlorphenamine (internal standard), respectively. The method was linear with a range of 4-400 ng/mL, and a 2 min run time. This method was applied in a switchability trial, where both formulations of DPN were bioequivalent.

Development of an ultra-performance liquid chromatography technique coupled with mass spectrometry for the measurement of tacrolimus in micro-samples of whole blood, and its application on a pharmacokiinetic trial.

OBJECTIVE: The aim was to develop a rapid, specific, sensitive and accurate chromatographic technique coupled with mass spectrometry for the measurement of tacrolimus (CAS 104987-11-3) in microsamples of whole blood, and its application on a pharmacokinetic pilot trial. METHODS: A fast gradient was designed in an ultra-performance liquid chromatography, and coupled with a mass spectrometer for the quantification of tacrolimus in 100 microl samples of EDTA whole blood. Multiple reaction monitoring was used for the measurement of tacrolimus (m/z(+1) 821.49-->4768.35 Th) and sirolimus as internal standard (m/z(+1) 931.69-->864.39 Th). The method was validated according to Mexican regulatory guidelines. Twenty-four young healthy male volunteers with similar hematocrit values participated in the pharmacokinetic trial; an oral single dose of one 5 mg tacrolimus capsule was administered and kinetic profiles were described since 0 h until 24 h post-dose. RESULTS: Method showed to be accurate, precise and linear over the range from 1 to 80 ng/ml, having an absolute recovery of 94%. Molecule was stable for two months at -70 degrees C, and heparin interfered with its quantification. Total run-time is around 1.5 min. Mean maximum blood concentration was 32.63 +/- 1.74 ng/ml, and was reached at 1 h post-dose; elimination half-life was 14.18 +/- 5.71 h. CONCLUSIONS: Method developed is not time-consuming, inexpensive, and sensitive enough for its application during pharmacokinetic trials, and can be suitable for therapeutic drug monitoring in transplanted patients. Pharmacokinetic data obtained in Mexican population are quite similar to previously reported in international literature.

Ciprofloxacin bioavailability is enhanced by oral co-administration with phenazopyridine: a pharmacokinetic study in a Mexican population.

BACKGROUND: and objective: In Mexico, urinary tract infections (UTIs) constitute the second most frequent type of infections treated at primary-care clinics. Ciprofloxacin has played a major role in the treatment of UTIs because it has a broad spectrum of antibacterial activity. In addition to antimicrobial agents, phenazopyridine has been used to alleviate symptoms that occur during episodes of UTI. Thus, the present study was designed to compare the pharmacokinetic behaviour of ciprofloxacin administered alone versus ciprofloxacin combined with phenazopyridine. PATIENTS AND METHODS: Twenty-four healthy male Mexican volunteers participated in this project. The study was carried out with a single oral dose of ciprofloxacin 500mg. The double-blind, crossover, randomised, balanced trial design comprised two treatments, two periods and two sequences. After administration of the study medication, serial blood samples were collected for a period of 12 hours. The harvested plasma was analysed for ciprofloxacin by high-performance liquid chromatography. The area under the concentration-time curve to last measurable concentration (AUC(t)), area under the concentration-time curve extrapolated to infinity (AUC(infinity)), peak plasma concentration (C(max)), time to reach C(max) (t(max)), mean residence time (MRT), elimination constant (k(e)) and elimination half-life (t(1/2)) were determined from plasma concentrations of both treatments and considered as primary variables for statistical analysis. RESULTS: While there were no differences between the two treatments in terms of C(max) and k(e), AUC(t )and AUC(infinity) were 35% and 29% higher, respectively, in the combined treatment arm. Moreover, a significant delay in t(max )(from 1 to 1.5 hours) and a statistical increase of 29% in MRT were also observed with phenazopyridine co-administration. CONCLUSION: Oral co-administration of phenazopyridine increases ciprofloxacin bioavailability with regard to the amount absorbed (AUC) and permanence in the body (MRT), which could be useful during treatment.