Considerations and perspectives on the practice of incurred sample reanalysis assessment for chiral drugs.

Given the inherent complexities of bioanalysis, the role of incurred sample reanalysis (ISR) is increasingly appreciated in regulatory bioanalysis. Incurred sample reanalysis has evolved as an integral part of an assay to ensure method reproducibility. The current regulatory ISR guidelines do not provide clarity regarding ISR assessment for chiral drugs comprising enantiomers. Because chiral assays evaluate two enantiomers, there are additional complexities associated with the ISR data generation and interpretation. Based on the current literature, the practices for conducting ISR in chiral methods were reviewed and assessed. While ISR was conducted in chiral methods for both enantiomers using the acceptance criteria prescribed for non-chiral methods, there may be a need to streamline the nuances of ISR data interpretation and define the ISR requirements for chiral methods. The article provides perspectives on the ISR of enantiomeric drugs, including strategy development, by providing various hypothetical scenarios and possible considerations for defining ISR evaluation for chiral assays.

Enantioselective in vitro ADME, absolute oral bioavailability, and pharmacokinetics of (-)-lumefantrine and (+)-lumefantrine in mice.

Lumefantrine (LFN) is a chiral antimalarial drug. Enantioselective in vitro attributes and absolute oral pharmacokinetics for (-)-LFN and (+)-LFN have been characterized in mice. No stereoselectivity was seen with either of the enantiomers when compared with rac-LFN in the executed in vitro studies (solubility, metabolic stability, protein binding, permeability and blood partitioning). Post intravenous or oral administration of rac-LFN, the AUC0-∞ and MRT of (+)-LFN was higher over (-)-LFN, which is reflected in higher clearance value for (-)-LFN. Following (-)-LFN intravenous administration to mice, the key PK parameters were comparable to (-)-LFN from rac-LFN; however, post intravenous administration of (+)-LFN alone to mice, the AUC0-∞ was 1.3-fold higher than (+)-LFN from rac-LFN. Similarly, post oral administration of (-)-LFN to mice, both AUC0-∞ and Cmax were 1.3-fold higher than (-)-LFN from rac-LFN. On other hand, (+)-LFN showed 1.4-fold higher AUC0-∞ and 1.7-fold higher Cmax post oral administration over (+)-LFN from rac-LFN.

A concise review of bioanalytical methods of small molecule immuno-oncology drugs in cancer therapy.

Immuno-oncology (IO) is an emerging option to treat cancer malignancies. In the last two years, IO has accounted for more than 90% of the new active drugs in various therapeutic indications of oncology drug development. Bioanalytical methods used for the quantitation of various IO small molecule drugs have been summarized in this review. The most commonly used are HPLC and LC-MS/MS methods. Determination of IO drugs from biological matrices involves drug extraction from the biological matrix, which is mostly achieved by simple protein precipitation, liquid-liquid extraction and solid-phase extraction. Subsequently, quantitation is usually achieved by LC-MS/MS, but HPLC-UV has also been employed. The bioanalytical methods reported for each drug are briefly discussed and tabulated for easy access. Our review indicates that LC-MS/MS is a versatile and reliable tool for the sensitive, rapid and robust quantitation of IO drugs.

Relevance of preclinical rodent pharmacokinetics in the selection of a companion antibiotic for combining with beta-lactamase inhibitor.

Recent approvals of beta-lactamase inhibitor (BLI) drug in combination with cephalosporins/penems have provided the right impetus for novel BLIs. One important research question, hitherto not addressed, is pertaining to the relevance of preclinical pharmacokinetics for pairing the antibiotic with existing/novel BLI.Two BLI combination drugs: (a) approved (i.e. ceftazidime/avibactam); (b) clinical development (i.e. cefepime/zidebactam) were explored to provide insights to address the research question.Individual intravenous dosing of ceftazidime, avibactam, cefepime and zidebactam was done at 1 mg/kg by intravenous route in Balb/c mice and Wistar rats. Serial blood samples were collected and analysed by LC-MS/MS method.Examination of the ratios of pharmacokinetic parameters (CL, VSS and T1/2) for individual drugs in combinations (for instance, CL (ceftazidime)/CL (avibactam); CL (cefepime)/CL (zidebactam)) suggested that the pharmacokinetic data gathered in rats were generally within 0.5- to 2-fold; but mouse data revealed larger disparity for VSS (0.11- to 8.25-fold) or CL (0.49- to 4.03-fold).The observed ratio for CL/VSS observed in rats agreed with corresponding human ratios for the pairwise comparison of the individual drugs in the combinations.Retrospectively, current pharmacokinetic findings suggest rat pharmacokinetic data may aid the combination of BLI with an appropriate antibiotic.

Uptake and pharmacokinetics of cefuroxime in rabbits after intravitreal, intracameral, and topical dosing: relevance to human ocular injection of cefuroxime.

Cefuroxime is one of the widely used antibiotics. The objective of this study was to determine pharmacokinetics and disposition in various ocular tissues following topical (TOP), intracameral (IC) and intravitreal (IVT) administration of cefuroxime to rabbits.Following TOP, IC and IVT dosing plasma and various ocular tissues (aqueous humor (AH), vitreous humor (VH), conjunctiva, trabecular mesh (TM), lens and retina-choroid (RC)) were collected and analyzed to understand the disposition of cefuroxime. Postintravenous administration plasma samples were collected to determine the systemic pharmacokinetics.Post-TOP dosing cefuroxime concentrations were observed only in conjunctiva up to 48 h. IC administration showed cefuroxime concentrations in AH up to 8 h; in conjunctiva, TM and plasma, the concentration lasted up to 4 h and in RC and VH till 1 h. IVT administration of cefuroxime showed concentrations in all ocular tissues (up to 8 h) and lasted up to 48 h except in conjunctiva and RC.There was evidence that the mechanism(s) of cefuroxime entry into the eye by via IVT, IC and TOP routes is clearly different. The present ocular tissue data may aid clinicians for considering appropriate choice in the treatment of post-operative ocular complications due to bacterial infections including endophthalmitis.

Stereoselective pharmacokinetics and tissue distribution of levodropropizine after administration of pure levodropropizine and the rac-dropropizine to Sprague-Dawley rats.

Levodropropizine (LDP) is a non-opioid anti-tussive. The stereoselective pharmacokinetics and tissue distribution (TD) of LDP vs. dextrodropropizine (DDP) have been characterized after oral and intravenous (IV) administration of LDP and rac-dropropozine in rats.Oral/IV doses of 50/5.0 mg/kg and 25/2.5 rac-dropropizine and LDP were employed. TD study focused on tissues such as liver, lung and kidney. Blood samples were collected for pharmacokinetic and TD evaluation. Validated methods were used to quantitate LDP, DDP and rac-dropropizine.No stereoselectivity in pharmacokinetics was observed between LDP vs. DDP following rac-dropropizine. However, LDP pharmacokinetics after LDP administration (oral/IV) appeared to be different compared to LDP derived from rac-dropropizine.TD data were similar between the two enantiomers regardless of oral/IV rac-dropropizine administration. When LDP alone was administered, levels were comparable to those derived for LDP from rac-dropropizine after oral/IV. However, in the lung and kidney tissues, the exposure after oral dosing was higher for LDP alone as compared to LDP from rac-dropropizine.In summary, complete characterization of stereoselective pharmacokinetics and TD of rac-dropropizine has been reported after oral/IV routes. It was evident that the presence of DDP, increased the plasma/tissue exposure of LDP which was evident after oral rac-dropropizine dosing.

Incurred sample reanalysis of cefuroxime in rabbit ocular tissues-A case study.

In this paper, we present the incurred sample reanalysis (ISR) data for cefuroxime in various ocular tissues of rabbits. Based on the cefuroxime concentration vs. time profile in various ocular tissues, three chosen time points enabled ISR assessment. Cefuroxime was quantitated in the ocular tissues using a published liquid chromatography-electrospray ionization/tandem mass spectrometry method operated under the multiple reaction-monitoring mode in positive ion mode. Regardless of the ocular tissue, the linearity range was 12.7-2760 ng/ml with a correlation coefficient (r2 ) of ≥0.996. All of the ISR samples representing various ocular tissues met the acceptance criteria. To the best of our knowledge, this is the first report showing the ISR of ocular tissues in any species.

A review of bioanalytical methods for chronic lymphocytic leukemia drugs and metabolites in biological matrices.

Quantitation of drugs used for the treatment of chronic lymphocytic leukemia in various biological matrices during both pre-clinical and clinical developments is very important, often in routine therapeutic drug monitoring. The first developed methods for quantitation were traditionally done on LC in combination with either UV or fluorescence detection. However, the emergence of LC with mass spectrometry in tandem in early 1990s has revolutionized the quantitation as it has provided better sensitivity and selectivity within a shorter run time; therefore it has become the choice of method for the analysis of various drugs. In this article, an overview of various bioanalytical methods (HPLC or LC-MS/MS) for the quantification of drugs for the treatment of chronic lymphocytic leukemia, along with applicability of these methods, is given.

Novel methodology to perform incurred sample reanalysis on dried blood spot cards: Experimental data using darolutamide and filgotinib.

Different options on performing incurred sample reanalysis (ISR) on dried blood spot (DBS) cards were investigated using drugs belonging to various therapeutic areas: (a) darolutamide (to treat prostate cancer) and (b) filgotinib (to treat rheumatoid arthritis). The proposed novel methodology included the generation of half-DBS and quarter-DBS discs after initial blood collection using the full-DBS discs. Accordingly, blood collection via DBS was performed in male BALB/c mice following intravenous and oral dosing of darolutamide; in male Sprague Dawley rats following intravenous and oral dosing of filgotinib. The ISR data generated from the full-DBS disc, half-DBS disc and quarter-DBS disc were compared for the assessment of the proposed methodology. Quantification of darolutamide and filgotinib was accomplished using liquid chromatography-electrospray ionization/tandem mass spectrometry methods. Darolutamide and filgotinib ISR samples, which were collected and prepared using full-, half- and quarter-DBS discs, met the acceptance criteria for ISR analysis. In conclusion, this is the first report showing a viable tool for the performance of ISR on DBS cards. The use of quarter- or half-DBS discs would aid in not only ISR but also in long-term storage experiments of analytes because it would avoid the need for additional blood sampling in patients.

Enantioselective LC-ESI-MS/MS method for quantitation of (-)-lumefantrine and (+)-lumefantrine in mice plasma and application to a pharmacokinetic study.

We developed and validated a simple, sensitive, selective, and reliable LC-MS/MS-ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(-)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 → 512.30 and 237.00 → 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39-895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03-6.14 and 6.36-8.70 and 2.03-4.88 and 5.82-11.5 for (-)-LFN and (+)-LFN, respectively. Both (-)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice.

Stability-indicating HPLC method for acyclovir and lidocaine in topical formulations.

A simple, rapid and accurate stability-indicating HPLC assay was developed for the determination of acyclovir and lidocaine in topical formulations. Chromatographic separation of acyclovir and lidocaine was achieved using a reversed-phase C18 column and a gradient mobile phase (20 mm ammonium acetate pH 3.5 in water and acetonitrile). The degradation products of acyclovir and lidocaine in the samples were analyzed by ultra performance liquid chromatography-time of flight mass spectrometry. The HPLC method successfully resolved the analytes from the impurities and degradation products in the topical formulation. Furthermore, the method detected the analytes from the human skin leachables following the extraction of the analytes in the skin homogenate samples. The method showed linearity over wide ranges of 5-500 and 10-200 µg/ml for acyclovir and lidocaine in the topical product, respectively, with a correlation coefficient (r2 ) >0.9995. The relative standard deviations for precision, repeatability, and robustness of the method validation assays were <2%. The skin extraction efficiency for acyclovir and lidocaine was 92.8 ± 0.7% and 91.3 ± 3.2%, respectively, with no interference from the skin leachables. Thus, simultaneous quantification of acyclovir and lidocaine in the topical formulations was achieved.

Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study.

A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d6 as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C18 column. The injection volume and flow rate were 5.0 µl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25-254 and 2.60-622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58-13.0 and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze-thaw cycles and long-term storage at -80°C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.

Strategy for the Prediction of Steady-State Exposure of Digoxin to Determine Drug-Drug Interaction Potential of Digoxin With Other Drugs in Digitalization Therapy.

Digoxin, a narrow therapeutic index drug, is widely used in congestive heart failure. However, the digitalization therapy involves dose titration and can exhibit drug-drug interaction. Ctrough versus area under the plasma concentration versus time curve in a dosing interval of 24 hours (AUC0-24h) and Cmax versus AUC0-24h for digoxin were established by linear regression. The predictions of digoxin AUC0-24h values were performed using published Ctrough or Cmax with appropriate regression lines. The fold difference, defined as the quotient of the observed/predicted AUC0-24h values, was evaluated. The mean square error and root mean square error, correlation coefficient (r), and goodness of the fold prediction were used to evaluate the models. Both Ctrough versus AUC0-24h (r = 0.9215) and Cmax versus AUC0-24h models for digoxin (r = 0.7781) showed strong correlations. Approximately 93.8% of the predicted digoxin AUC0-24h values were within 0.76-fold to 1.25-fold difference for Ctrough model. In sharp contrast, the Cmax model showed larger variability with only 51.6% of AUC0-24h predictions within 0.76-1.25-fold difference. The r value for observed versus predicted AUC0-24h for Ctrough (r = 0.9551; n = 177; P < 0.001) was superior to the Cmax (r = 0.6134; n = 275; P < 0.001) model. The mean square error and root mean square error (%) for the Ctrough model were 11.95% and 16.2% as compared to 67.17% and 42.3% obtained for the Cmax model. Simple linear regression models for Ctrough/Cmax versus AUC0-24h were derived for digoxin. On the basis of statistical evaluation, Ctrough was superior to Cmax model for the prediction of digoxin AUC0-24h and can be potentially used in a prospective setting for predicting drug-drug interaction or lack of it.

Differential pharmacokinetic drug-drug interaction potential of eletriptan between oral and subcutaneous routes.

1. Pharmacokinetic drug-drug interaction (DDI) data is important from a label claim either in combination drug usage or in polypharmacy situation. 2. Eletriptan undergoes first pass related metabolism through CYP3A4 enzyme to form pharmacologically active N-desmethyl metabolite. 3. Differential DDI interaction of the concomitant oral dosing of ketoconazole (20.1 mg/kg), a CYP3A4 inhibitor, with oral (4.2 mg/kg) or subcutaneous dose (2.1 mg/kg) of eletriptan was evaluated in male Sprague Dawley rats. Serial pharmacokinetic samples were collected and simultaneously analysed for eletriptan/N-desmethyl eletriptan using validated assay. Non-compartmentally derived pharmacokinetic parameters for various treatments were analysed statistically. 4. After oral eletriptan in presence of ketoconazole, Cmax (40 vs. 32 ng/mL alone) and AUCinf (81 vs. 24 ng.h/mL alone) of eletriptan increased; the formation of N-desmethyl eletriptan decreased (Cmax=1.1 ng/mL, 3.9%) with ketoconazole as compared to without treatment (Cmax=3.7 ng/mL, 11.2%). After subcutaneous eletriptan in presence of ketoconazole, there was no change in Cmax (153 vs.152 ng/mL) or AUCinf (267 vs. 266 ng.h/mL) of eletriptan. Formation of N-desmethyl eletriptan after the subcutaneous dose was determined at few intermittent time points with/without ketoconazole. 5. Preclinical data support differential DDI of eletriptan when dosed oral vs. subcutaneous, which need to be evaluated in a clinical setting.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of ZYTP1, a novel poly (ADP-ribose) polymerase inhibitor.

ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications. The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1. ZYTP1 IC50 for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC50 of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam. The mean IC50 values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3-58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC50 of depropylated metabolite was 11.95-24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC50 shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075 min-1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively. Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.

Rats and rabbits as pharmacokinetic screening tools for long acting intramuscular depots: case study with paliperidone palmitate suspension.

Development of prodrug of 9-hydroxyrisperidone (paliperidone) long-acting intramuscular injection has enabled delivery over four-week time period with improved compliance. The key aim of this work was to establish a reliable preclinical model which may potentially serve as a screening tool for judging the pharmacokinetics of paliperidone formulation(s) prior to human clinical work. Sparse sampling composite study was used in rats, (Wistar/Sprague-Dawley (SD; n = 10)) and a serial blood sampling study design was used in rabbits (n = 4). Animals received intramuscular injection of paliperidone palmitate in the thigh muscle at dose of 16 (rats) and 4.5 mg/kg (rabbits). Samples were drawn in rats (retro-orbital sinus) and rabbits (central ear artery) and were analysed for paliperidone using liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) assay. The plasma data was subjected to pharmacokinetic analysis. Following intramuscular injection of depot formulation in Wistar/SD rats and rabbits, absorption of paliperidone was slow and gradual with median value of time to reach maximum concentration (Tmax) occurring on day 7. The exposures (i.e. area under the curve (AUC; 0-28) days) were 18,597, 21,865 and 18,120 ng.h/mL, in Wistar, SD and rabbits, respectively. The clearance was slow and supported long half-life (8-10 days). Either one of the two models can serve as a research tool for establishing pharmacokinetics of paliperidone formulation(s).

Impact of collagen-induced arthritis on the pharmacokinetic disposition of voriconazole, a widely used antifungal agent: in vitro and in vivo investigations in DBA/1J mice.

Pharmacokinetics of voriconazole, an anti-fungal agent, was determined in collagen-induced arthritic (CIA) and healthy DBA/1J mice. CIA was confirmed in DBA/1J mice by clinical scoring and histological analysis. In vivo oral pharmacokinetic study (3 mg/kg) and in vitro stability assessment in liver microsomes were performed in CIA vs. healthy DBA/1J mice. Additionally, hepatic portal vein cannulated (HPVC) CIA and healthy mice were used to clarify the role of gut first-pass effect. Voriconazole/N-oxide metabolite was measured in plasma and in vitro samples using liquid chromatography tandem-mass spectrometry method. Voriconazole exposure was reduced in CIA by 27% as compared to healthy mice. Formation of voriconazole N-oxide was higher in CIA mice as evidenced by higher molar Cmax ratio (i.e. metabolite/parent) of 2.08 vs. 1.66 in healthy mice. Because voriconazole was stable in microsomes, involvement of presystemic gut metabolism was suspected for decreased voriconazole exposure and formation of higher molar ratio of metabolite. HPVC work revealed higher formation of voriconazole N-oxide in CIA relative to healthy mice resulting in Cmax/AUC ratios of 0.41/0.54 and 0.08/0.17, respectively, confirming first-pass effect. The findings may have implications in the clinical therapy of arthritis patients who are concomitantly given voriconazole for the management of fungal infections.

Incurred sample reanalysis in drug discovery bioanalysis.

Bioanalysis plays a key role during the drug discovery process to generate the pharmacokinetic data to facilitate unbiased evaluation of leads, optimized leads and drug candidates. Such pharmacokinetic data are used to enable key decisions in the drug discovery process. The aim of the work is to put forward a new strategy of performing the incurred sample reanalysis for select small molecule novel chemical entities at different stages of drug discovery prior to candidate selection. Three discovery programs representing hits, leads and optimized lead candidates were selected for the incurred sample reanalysis (ISR) analysis. From each discovery program, two novel chemical entities were selected for the ISR analysis. The time points considered for ISR generally varied among the programs; however, samples coinciding with drug absorption, distribution and elimination were considered in the ISR assessment. With the exception of a single ISR value that gave a high deviation (about 63%), the observed ISR values supported the discovery bioanalytical assays. While the individual bioanalytical laboratory can draw an algorithm for selecting novel chemical entities and fixing the acceptance criteria for the ISR data, it is proposed that the percentage difference between ISR vs. original concentration for 67% of the repeat samples is contained within ±30% for discovery bioanalysis.

Review of DBS methods as a quantitative tool for anticancer drugs.

An overview of published dried blood spot (DBS) methods for the quantitation of various classes of anticancer drugs from clinical and preclinical studies is presented. The increased reporting of DBS methods in the literature for quantitation of various classes of drugs is a testimony to their utility in bioanalytical applications. While DBS offers several advantages as compared with conventional wet sampling techniques, there remain a number of nuances that may impede the assay adaptability of DBS method in routine quantitative bioanalysis. This review covers several case studies of DBS application in the quantitation of anticancer drugs. Some perspectives are provided on the optimization of the DBS method with respect to the selection of DBS card, spot volume, hematocrit effect and other regular validation parameters, which are essential in quantitative bioanalysis. Some thoughts are provided on the existing gaps in the DBS method and possible remedial measure(s) to address such gaps. Although DBS methods have great potential, there is the need for a global consensus including regulatory support on the type of validation experiments to be performed to support quantitative data.