Tool For Pharmacokinetic Studies Research Articles

Application of a Volumetric Absorptive Microsampling (VAMS)-Based Method for the Determination of Paracetamol and Four of its Metabolites as a Tool for Pharmacokinetic Studies in Obese and Non-Obese Patients.

The pharmacokinetic (PK) profile of a drug is influenced by several factors, which can lead to a suboptimal dosing regimen in specific patient populations. As obesity becomes increasingly prevalent, it is important that optimized dosing schemes are available for these patients. To set up such dosing schemes, PK studies should be performed in this population. Regarding paracetamol (acetaminophen [APAP]), obese patients would benefit from a tailored dosing scheme, as both the volume of distribution and metabolism are increased compared with non-obese patients. This includes metabolism by cytochrome P450 2E1, which is involved in APAP-associated hepatotoxicity. To decrease the burden for patients in these PK studies, finger-prick sampling could be used. The aim of this study was to compare the quantitative determination of APAP and four metabolites in different blood-based matrices and to determine if capillary dried blood samples, collected directly following finger-prick, could serve as a tool to investigate APAP PK in obese and non-obese patients. In this study, we performed a clinical validation of methods for the determination of APAP and four of its metabolites (APAP-glucuronide, APAP-sulfate, APAP-mercapturate, and APAP-cysteine) in blood, plasma, and dried blood. The latter was obtained by volumetric absorptive microsampling (VAMS), either starting from the venous blood or collected directly following a finger-prick. Results were compared between the different matrices and, in addition, blood:plasma (B:P) ratios were determined for the different analytes. Liquid and dried venous blood results were in good agreement. Furthermore, differences between capillary (finger-prick) and venous VAMS blood samples remained limited for most analytes. However, for APAP-cysteine, caution should be paid to the interpretation of concentrations in (dried) blood. With the exception of APAP, concentrations were higher in plasma compared with blood, with B:P ratios ranging between 0.52 and 0.65. A time-dependent change in median B:P ratio was observed for APAP and APAP-cysteine. Additionally, a time-dependent trend was seen for APAP, as well as for APAP-glucuronide and APAP-mercapturate, for the distribution between capillary and venous blood. We demonstrated that finger-prick sampling is a viable alternative to conventional venous blood sampling to investigate the PK of APAP and its metabolites in obese and non-obese patients.

An Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Brigatinib and Gilteritinib Using a Specific Polyclonal Antibody.

Brigatinib and gilteritinib are oral tyrosine kinase inhibitors (TKIs). We aimed to develop a simple and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to quantify brigatinib and gilteritinib in various biological matrices. Antiserum against these TKIs was obtained from mice by using 3-methoxy-4-(-4-(4-methylpiperazin-1-yl) piperidin-1-yl) aniline as a hapten, which has a common substructure with these TKIs. The generated antibody was used to develop an indirect competitive ELISA for these TKIs in human serum. The lower limit of quantification of brigatinib and gilteritinib in human serum was 6.2 and 6.8 ng/mL, respectively. The developed ELISA was used to examine the pharmacokinetics of these TKIs after oral administration in mice and rats. This ELISA is expected to be a valuable tool in pharmacokinetic studies of these TKIs.

Determination of paracetamol and its metabolites via LC-MS/MS in dried blood volumetric absorptive microsamples: A tool for pharmacokinetic studies

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic and antipyretic worldwide. Nonetheless, APAP induced hepatotoxicity is the most common cause of acute liver failure in the western world. This hepatotoxicity is related to the metabolism of APAP, via the formation of the electrophilic oxidation product N-acetyl-para-benzoquinone imine. To investigate differences in APAP metabolism in specific patient populations and to optimize dosing regimens, quantification of metabolites from the different metabolic pathways is needed to perform pharmacokinetic (PK) studies. For this purpose, sensitive and short liquid chromatography-tandem mass spectrometry methods were developed for the quantitation of APAP and four of its metabolites (APAP-glucuronide, APAP-sulfate, APAP-mercapturate, and APAP-cysteine) in plasma, whole blood and dried blood microsamples collected via 10 µL volumetric absorptive microsampling (VAMS) devices. The methods were successfully validated based on internationally accepted guidelines (EMA, FDA), encompassing selectivity, evaluation of the calibration model, matrix effect and recovery, accuracy and precision, stability, and dilution integrity. In addition, for the VAMS samples, the effect of the hematocrit on the recovery was evaluated. Successful application on whole blood and plasma, as well as on VAMS samples prepared from venous or capillary blood of patients, demonstrated that the methods were fit-for-purpose and can be used for future PK studies.

Development and Validation of a Method for Simultaneous Estimation of Sitagliptin and Ertugliflozin in Rat Plasma by LC-MS method

Background: The development of sound bioanalytical LC-MS (liquid chromatography-mass spectroscopy) method(s) is of paramount importance during the process of drug discovery, development and culminating in a marketing approval. The use of oral antidiabetic agents has been increased significantly from the last decades and till now no bioanalytical method is available for quantitation of sitagliptin (SG) and ertugliflozin (EG) in biological matrix which can be applied to pharmacokinetic studies using LC-MS/MS. Objective:To develop a new, rapid and sensitive LC–MS/MS method for the simultaneous estimation of sitagliptin (SG) and ertugliflozin (EG) in rat plasma by liquid–liquid extraction method (LLE) using deutereated sitagliptin (SGd6) and ertugliflozin (EGd6). Methods: Chromatographic separation was carried out on a reverse phase Waters, Xetrra C18 (150mm x 4.6mm, 2μm) column using a mixture of acetonitrile and OPA buffer (50:50v/v) at a flow rate of 1ml/min in isocratic mode. Quantification was achieved using an electrospray ion interface operating in positive mode, under Multiple Reaction Monitoring (MRM) conditions. Results:The method showed excellent linearity over the concentration range of 5.00- 75.00pg/mL for sitagliptin and 0.75- 11.35pg/mL ertugliflozin. The intra-batch and inter batch precision (%CV) was ≤ 4.3% and matrix effect (%CV) was 0.02% and 0.12% for sitagliptin at HQC and LQC, respectively. Matrix effect (%CV) was 0.08% and 0.33% for ertugliflozin at HQC and LQC, respectively. Conclusion: The simplicity of the method allows for application in laboratories, presents a valuable tool for pharmacokinetic studies. The particular assay has been proficiently put on pharmacokinetic study in rats subjects.

Estimating Efflux Transporter-Mediated Disposition of Molecules beyond the Rule of Five (bRo5) Using Transporter Gene Knockout Rats.

Transporter gene knockout models are a practical and widely used tool for pharmacokinetic studies in drug discovery. P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are major efflux transporters that control absorption and bioavailability, and are important when determining oral drug disposition. To the best of our knowledge, beyond the rule of five (bRo5) molecules launched on the market to date tend to be substrates for efflux transporters. The purpose of this study is to evaluate in vivo the impact of efflux transporters on the oral absorption process and systemic clearance using rats which lack P-gp and/or Bcrp expression. We administered five bRo5 substrates (asunaprevir, cyclosporine, danoprevir, ledipasvir, and simeprevir) intravenously or orally to wild-type and Mdr1a, Bcrp, and Mdr1a/Bcrp knockout rats, calculated the clearance, oral bioavailability, and absorption rate profile of each substrate, and compared the results. Systemic clearance of the substrates in knockout rats changed within approximately ±40% compared to wild-types, suggesting the efflux transporters do not have a significant influence on clearance in rats. On the other hand, the oral absorption of substrates in the knockout rats, especially those lacking Mdr1a, increased greatly-between 2- and 5-fold more than in wild-types. This suggests that rat efflux transporters, especially P-gp, greatly reduce the oral exposure of these substrates. Moreover, results on the absorption rate-time profile suggest that efflux transporters are constantly active during the absorption period in rats. Transporter knockout rats are a useful in vivo tool for estimating the transporter-mediated disposition of bRo5 molecules in drug discovery.

Two new convenient syntheses of 14C-squalene from turbinaric acid.

Carbon-14 labeled (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, also known as squalene, was synthesized as a tool for pharmacokinetic studies. Two simple and efficient labeling approaches were developed to give [2-14 C]-squalene and [3-14 C]-squalene from a halogenated precursor derived from turbinaric acid. They were obtained in 13.5% radiochemical yield in 6 steps and in 38% radiochemical yield in 3 steps respectively from carbon-14 labeled potassium cyanide with a radiochemical purity higher than 98% in both cases.

A Rapid HPLC Method for the Simultaneous Determination of Amiodarone and its Major Metabolite in Rat Plasma and Tissues: A Useful Tool for Pharmacokinetic Studies

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed and validated in rat plasma and tissue (heart, liver, kidney and lung) homogenates for the determination of amiodarone and its primary metabolite (desethylamiodarone), using tamoxifen as internal standard. Chromatographic separation was achieved within less than 5 min on a LiChroCART Purospher Star C18 column (55 × 4 mm, 3 µm). The mobile phase, consisting of phosphate buffer (50 mM) with 0.1% formic acid (pH 3.1)-methanol-acetonitrile (45:5:50, v/v/v), was pumped isocratically at a flow rate of 1.2 mL/min. The detection was conducted at 254 nm for all compounds. Calibration curves were linear (r(2) ≥ 0.995) in the range of 0.1-15 µg/mL for amiodarone and desethylamiodarone. The limits of quantification were established at 0.1 µg/mL for both analytes. The overall data of precision and accuracy were in accordance with international guidelines for bioanalytical method validation. Amiodarone and desethylamiodarone were extracted from rat matrices by a liquid-liquid extraction procedure and the mean recovery ranged from 59.9 to 97.6%. This novel HPLC method allows the fast and reliable determination of amiodarone and desethylamiodarone from several rat matrices (plasma, liver, kidneys, lungs and heart) and was successfully applied in a preliminary pharmacokinetic study.

Reverse iontophoresis as a noninvasive tool for lithium monitoring and pharmacokinetic profiling.

Transdermal iontophoresis was investigated as a noninvasive tool for drug monitoring and pharmacokinetic profiling. Lithium, a frequently monitored drug, was used as a model. The objectives were a) to demonstrate the linear dependence of the iontophoretic extraction flux of lithium on the subdermal concentration of the drug, b) to evaluate the capacity of iontophoresis to monitor sudden changes in the subdermal level, c) to investigate the utility of reverse iontophoresis as a tool in pharmacokinetic studies, and d) to examine the validity of an internal standard calibration procedure to render the method completely noninvasive. Transdermal, iontophoretic extraction was performed in vitro using dermatomed pig-ear skin. The subdermal solution consisted of a physiological buffer containing lithium chloride at concentrations in the therapeutic range and two putative internal standards, sodium and potassium, at fixed physiological levels. The subdermal concentration of lithium was changed either in a stepwise fashion or by simulating one of two pharmacokinetic profiles. Lithium was extracted via electromigration to the cathode. A excellent correlation between subdermal lithium concentration and iontophoretic extraction flux was observed. Iontophoresis tracked sudden concentration changes and followed kinetic profiles. In addition, the effective elimination rate constant could be directly, and noninvasively, estimated from the extraction flux data. Reverse iontophoresis is a potentially useful and noninvasive tool for lithium monitoring.

Determination of Oxycodone and Its Metabolite, Noroxycodone, in Human Plasma by HPLC with Post‐column Chemiluminescence Detection Using Electrogenerated Tris(2,2′‐Bipyridyl)ruthenium(III)

A sensitive high performance liquid chromatography (HPLC) method with post‐column chemiluminescence detection was developed for the simultaneous determination of oxycodone (OXC) and its metabolite, noroxycodone (NOC), in human plasma. Sample preparation for human plasma was performed by means of solid extraction with Sep‐Pak Vac C18 (100 mg/mL). Separation was performed by reversed‐phase HPLC using a 5‐µm Develosil ODS‐HG‐5 column (150 mm × 4.6 mm) and a mobile phase consisting of a mixture of 0.1 mol/L phosphate buffer (pH 2.6) containing 5 mmol/L sodium dodecylsulfate and methanol (53:47, v/v). Quantitation was performed by the measurement of chemiluminescence using electrogenerated tris(2,2′‐bipyridyl)ruthenium(III). The lower and upper limits of quantitation for OXC and NOC were 0.5–250 ng/mL and 1–500 ng/mL, respectively. The calibration curve for this assay showed good linearity for the concentration range of 0.5–50 ng/mL for OXC and 1–100 ng/mL for NOC. Recoveries of the studied compounds were excellent at the individual assay ranges (102.9–108.7%), and validation of the assay gave results that were satisfactory in terms of within‐run or between‐run precision (1.9–11.9% as RSD) and accuracy (−13.5% to +9.2% as bias). Stability studies showed that OXC and NOC were stable in assay solutions for at least 6 days at 4°C storage in the autosampler. OXC and NOC were also stable in human plasma for at least 48 hr at room temperature, for at least 8 months at −20°C and after five repeated freeze–thaw cycles. Furthermore, OXC and NOC were stable in human whole blood for up to 5 days at 4°C and for up to 5 hr at room temperature. This system was applied to the clinical study of cancer patients after single oral administration doses of controlled‐released tablets of OXC hydrochloride. Consequently, this method should serve as a useful tool for pharmacokinetic studies of OXC and NOC.

An improved HPLC-ED method for monitoring plasma levels of clozapine and its active metabolites in schizophrenic patients

An HPLC method with electrochemical detection has been developed for the determination of clozapine and its main metabolites, desmethylclozapine and clozapine N-oxide, in human plasma. An accurate pretreatment of the biological samples was implemented by means of solid phase extraction (SPE) on HLB cartridges. This improved pretreatment, together with a new mobile phase, allows for the accurate determination of clozapine N-oxide, which could not be quantitated by a previous method. The method uses only 100 μL of plasma for one complete analysis and shows good recovery values for all three analytes. The eluates from the SPE procedure were chromatographed in a reversed phase C18 column using a mobile phase composed of phosphate buffer, acetonitrile and methanol. Clozapine, desmethylclozapine and clozapine N-oxide were eluted in less than 10 minutes, without any interference from the biological matrix. Linearity was observed over the 2.50–150 ng mL−1 (clozapine and desmethylclozapine) or 1.25–75 ng mL−1 clozapine N-oxide) range for the three analytes, with satisfactory repeatability values. The limit of detection was 0.3 ng mL−1 for clozapine and desmethylclozapine, samples of patients treated with Leponex gave good results. No interference from other common central nervous system drugs was found. This method seems to be a useful tool for pharmacokinetic studies and for clinical monitoring, because of its need for small plasma samples and its high sensitivity and selectivity.

Human Pharmacokinetics of l-3,4-Dihydroxyphenylalanine Studied with Microdialysis

Intravenous and subcutaneous microdialysis was performed to compare the free concentrations and pharmacokinetics of L-3, 4-dihyroxyphenylalanine (L-dopa) in blood and tissue in healthy subjects and in patients with Parkinson disease. Nine healthy volunteers and 10 patients with Parkinson disease, stage 1. 5-2 according to the Hoehn-Yahr rating scale, took part of the study. In the patient group subcutaneous microdialysis and ordinary blood sampling were performed, whereas in the control group intravenous microdialysis was also performed. Microdialysis samples were collected in fractions of 15 min. The first two fractions were collected for analysis of basal concentrations. A blood sample was also taken. The patients were then given one tablet of Madopar((R)) (100 mg of L-dopa and 25 mg of benserazide), and the microdialysis was continued for another 210 min. Blood samples were obtained at 30-min intervals. The serum samples gave a significantly higher mean area under the curve (AUC; 491 +/- 139 micromol. min/L) than that for intravenous dialysates (235 +/- 55.3 micromol. min/L), suggesting a protein binding of 50%. The L-dopa concentrations from the subcutaneous dialysates matched those from the intravenous dialysates, indicating rapid distribution of L-dopa to the tissues. Parkinsonian patients in early stages of the disease have a pharmacokinetic pattern of free L-dopa similar to that of healthy subjects. Comparison of AUCs from microdialysis with ordinary serum analysis revealed data indicating significant protein binding. Microdialysis is a suitable and easily applied tool in pharmacokinetic studies.

Capillary electrophoresis with laser-induced fluorescence: a routine method to determine moxifloxacin in human body fluids in very small sample volumes

The feasibility of capillary electrophoresis with HeCd laser-induced fluorescence detection as a validated routine method for bioanalytical analysis is reported. Method evaluation, validation and results of the determination of moxifloxacin (BAY 12-8039), a new antimicrobially active 8-methoxy-quinolone, in plasma and microdialysate are described. After a one step sample preparation the samples can be injected directly into the capillary. The volume of microdialysate and plasma, respectively, needed for more than 50 injections is only 10 μl and 20 μl. Total run time is less than 7 min using a 27 cm capillary on commercial instrumentation. An analysis time of less than 1 min was shown to be possible, however it could not be used routinely since appropriate instrumentation was not available. Evaluation is based on the relative corrected peak area (analyte/I.S.). The method's dynamic range comprises three orders of magnitude (plasma: 2.5–5000 μg/l; microdialysate: 5–5000 μg/l). Validation according to international guidelines yielded data on accuracy and precision of the method throughout the entire working range of inter-day precision: plasma <6%, microdialysate <5% and inter-day accuracy: plasma <2%, microdialysate <4%. The crossvalidation with an existing HPLC method utilizing clinical study samples shows linear correlation. In view of its adequate sensitivity and high selectivity capillary electrophoresis with laser-induced fluorescence is a very versatile tool in pharmacokinetic studies of quinolones, especially in situations with limited sample volumes: e. g. pediatrics, patients at risk, animal-, microdialysis- and tissue-kinetic studies. Validation parameters and other features, like high sample throughput and robustness, are comparable to or even better than HPLC. Further necessary improvements of the capillary electrophoresis with laser-induced fluorescence instrumentation (autosampler, vials, parallel capillaries) and its use in bioanalytical routine analysis are discussed.

In vivo microdialysis sampling for pharmacokinetic investigations.

In vivo microdialysis sampling coupled to liquid chromatography was used to study acetaminophen disposition in anesthetized rats. The pharmacokinetics of acetaminophen and its sulfate and glucuronide metabolites were determined using both microdialysis sampling and collection of whole blood. For microdialysis, samples were continuously collected for over 5 hr without fluid loss using a single experimental animal. Microdialysis sampling directly assesses the free drug concentration in blood. The pharmacokinetic results obtained with microdialysis sampling were the same as those obtained from blood collection. The administration of heparin, necessary when collecting blood samples, was found to double the elimination half-life of acetaminophen. Microdialysis sampling is a powerful tool for pharmacokinetic studies, providing accurate and precise pharmacokinetic data.

Determination of the serum concentration of spironolactone and its metabolites by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic assay is described for the simultaneous determination in serum of the aldosterone antagonist spironolactone and its metabolites 7α-thiomethylspirolactone, 6β-hydroxy-7α-thiomethylspirolactone and canrenone. Ultraviolet absorption at 240 nm was used to detect the different compounds after elution on a normal-phase column. Endogenous serum substances did not interfere with the assay. This method provides a convenient tool in pharmacokinetic studies of spironolactone, in contrast to previously reported aspecific fluorimetric assays or time-consuming thin-layer chromatographic analyses of radioactive biological material.