Organic anion transporting polypeptides OATP1B1 and OATP1B3 and their genetic variants influence the pharmacokinetics and pharmacodynamics of raloxifene.

BACKGROUND: Raloxifene, a selective estrogen receptor modulator, exhibits quite large and unexplained interindividual variability in pharmacokinetics and pharmacodynamics. The aim of this study was to determine the role of organic-anion transporting polypeptides OATP1B1 and OATP1B3 and their genetic variants in the pharmacokinetics and pharmacodynamics of raloxifene. METHODS: To test the role of OATP1B1 and OATP1B3 transporters on hepatic uptake of raloxifene and its metabolites an in vitro model of Chinese Hamster Ovary cells expressing OATP1B1 or OATP1B3 was employed. The influence of OATP1B1 and OATP1B3 genetic variants on in vivo pharmacokinetics and pharmacodynamics was evaluated in 53 osteoporotic postmenopausal women treated with raloxifene. RESULTS: Our in vitro results showed that raloxifene and two of the three metabolites, raloxifene-4'-ß-glucuronide (M2) and raloxifene-6,4'-diglucuronide (M3), interact with OATP1B1 and OATP1B3. Higher M3 and total raloxifene serum concentrations in patients correlated with lower serum levels of bone resorption marker, serum C-terminal telopeptide fragments of type I collagen, indicating a higher antiresorptive effect of raloxifene. Higher concentrations of M2 correlated with higher increase of lumbar spine bone mineral density supporting the raloxifene vertebral fracture specific protection effect. Finally, raloxifene, M3 and total raloxifene serum concentrations were significantly higher in patients with SLCO1B1 c.388A > G polymorphism and *1b haplotype implicating a considerable genetic effect on pharmacokinetics and pharmacodynamics of raloxifene. CONCLUSIONS: These findings indicate that SLCO1B1 c.388A > G polymorphism could play an important role in pharmacokinetics and pharmacodynamics of raloxifene.

Development of an Analytical Method for Determination of Related Substances and Degradation Products of Cabotegravir Using Analytical Quality by Design Principles.

Cabotegravir is one of the newly approved human immunodeficiency virus (HIV) integrase enzyme inhibitors used for the prevention and treatment of HIV infection. It is the first approved long-acting injectable antiretroviral therapy for HIV and is also very effective in combination with rilpivirine, a non-nucleoside reverse transcriptase inhibitor. Therefore, future drug development involving cabotegravir can be expected. We developed an ultrahigh performance liquid chromatography (UHPLC) method compatible with mass spectrometry for the determination of eight cabotegravir impurities. The described method is able to differentiate cabotegravir and its related substances as well as its degradation products. Analytical quality by design principles were used for method development. The method is robust within the defined method operable design region: flow rate = 0.32-0.40 mL/min; column temperature = 30-40 °C; pH of mobile phase A = 3.25-3.75, and the final percent of acetonitrile in gradient = 50.0-60.0%. Inside the method operable design region, a working optimal point was selected: pump flow rate = 0.36 mL/min; column temperature = 35 °C; pH of mobile phase A = 3.5, and final percent of acetonitrile in gradient = 55%. Method validation was performed, and the following parameters were verified: accuracy, repeatability, linearity, response factors, detection limit, and quantification limit. All method validation results were within selected criteria. The presented method could be used for the development of new pharmaceutical products based on cabotegravir.

Raloxifene glucuronidation in human intestine, kidney, and liver microsomes and in human liver microsomes genotyped for the UGT1A1*28 polymorphism.

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-ß-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-ß-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.

Understanding of cabotegravir degradation through isolation and characterization of key degradation products and kinetic studies.

Cabotegravir is a novel human immunodeficiency virus integrase enzyme inhibitor used for prevention and treatment of HIV infection. The combinational final dosage form, as extended release injection suspension in combination with rilpivirine and as cabotegravir tablets (for lead-in therapy), was recently approved in Canada, EU and in USA and is currently seeking approval also in other countries. The subject of this investigation was to study the degradation of cabotegravir under different stress conditions as per the International Council for Harmonization (ICH) guidelines. The drug substance was found to be stable in thermal, photolytic and basic stress conditions, but degraded under acidic and oxidative stress conditions. It was determined that four main degradation products of cabotegravir are formed in forced degradation studies. All four main degradation products were isolated using preparative chromatography and subjected to NMR and HRMS analysis in order to determine their structure. We proposed degradation pathways of cabotegravir under acidic stress conditions in solution based on the structure of isolated degradation products, cabotegravir degradation kinetic studies and degradation studies on two isolated key degradation products. Moreover, degradation pathway to predominant oxidation degradation product is proposed based on the adduct of cabotegravir and peroxide species, which was identified by LC-HRMS analysis. This is the first report to the best of our knowledge that describes characterized cabotegravir forced degradation impurities and provides insights into its degradation pathways.

Co-crystals, Salts or Mixtures of Both? The Case of Tenofovir Alafenamide Fumarates.

Tenofovir alafenamide fumarate (TAF) is the newest prodrug of tenofovir that constitutes several drug products used for the treatment of HIV/AIDS. Although the solid-state properties of its predecessor tenofovir disoproxil fumarate have been investigated and described in the literature, there are no data in the scientific literature on the solid state properties of TAF. In our report, we describe the preparation of two novel polymorphs II and III of tenofovir alafenamide monofumarate (TA MF2 and TA MF3). The solid-state structure of these compounds was investigated in parallel to the previously known tenofovir alafenamide monofumarate form I (TA MF1) and tenofovir alafenamide hemifumarate (TA HF). Interestingly, the single-crystal X-ray diffraction of TA HF revealed that this derivative exists as a co-crystal form. In addition, we prepared a crystalline tenofovir alafenamide free base (TA) and its hydrochloride salt (TA HCl), which enabled us to determine the structure of TA MF derivatives using 15N-ssNMR (15N-solid state nuclear magnetic resonance). Surprisingly, we observed that TA MF1 exists as a mixed ionization state complex or pure salt, while TA MF2 and TA MF3 can be obtained as pure co-crystal forms.

Fast and Simple LC-MS/MS Method for Rifampicin Quantification in Human Plasma.

A simple, fast, and cost-effective LC-MS/MS method for quantification of rifampicin in human plasma was developed and fully validated. The plasma samples containing rifampicin and isotopically labelled internal standard rifampicin D8, were cleaned up using a Captiva ND Lipids filtration plate. Chromatographic separation was achieved on an 1290 Infinity liquid chromatograph coupled to 6460 Triple Quadrupole operated in positive mode on a core-shell Kinetex C18 column (50 × 2.1 mm, 2.6 µm) by gradient elution using 0.1% formic acid in water and acetonitrile as a mobile phase. The proposed method is the fastest method published by now, both in terms of sample preparation (approximately one minute per sample) and chromatographic analysis (total run time 2.4 min). Another key benefit is the outstanding sensitivity and wide analytical range (5-40000 µg/L) with good linearity, accuracy, and precision. The method showed almost complete recovery (92%) and absence of any significant matrix effect as demonstrated by uniform responses from QC samples prepared in blood plasma from 6 volunteers (RSD <5%). The proposed method was successfully applied to rifampicin quantification in 340 patients' plasma samples, thus demonstrating its suitability for both therapeutic drug monitoring and pharmacokinetic analysis.

Evaluation of bisphenol A glucuronidation according to UGT1A1*28 polymorphism by a new LC-MS/MS assay.

The endocrine disruptor bisphenol A (BPA) is a frequently used chemical in the manufacture of consumer products. In humans, BPA is extensively metabolized to BPA glucuronide (BPAG) by different UDP-glucuronosyltransferase (UGT) isoforms. The study has been performed with the intention to improve the accuracy of published physiologically based pharmacokinetic models and to improve regulatory risk assessments of BPA. In order to gain insight into intestine, kidney, liver, and lung glucuronidation of BPA, human microsomes of all tested organs were used. BPAG formation followed Michaelis-Menten kinetics in the intestine and kidney, but followed substrate inhibition kinetics in the liver. Human lung microsomes did not show glucuronidation activity towards BPA. While the liver intrinsic clearance was very high (857 mL min(-1)kg body weight(-1)), the tissue intrinsic clearances for the kidney and intestine were less than 1% of liver intrinsic clearance. Since BPA is a UGT1A1 substrate, we postulated that the common UGT1A1*28 polymorphism influences BPA glucuronidation, and consequently, BPA detoxification. Hepatic tissue intrinsic clearances for UGT1A1*1/*1, UGT1A1*1/*28, and UGT1A1*28/*28 microsomes were 1113, 1075, and 284 mL min(-1)kg body weight(-1), respectively. Prior to microsomal experiments, the bioproduction of BPAG and stable isotope-labeled BPAG (BPAG(d16)) was performed for the purpose of the reliable and accurate quantification of BPAG. In addition, a sensitive LC-MS/MS analytical method for the simultaneous determination of BPA and BPAG based on two stable isotope-labeled internal standards was developed and validated. In conclusion, our in vitro results show that the liver is the main site of BPA glucuronidation (K(m) 8.9 µM, V(max) 8.5 nmol min(-1) mg(-1)) and BPA metabolism may be significantly influenced by a person's genotype (K(m) 10.0-13.1 µM, V(max) 3.4-16.2 nmol min(-1) mg(-1)). This discovery may be an important fact for the currently on-going worldwide BPA risk assessments and for the improvement of physiologically based pharmacokinetic models.

Influence of hepatic and intestinal efflux transporters and their genetic variants on the pharmacokinetics and pharmacodynamics of raloxifene in osteoporosis treatment.

Raloxifene exhibits a large and unexplained interindividual variability in its pharmacokinetics and pharmacodynamics. The aim of our study was to identify transporters involved in the efflux of raloxifene and its glucuronide metabolites by various in vitro models and by an in vivo study to explore the possible involvement of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP)1, MRP2, MRP3, and breast cancer resistance protein in the observed high interindividual variability. Experiments with the parallel artificial membrane permeability assay showed the highest passive permeability for raloxifene, followed by raloxifene-6-ß-glucuronide (M1), raloxifene-4'-ß-glucuronide (M2), and raloxifene-6,4'-diglucuronide (M3). Caco-2 cell monolayer experiments indicated an interaction of raloxifene with Pgp. The ATPase assay confirmed the raloxifene interaction with Pgp and indicated interactions of all raloxifene species with MRP1, MRP2, MRP3, and breast cancer resistance protein, except for M1, which did not show any interactions with MRP2. Furthermore, the vesicular experiments confirmed the interaction of M2 and M3 with MRP2. Although the in vivo study on osteoporotic postmenopausal women on raloxifene could not confirm a significant influence of ABCB1 and ABCC2 genetic polymorphisms on its pharmacokinetics, a clear trend toward higher total raloxifene concentrations was observed in carriers of at least 1 ABCB1 c.3435T allele. Moreover, the same polymorphism effect was also observed as a significant increase in total hip bone mineral density after 1 year of treatment. The results of our study support the involvement of efflux transporters in disposition of raloxifene and its metabolites and may partially explain the observed raloxifene variability by the influence of the ABCB1 c.3435C>T polymorphism.