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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--glucuronide (M1), raloxifene-4'--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. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 AbstractRaloxifene exhibits quite 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 Pgp, MRP1, MRP2, MRP3, and BCRP in the observed high interindividual variability. Experiments with PAMPA (Parallel Artificial Membrane Permeability Assay) showed the highest passive permeability for raloxifene, followed by raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2) and raloxifene-6,4'-diglucuronide (M3). Caco-2 cell monolayer experiments indicated an inte...

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BackgroundRaloxifene, 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.MethodsTo 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.ResultsOur in vitro results showed that raloxifene and two of the three metabolites, raloxifene-4'-β-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.ConclusionsThese findings indicate that SLCO1B1 c.388A > G polymorphism could play an important role in pharmacokinetics and pharmacodynamics of raloxifene.

ABSTRACT: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-␤-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4-␤-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 ⅐ min ؊1 ⅐ 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.

An ultra-high performance liquid chromatography method for simultaneous determination of tacrolimus impurities in pharmaceutical dosage forms has been developed. Appropriate chromatographic separation was achieved on a BEH C18 column using gradient elution with a total run time of 14 min. The method was applied to analyses of commercial samples and was validated in terms of linearity, precision, accuracy, sensitivity and specificity. It was found to be linear, precise and accurate in the range of 0.05 to 0.6 % of the impurities level in pharmaceutical dosage forms. Stability indicating power of the method was demonstrated by the results of forced degradation studies. The forced degradation study in solution revealed tacrolimus instability under stress alkaline, thermal, light and photolytic conditions and in the presence of a radical initiator or metal ions. The drug was stable at pH 3–5. Solid-state degradation studies conducted on amorphous tacrolimus demonstrated its sensitivity to light, elevated temperature, humidity and oxidation.

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.

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.