Evaluation of drug-drug interactions of pemigatinib in healthy participants.

PURPOSE: Pemigatinib (INCB054828), a potent and selective oral fibroblast growth factor receptor 1-3 inhibitor, is a Biopharmaceutical Classification System class II compound with good permeability and pH-dependent solubility that is predominantly metabolized by cytochrome P450 (CYP) 3A. Two drug-drug interaction studies, one with acid-reducing agents, esomeprazole (proton pump inhibitor [PPI]) and ranitidine (histamine-2 [H2] antagonist), and the other with potent CYP3A-modulating agents, itraconazole (CYP3A inhibitor) and rifampin (CYP3A inducer), were performed. METHODS: Both were open-label, fixed-sequence studies conducted in up to 36 healthy participants each, enrolled into two cohorts (n = 18 each). Pemigatinib plasma concentration was measured, and pharmacokinetic parameters were derived by non-compartmental analysis. RESULTS: There was an 88% and 17% increase in pemigatinib area under the plasma drug concentration-time curve (AUC) and maximum plasma drug concentration (Cmax), respectively, with itraconazole, and an 85% and 62% decrease in pemigatinib AUC and Cmax with rifampin coadministration. There was a 35% and 8% decrease in pemigatinib AUC and Cmax, respectively, with esomeprazole, and a 2% decrease in Cmax and 3% increase in AUC with ranitidine coadministration. In both studies, all adverse events reported were grade ≤ 2. CONCLUSION: Coadministration with itraconazole or rifampin resulted in a clinically significant change in pemigatinib exposure. Therefore, coadministration of strong CYP3A inducers with pemigatinib should be avoided, and the dose of pemigatinib should be reduced if coadministration with strong CYP3A inhibitors cannot be avoided. The effect of PPIs/H2 antagonists on pemigatinib exposure was modest, and pemigatinib can be administered without regard to coadministration of PPIs/H2 antagonists.

Hemoadsorption eliminates remdesivir from the circulation: Implications for the treatment of COVID-19.

Both antiviral treatment with remdesivir and hemoadsorption using a CytoSorb® adsorption device are applied in the treatment of severe COVID-19. The CytoSorb® adsorber consists of porous polymer beads that adsorb a broad range of molecules, including cytokines but also several therapeutic drugs. In this study, we evaluated whether remdesivir and its main active metabolite GS-441524 would be adsorbed by CytoSorb® . Serum containing remdesivir or GS-441524 was circulated in a custom-made system containing a CytoSorb® device. Concentrations of remdesivir and GS-441524 before and after the adsorber were analyzed by liquid chromatography-tandem mass spectrometry. Measurements of remdesivir in the outgoing tube after the adsorber indicated almost complete removal of remdesivir by the device. In the reservoir, concentration of remdesivir showed an exponential decay and was not longer detectable after 60 mins. GS-441524 showed a similar exponential decay but, unlike remdesivir, it reached an adsorption-desorption equilibrium at ~48 µg/L. Remdesivir and its main active metabolite GS-441524 are rapidly eliminated from the perfusate by the CytoSorb® adsorber device in vitro. This should be considered in patients for whom both therapies are indicated, and simultaneous application should be avoided. In general, plasma levels of therapeutic drugs should be closely monitored under concurrent CytoSorb® therapy.

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum.

BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 µL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.

Validation of LC-MS/MS methods for determination of remdesivir and its metabolites GS-441524 and GS-704277 in acidified human plasma and their application in COVID-19 related clinical studies.

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies.

In Vitro and In Vivo Rat Model Assessments of the Effects of Vonoprazan on the Pharmacokinetics of Venlafaxine.

PURPOSE: The purpose of the present study was to investigate the effects of vonoprazan on the pharmacokinetics of venlafaxine in vitro and in vivo. METHODS: The mechanism underlying the inhibitory effect of vonoprazan on venlafaxine was investigated using rat liver microsomes. In vitro, the inhibition was evaluated by determining the production of O-desmethylvenlafaxine. Eighteen male Sprague-Dawley rats were randomly divided into three groups: control group, vonoprazan (5 mg/kg) group, and vonoprazan (20 mg/kg) group. A single dose of 20 mg/kg venlafaxine was administrated to rats orally without or with vonoprazan. Plasma was prepared from blood samples collected via the tail vein at different time points and concentrations of venlafaxine and its metabolite, O-desmethylvenlafaxine, were determined by ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: We observed that vonoprazan could significantly decrease the amount of O-desmethylvenlafaxine (IC50 = 5.544 µM). Vonoprazan inhibited the metabolism of venlafaxine by a mixed inhibition, combining competitive and non-competitive inhibitory mechanisms. Compared with that in the control group (without vonoprazan), the pharmacokinetic parameters of venlafaxine and its metabolite, O-desmethylvenlafaxine, were significantly increased in both 5 and 20 mg/kg vonoprazan groups, with an increase in MRO-desmethylvenlafaxine. CONCLUSION: Vonoprazan significantly alters the pharmacokinetics of venlafaxine in vitro and in vivo. Further investigations should be conducted to check these effects in humans. Therapeutic drug monitoring of venlafaxine in individuals undergoing venlafaxine maintenance therapy is recommended when vonoprazan is used concomitantly.

Effects of Concomitant Administration of Vonoprazan Fumarate on the Tacrolimus Blood Concentration in Kidney Transplant Recipients.

Vonoprazan fumarate (vonoprazan) is a new kind of acid suppressant with potent acid inhibitory effects. Therefore, it has been administered to kidney transplant recipients for treatment or prophylaxis of steroid ulcers, refractory peptic ulcers, and gastroesophageal reflux disease. Because tacrolimus, which is a well-established immunosuppressant for kidney transplantation, and vonoprazan share the CYP3A4 system for metabolism, drug interactions are anticipated upon simultaneous administration. We retrospectively analyzed 52 kidney transplant recipients who were converted from rabeprazole, which has a small effect on the tacrolimus trough blood concentration (C0), to vonoprazan between August 2016 and July 2019. We compared the tacrolimus C0/tacrolimus dose (C0/D) before and after conversion and serum liver enzymes, serum total bilirubin, and the estimated glomerular filtration rate (eGFR). As a result, mean tacrolimus C0/D before and after conversion was 1.98 ± 1.02 and 2.19 ± 1.15 (ng/mL)/(mg/d), respectively, (p < 0.001). Additionally, mean aspartate transaminase (AST) before and after conversion was 18.6 ± 4.2 and 19.6 ± 5.2 IU/L, respectively, (p = 0.037). Mean alanine transaminase (ALT) before and after conversion was 15.8 ± 5.5 and 17.6 ± 7.1 IU/L, respectively, (p = 0.007). Mean eGFR before and after conversion was 50.6 ± 14.4 and 51.4 ± 14.7 mL/min/1.73 m2, respectively (p = 0.021). Mean AST, ALT, and eGFR were slightly but significantly elevated within normal ranges after conversion. In conclusion, our study suggests that the mean tacrolimus C0/D was elevated significantly by converting from rabeprazole to vonoprazan, but it had little clinical significance. Vonoprazan can be administered safely to kidney transplant recipients receiving tacrolimus.

Pharmacokinetic interactions of esaxerenone with amlodipine and digoxin in healthy Japanese subjects.

BACKGROUND: To investigate the effects of coadministration of esaxerenone with amlodipine on the pharmacokinetics (PK) of each drug, and of esaxerenone on the PK of digoxin. METHODS: In three open-label, single-sequence, crossover studies, healthy Japanese males received single oral doses of esaxerenone 2.5 mg (Days 1, 15), with amlodipine 10 mg/day (Days 8-18) (Study 1, N = 24); single doses of amlodipine 2.5 mg (Days 1, 21), with esaxerenone 5 mg/day (Days 8-25) (Study 2; N = 20); or digoxin 0.25 mg/day (Days 1-15) with esaxerenone 5 mg/day (Days 11-15) (Study 3; N = 20). PK parameters and safety were assessed. RESULTS: Study 1: esaxerenone peak plasma concentration (Cmax) and time to Cmax were unaltered by amlodipine coadministration, but mean half-life was slightly prolonged from 18.5 to 20.9 h. Geometric least-squares mean (GLSM) ratios for Cmax, area under the plasma concentration-time curve (AUC) from zero to last measurable concentration and from zero to infinity for esaxerenone + amlodipine versus esaxerenone were 0.958, 1.154, and 1.173, respectively. Study 2: corresponding GLSM ratios for amlodipine + esaxerenone versus amlodipine were 1.099, 1.185, and 1.214. Study 3: esaxerenone did not markedly alter digoxin PK. GLSM ratios for Cmax, trough plasma concentration, and AUC during a dosing interval for digoxin versus esaxerenone + digoxin were 1.130, 1.088, and 1.072, respectively. CONCLUSIONS: No drug-drug interactions are expected during combination therapy with esaxerenone and either amlodipine or digoxin, based on a lack of any clinically relevant PK changes. TRIAL REGISTRATION: Studies 1 and 2: JapicCTI-163379 (registered on 20 September 2016); Study 3: JapicCTI-163443 (registered on 24 November 2016).

A validated LC-MS/MS method for the quantification of capivasertib in dog plasma: Application to its pharmacokinetics study.

In this study, a simple and reliable liquid chromatography tandem mass spectrometric method was first developed for the determination of capivasertib in dog plasma with ipatasertib as internal standard. The plasma samples were deproteinated using acetonitrile. An Acquity BEH C18 column (1.7 µm, 2.1 × 50 mm) maintained at 40°C was used for chromatographical separation, with water containing 0.1% formic acid and acetonitrile as mobile phase. Multiple reaction monitoring transitions were m/z 429.2 > 135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. Excellent linearity was achieved in the concentration range of 1-1,000 ng/ml with a correlation coefficient of >0.9981. The lower limit of quantification was 1 ng/ml. The extraction recovery of capivasertib from dog plasma was >85.81% and no significant matrix effect was found. The intra- and inter-day precision was <9.58% and the accuracy ranged from -10.60% to 12.50%. The validated method was further applied to the pharmacokinetic study of capivasertib in dog plasma after single oral (5 mg/kg) and intravenous (1 mg/kg) administrations. The results revealed that capivasertib was rapidly absorbed into plasma with good bioavailability (47.04%) and low clearance.

Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient.

Objectives: A method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524. Methods: A simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 â†’ m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 â†’ m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 â†’ m/z 206.0 for remdesivir-13C6. Results: Calibration curves were linear in the 1-5000 µg/L range for remdesivir and 5-2500 for GS-441524, with limit of detection set at 0.5 and 2 µg/L and limit of quantification at 1 and 5 µg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 µg/L for remdesivir and 12.5, 125, 2000 µg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6-110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h. Conclusions: This method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

Effects of naringenin on the pharmacokinetics of tofacitinib in rats.

Context: Naringenin and tofacitinib are often used together for treatment of rheumatoid arthritis in Chinese clinics.Objective: This experiment investigates the effect of naringenin on the pharmacokinetics of tofacitinib in rats.Materials and methods: Twelve Sprague-Dawley rats were randomly divided into two groups (experimental group and control group). The experimental group was pre-treated with naringenin (150 mg/kg/day) for two weeks before dosing tofacitinib, and equal amounts of CMC-Na solution in the control group. After a single oral administration of 5 mg/kg of tofacitinib, 50 µL blood samples were directly collected into 1.5 mL heparinized tubes via the caudal vein at 0.083, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h. The plasma concentration of tofacitinib was quantified by UPLC/MS-MS.Results: Results indicated that naringenin could significantly affect the pharmacokinetics of tofacitinib. The AUC0-24 of tofacitinib was increased from 1222.81 ± 222.07 to 2016.27 ± 481.62 ng/mL/h, and the difference was significant (p < 0.05). Compared with the control group, the Tmax was increased from 0.75 ± 0.29 to 3.00 ± 0.00 h (p < 0.05), and the MRT(0-24) was increased from 4.90 ± 0.51 to 6.57 ± 0.66 h (p < 0.05), but the clearance was obviously decreased from 4.10 ± 0.72 to 2.42 ± 0.70 L/h/kg (p < 0.05) in experimental group. Although the Cmax and t1/2 of tofacitinib were increased, there were no significant differences (p > 0.05).Conclusions: This research demonstrated a drug-drug interaction between naringenin and tofacitinib possibly when preadministered with naringenin; thus, we should pay attention to this possibility in the clinic.

Simultaneous determination of Rivaroxaban and TAK-438 in rat plasma by LC-MS/MS: application to pharmacokinetic interaction study.

Aim: A sensitive and reliable LC-MS/MS method has been established and validated to the quantitation of rivaroxaban (RIV) and TAK-438 in rat plasma using carbamazepine as internal standard. Results: The procedure of method validation was conducted according to the guidelines of EMA and US FDA. At the same time, the method was applied to pharmacokinetic interactions study between RIV and TAK-438 for the first time. When RIV and TAK-438 co-administration to rats, main pharmacokinetic parameters of TAK-438 like AUC(0-t), AUC(0-∞) and Cmax had statistically significant increase. The main pharmacokinetic parameters of RIV have no statistically significant difference (p > 0.05) when co-administered except for t1/2 (p < 0.01). Conclusion: The results indicated that drug-drug interactions occurred between RIV and TAK-438 when co-administered to rats.

Method of Determining Pyrrole-Imidazole Polyamide in Rat Plasma Using Ultra-Fast Liquid Chromatography-Ultraviolet Spectrometry.

To improve the efficiency of drug-discovery research on pyrrole-imidazole polyamides (PIs), a more rapid method for quantitative and qualitative measurement of PI in rat plasma samples was developed here using ultra-fast liquid chromatography-ultraviolet spectrometry (UFLC-UV) in order to shorten the measurement time. A measurement method of PIs by HPLC developed until now takes 45 min for one sample measurement. This method was inefficient to investigate extraction conditions from biological samples and measurement of animal experimental samples. In the developed method of this study, PI and phenacetin (internal standard, IS) were separated with an ACQUITY UPLC HSS T3 (1.8 µm, 2.1 × 50 mm; Nihon Waters K.K., Japan) column using a mobile phase of 0.1% acetic acid (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min with a linear gradient. The detection wavelength was 310 nm. The calibration curve was linear in the range of 0.225-4.5 µg/mL (correlation coefficients ≥0.9995, n = 5). The intra- and inter-day accuracies were in the range of -6.04 to 12.2%, and the precision was less than 2.99%. The measurement time of this method (7 min per injection) was markedly shortened to about one-sixth of the previous measurement time (45 min per injection). This is the first report describing the quantitative and qualitative measurement of PI in plasma using UFLC-UV. The present method will be very useful for the drug-discovery research of PIs.

A new high-performance liquid chromatography-tandem mass spectrometry method for the determination of sunitinib and N-desethyl sunitinib in human plasma: Light-induced isomerism overtaking towards therapeutic drug monitoring in clinical routine.

Sunitinib is approved for advanced renal cell cancer, imatinib-resistant or -intolerant gastrointestinal stromal tumors and pancreatic neuroendocrine cancers. It is prescribed at a fixed dose but its plasma exposure shows large inter-individual variations. Taking into account the narrow therapeutic window and the positive exposure-efficacy relationship, there is a robust rationale for its therapeutic drug monitoring. In fact, a target plasma concentration of sunitinib plus its active metabolite, N-desethyl sunitinib, ≥50 ng/mL was suggested. In order to quantify sunitinib and N-desethyl sunitinib in patients' plasma, we developed and validated a new LC-MS/MS method applicable to clinical routine. In solution, sunitinib and N-desethyl sunitinib undergo to photo-isomerization and many published methods overcome this problem by conducting the entire procedures of samples collection and handling under strictly light-protection. Our method is based on a simple and fast procedure that quantitatively reconverts the E-isomer of both analytes, obtained during sample draw and processing without light-protection, into their Z-forms. Moreover, our method uses a small plasma volume (30 µL) and the analytes are extracted by a rapid protein precipitation. It was validated according to EMA-FDA guidelines. The calibration curves resulted linear (R2 always >0.993) over the concentration ranges (0.1-500 ng/mL for sunitinib, 0.1-250 ng/mL for N-desethyl sunitinib) with a good precision (within 7.7 % for sunitinib and 10.8% for N- desethyl sunitinib) and accuracy (range 95.8-102.9% for sunitinib and 92.3-106.2% for N-desethyl sunitinib). This method was applied to a pharmacokinetic study in one patient treated with sunitinib. Moreover, as incurred samples reanalysis is an established part of the bioanalytical process to support clinical studies, its assessment was performed early in order to assure that any reproducibility issues was detected as soon as possible. The percentage difference between the two runs resulted within ±20% in all the re-analysed samples for both sunitinib and N- desethyl sunitinib.

Quantitative tracking of inflammatory activity at the peak and trough plasma levels of tofacitinib, a Janus kinase inhibitor, via in vivo 18 F-FDG PET.

PURPOSE: To assess the capability of in vivo positron emission tomography (PET) using 18 F-fluorodeoxyglucose (18 F-FDG) to quantify changes in inflammatory activity in response to tofacitinib, a Janus kinase (JAK) inhibitor, over a timeframe of a few hours to few days in a preclinical model of rheumatoid arthritis (RA). METHODS: Twenty-four mice with collagen-induced arthritis in the following groups were assessed: Group 1, where the changes in PET measures for the extremity joints were evaluated at the peak and trough plasma drug levels after administration of a single dose of tofacitinib (4 hours apart); Group 2, where joint PET measures were assessed before treatment and after 6 days of administration of a daily dose of tofacitinib; and group 3 (controls), where joint PET measures were derived from the same mice, 6 days apart. RESULTS: At about peak plasma levels of the drug after a single tofacitinib administration, there was a reduction in PET measures compared to pretreatment values, suggesting decreased inflammatory activity. These measures were equivalent to those obtained after 6 days of daily dosing by tofacitinib. However, PET measures at trough plasma levels of the drug from tofacitinib administration were significantly higher than those at peak plasma drug levels and equivalent to pretreatment measures. There were insignificant changes in PET measures for the control animals. CONCLUSION: 18 F-FDG PET can detect changes in inflammatory activity occurring in response to the JAK inhibitor tofacitinib: (a) during peak and trough plasma drug levels, that is within mere hours of treatment; and (b) over a span of days.

Pharmacokinetic study of imrecoxib in patients with renal insufficiency.

OBJECTIVE: Renal insufficiency may influence the pharmacokinetics of drugs. We have investigated the pharmacokinetic parameters of imrecoxib and its two main metabolites in individuals with osteoarthritis (OA) with normal renal function and renal insufficiency, respectively. METHODS: This was a prospective, parallel, open, matched-group study in which 24 subjects were enrolled (renal insufficiency group, n = 12; healthy control group, n = 12). Blood samples of subjects administered 100 mg imrecoxib were collected at different time points and analyzed. Plasma concentrations of imrecoxib and its two metabolites (M1 and M2) were determined by the liquid chromatography-tandem mass spectrometry method, and pharmacokinetic parameters (clearance [CL], apparent volume of distribution [Vd], maximum (or peak) serum concentration [Cmax], amount of time drug is present in serum at Cmax [Tmax], area under the curve [AUC; total drug exposure across time], mean residence time [MRT] and elimination half-life [t1/2]) were calculated. RESULTS: The demographic characteristics of the two groups were not significantly different, with the exception of renal function. The mean Cmax and AUC0-t (AUC from time 0 to the last measurable concentration) of imrecoxib in the renal insufficiency group were 59 and 70%, respectively, of those of the healthy control volunteers with normal renal function, indicating a significant decline in the former group (P < 0. 05). The mean pharmacokinetic parameters of Ml in the renal insufficiency and healthy control groups did not significantly differ. In contrast, the mean Cmax and AUC0-t of M2 in the renal insufficiency group were 233 and 367%, respectively, of those of the normal renal function group, indicating a significant increase in the former group (P < 0.05). The mean CL/F (clearance/bioavailability) of M2 of the renal insufficiency group was 37% of that of the normal renal function group, indicating a notable reduction in the former group (P < 0.05). CONCLUSION: The exposure of imrecoxib in OA patients with renal insufficiency showed a decline compared to that in healthy subjects. However, in patients with renal insufficiency the exposure of M2 was markedly increased and the CL was noticeably reduced. These results indicate that the dosage of imrecoxib should be reduced appropriately in patients with renal insufficiency.

Simultaneous determination of imrecoxib and its two active metabolites in plasma of hepatic impairment patients by liquid chromatography-tandem mass spectrometry.

Imrecoxib is a specific inhibitor of cyclooxygenase-2. Its hydroxymethyl (M1) and carboxylic acid (M2) metabolites are the major circulating components in human plasma. It has been demonstrated that the anti-inflammatory activities of M1 and M2 are both equal to the parent drug. In the current study, a highly sensitive and rapid method was established and validated for the determination of imrecoxib, M1 and M2 in human plasma via liquid chromatography-tandem mass spectrometry technique. To our knowledge, this is the first study that simultaneously analyzed imrecoxib and its two active metabolites following a rapid protein-precipitation clean-up. Imrecoxib and its metabolites were separated on a reversed-C18 column (3.5 µm; 100 × 4.6 mm), and the mobile phase was optimized as 5 mM ammonium acetate: acetonitrile: formic acid (35: 65: 0.1, v/v/v), based on the pKa values of analytes. Mass spectrometric detection was conducted in a positive multiple reaction monitoring mode. The m/z transitions of imrecoxib (370.2 → 278.2), M1 (386.2 → 278.2) and M2 (400.2 → 236.2) were selected for an effective balance between sensitivity and selectivity. An excellent linearity was demonstrated over the concentration ranges of 0.100-40.0, 0.200-80.0 and 2.00-800 ng/mL for imrecoxib, M1 and M2, respectively. The method validation was carried out in agreement with the FDA guidance. Furthermore, the pharmacokinetic properties of imrecoxib and its two active metabolites were characterized in patients with moderate hepatic impairment, by using the developed and validated method.

Pyrrole adducts in globin and plasma of workers exposed to hexane.

OBJECTIVES: Urinary excretion of 2,5-hexanedione is currently used to estimate the exposure levels of hexane occurring to an individual during the previous work shift. However, because hexane exposures and urinary 2,5-hexanedione levels can vary considerably from day to day, and subchronic to chronic exposures to hexane are required to produce neuropathy, this biomarker may not accurately reflect the risk of an individual for developing hexane neuropathy. This investigation examines the potential of hexane-derived pyrrole adducts produced on globin and plasma proteins as markers for integrating cumulative exposures. Because the pyrrole markers incorporate bioactivation of hexane to 2,5-hexandione and the initial step of protein adduction involved in hexane-induced neuropathy, they potentially can serve as biomarkers of effect through reflecting pathogenetic events within the nervous system. Additionally, pyrrole formation is an irreversible reaction suggesting that hexane-derived protein pyrroles can be used to assess cumulative exposures to provide a better characterization of individual susceptibilities. METHODS: To examine the utility of the proposed markers, blood samples were obtained from eleven workers who used hexane for granulating metal powders in a slurry to produce metal machining die tools and four non-exposed volunteers. Globin and plasma were isolated, and the proteins were digested using pepsin, reacted with Ehrlich's reagent and the level of pyrrole adducts were determined by absorbance at 530 nm. To determine the dose-response curve and dynamic range of the assay, erythrocytes were incubated with a range of 2,5-hexanedione concentrations and the net absorbance at 530 nm of isolated globin was measured. RESULTS: Pyrrole was detected in both the globin and plasma samples of the workers exposed to hexane and the levels of pyrroles in plasma were positively correlated with the levels of pyrroles in globin for most of the workers. CONCLUSIONS: This investigation demonstrates that detectable levels of hexane-derived protein pyrrole adducts are produced on peripheral proteins following occupational exposures to hexane and supports the utility of measuring pyrroles for integrating cumulative exposures to hexane.

Pyrrole-Hemoglobin Adducts, a More Feasible Potential Biomarker of Pyrrolizidine Alkaloid Exposure.

Pyrrolizidine alkaloids (PAs) are naturally occurring phytotoxins widely distributed in about 3% of flowering plants. The formation of PA-derived pyrrole-protein adducts is considered as a primary trigger initiating PA-induced hepatotoxicity. The present study aims to (i) further validate our previous established derivatization method using acidified ethanolic AgNO3 for the analysis of pyrrole-protein adducts and (ii) apply this method to characterize the binding tendency, dose-response, and elimination kinetics of pyrrole-protein adducts in blood samples. Two pyrrole-amino acid conjugates, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-cysteine (7-cysteine-DHP) and 9-histidine-DHP, were synthesized and used to demonstrate that acidified ethanolic AgNO3 derivatization can cleave both S-linkage and N-linkage of pyrrole-protein adducts. Subsequently, using precolumn AgNO3 derivatization followed by ultra-high-pressure liquid chromatography/mass spectrometry analysis, we quantified pyrrole-protein adducts in monocrotaline-treated rat blood protein fractions, including hemoglobin (Hb), plasma, albumin, and plasma residual protein fractions, and found that the amount of pyrrole-Hb adducts was significantly higher than that in all plasma fractions. Moreover, elimination half-life of pyrrole-Hb adducts was also significantly longer than pyrrole-protein adducts in plasma fractions (12.08 vs 2.54-2.93 days). In addition, we also tested blood samples obtained from five PA-induced liver injury patients and found that the amount of pyrrole-protein adducts in blood cells was also remarkably higher than that in plasma. In conclusion, our findings for the first time confirmed that the AgNO3 derivatization method could be used to measure both S- and N-linked pyrrole-protein adducts and also suggested that pyrrole-Hb adducts with remarkably higher level and longer life span could be a better biomarker of PA exposure.

Characterization of Disulfide Bond Rebridged Fab-Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload.

We describe the characterization of antigen binding fragments (Fab)-drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross-linkers, are a class of payloads used in antibody-drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug-to-Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti-HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti-HER2 trastuzumab Fab-rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen-dependent cancer cell-killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab-pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.