Humanization of SLCO2B1 in Rats Increases rCYP3A1 Protein Expression but Not the Metabolism of Erlotinib to OSI-420.

The organic anion transporting polypeptide (OATP)2B1 [(gene: solute carrier organic anion transporter family member 2B1 (SLCO2B1)] is an uptake transporter that facilitates cellular accumulation of its substrates. Comparison of SLCO2B1+/+ knockin and rSlco2b1-/- knockout rats showed a higher expression of rCYP3A1 in the humanized animals. We hypothesize that humanization of OATP2B1 not only affects cellular uptake but also metabolic activity. To further investigate this hypothesis, we used SLCO2B1+/+ and rSlco2b1-/ - rats and the OATP2B1 and rCYP3A1 substrate erlotinib, which is metabolized to OSI-420, for in vivo and ex vivo experiments. One hour after administration of a single dose of erlotinib, the knockin rats exhibited significantly lower erlotinib serum levels, but no change was observed in metabolite concentration or the OSI-420/erlotinib ratio. Similar results were obtained for liver tissue levels comparing SLCO2B1+/+ and rSlco2b1-/- rats. Liver microsomes isolated from the erlotinib-treated animals were characterized ex vivo for rCYP3A activity using testosterone, showing higher activity in the knockin rats. The contrary was observed when microsomes isolated from treatment-naïve animals were assessed for the metabolism of erlotinib to OSI-420. The latter is in contrast to the higher rCYP3A1 protein amount observed by western blot analysis in rat liver lysates and liver microsomes isolated from untreated rats. In summary, rats humanized for OATP2B1 showed higher expression of rCYP3A1 in liver and reduced serum levels of erlotinib but no change in the OSI-420/erlotinib ratio despite a lower OSI-420 formation in isolated liver microsomes. Studies with CYP3A-specific substrates are warranted to evaluate whether humanization affects not only rCYP3A1 expression but also metabolic activity in vivo. SIGNIFICANCE STATEMENT: Humanization of rats for the organic anion transporting polypeptide (OATP)2B1 increases rCYP3A1 expression and activity in liver. Using the OATP2B1/CYP3A-substrate erlotinib to assess the resulting phenotype, we observed lower erlotinib serum and liver concentrations but no impact on the liver/serum ratio. Moreover, there was no difference in the OSI-420/erlotinib ratio comparing humanized and knockout rats, suggesting that OSI-420 is not applicable to monitor differences in rCYP3A1 expression as supported by data from ex vivo experiments with rat liver microsomes.

Influence of Slco2b1-knockout and SLCO2B1-humanization on coproporphyrin I and III levels in rats.

BACKGROUND AND PURPOSE: Coproporphyrin (CP) I and III are byproducts of haem synthesis currently investigated as biomarkers for drug-drug interactions involving hepatic organic anion transporting polypeptide (OATP) 1B transporters. Another hepatically expressed OATP-member is OATP2B1. The aim of this study was to test the impact of OATP2B1, which specifically transports CPIII, on CP serum levels, applying novel rat models. EXPERIMENTAL APPROACH: CPIII transport kinetics and the interplay between OATP2B1 and multidrug resistance-associated proteins (MRPs) were determined in vitro using the vTF7 expression system. Novel rSlco2b1-/- and SLCO2B1+/+ rat models were characterized for physiological parameters and for CP serum levels. Hepatic and renal expression of transporters involved in CP disposition were determined by real-time qPCR, Western blot analysis, and immunohistochemistry. KEY RESULTS: In vitro experiments revealed differences in transport kinetics comparing human and rat OATP2B1 and showed a consistent, species-specific interplay with hMRP3/rMRP3. Deletion of rOATP2B1 was associated with a trend towards lower CPI serum levels compared with wildtype rats, while CPIII remained unchanged. Comparing SLCO2B1+/+ with knockout rats revealed an effect of sex: only in females the genetic modification influenced CP serum levels. Analysis of hepatic and renal transporters revealed marginal, but in part, statistically significant differences in rMRP2 abundance, which may contribute to the observed changes in CP serum levels. CONCLUSION AND IMPLICATIONS: Our findings support that factors other than OATP1B transporters are of relevance for basal CP levels. Only in female rats, humanization of SLCO2B1 affects basal CPI and CPIII serum levels, despite isomer selectivity of OATP2B1.

St. John's Wort Formulations Induce Rat CYP3A23-3A1 Independent of Their Hyperforin Content.

The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. SIGNIFICANCE STATEMENT: Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.

Various effects of repeated rifampin dosing on coproporphyrin levels in humans.

In recent years, the identification of endogenous substrates as biomarkers became an uprising topic. Particularly coproporphyrins (CPs), byproducts of heme biosynthesis, are intensely investigated as biomarkers for predicting interactions with the organic anion transporting polypeptide (OATP) 1B transporters. In the context of drug-drug interactions, several preclinical and clinical studies assessed the effect of the OATP1B-index inhibitor rifampin on CPI levels. However, rifampin is not only a "perpetrator" drug of transporters but is also known for its interaction with the nuclear receptor pregnane X receptor (PXR) leading to the efficient induction of PXR-target genes. These include hemoproteins like cytochrome P450 enzymes but also the δ-aminolevulinate synthase 1, which is the rate-limiting enzyme in heme biosynthesis. In this study, we showed that quantification of CPs in clinical serum samples was possible after long-term storage at -20°C. We quantified CPI, CPIII, and heme levels in clinical serum samples (at selected timepoints) that originated from a trial investigating the interaction potential of repeated rifampin administration in 12 healthy participants. In samples collected at the assumed time to maximum concentration of rifampin, higher CP levels were observed compared to baseline. Increased levels persisted even 14 h after discontinuation of rifampin. No impact on heme serum levels was observed. We found a correlation between CP isomers at baseline and at 14 h after rifampin intake. In summary, we show that multiple doses of rifampin affect CP levels. However, besides inhibition of hepatic OATP function there is evidence for an interaction with CP levels beyond the transporter level.

Simultaneous quantification of atorvastatin, erlotinib and OSI-420 in rat serum and liver microsomes using a novel liquid chromatography-mass spectrometry method.

Erlotinib is an epidermal growth factor receptor tyrosine kinase inhibitor used in the treatment of cancer. Atorvastatin is a statin commonly applied to treat hypercholesterolemia. In humans, both compounds are metabolized by CYP3A4 and are transported by OATP2B1, ABCB1 and ABCG2. We aimed to generate and validate a bioanalytical method for simultaneous determination of atorvastatin, erlotinib and its major metabolite OSI-420 applicable to biological samples. Quantification of erlotinib, OSI-420, and atorvastatin was achieved with an Agilent high-performance liquid chromatography system 1100/1200 coupled to a triple quadrupole G6410B. The method involved separation over the column Kinetex C8 (100 × 3 mm, 2.6 µm) using 2 mM ammonium acetate (pH 4.0) and acetonitrile as eluent. The method was assessed for selectivity, accuracy, recovery, matrix effect, and stability over a range from 1 to 4,000 ng/mL according to the respective guidelines. We applied the bioanalytical method to quantify the formation of OSI-420 in liver microsomes isolated from male and female Wistar rats. The optimized experiment revealed slower formation in microsomes of female compared to male rats, in which we observed lower amounts of CYP3A1 by Western blot analysis. Moreover, the presence of atorvastatin inhibited the CYP3A-mediated metabolism of erlotinib. Serum obtained from a drug-drug interaction study performed in male rats was also analyzed using the validated method. Non-compartmental pharmacokinetic analysis revealed a lower clearance of erlotinib when atorvastatin was co-administered. However, for atorvastatin we observed a lower systemic exposure in presence of erlotinib. In summary, we report a method to detect OSI-420, erlotinib and atorvastatin applicable to samples from ex vivo and in vivo studies.

The influence of OATP2B1 and atorvastatin on coproporphyrin isomers in rats.

Coproporphyrin I (CPI) and III (CPIII) are discussed as biomarkers for organic anion transporting polypeptides (OATPs). We report on CPI and CPIII levels in wildtype, rSlco2b1-knockout, and SLCO2B1-humanized rats at baseline and after administration of atorvastatin, an inhibitor of the CPIII-specific rOATP2B1/hOATP2B1 and the CPI/CPIII-transporting rOATP1B2. OATP-inhibition by atorvastatin leads to significantly increased CPI and CPIII serum levels. However, basal CP serum levels in rSlco2b1-knockout animals were significantly lower (CPI), or unaffected (CPIII). In the presence of atorvastatin, this genotype effect was abolished. In conclusion, our results indicate an unexpected impact of OATP2B1 on CP serum levels in rats.

Pharmacogenetic Analysis of Voriconazole Treatment in Children.

Voriconazole is among the first-line antifungal drugs to treat invasive fungal infections in children and known for its pronounced inter- and intraindividual pharmacokinetic variability. Polymorphisms in genes involved in the metabolism and transport of voriconazole are thought to influence serum concentrations and eventually the therapeutic outcome. To investigate the impact of these genetic variants and other covariates on voriconazole trough concentrations, we performed a retrospective data analysis, where we used medication data from 36 children suffering from invasive fungal infections treated with voriconazole. Data were extracted from clinical information systems with the new infrastructure SwissPKcdw, and linear mixed effects modelling was performed using R. Samples from 23 children were available for DNA extraction, from which 12 selected polymorphism were genotyped by real-time PCR. 192 (49.1%) of 391 trough serum concentrations measured were outside the recommended range. Voriconazole trough concentrations were influenced by polymorphisms within the metabolizing enzymes CYP2C19 and CYP3A4, and within the drug transporters ABCC2 and ABCG2, as well as by the co-medications ciprofloxacin, levetiracetam, and propranolol. In order to prescribe an optimal drug dosage, pre-emptive pharmacogenetic testing and careful consideration of co-medications in addition to therapeutic drug monitoring might improve voriconazole treatment outcome of children with invasive fungal infections.

Differences in transport function of the human and rat orthologue of the Organic Anion Transporting Polypeptide 2B1 (OATP2B1).

The human drug transporter Organic Anion Transporting Polypeptide (hOATP)2B1 facilitates cellular uptake of its substrates. Various studies suggest that hOATP2B1 is involved in intestinal absorption, but preclinical evaluations performed in rodents do not support this. Thus, our study aimed to compare the expression and function of hOATP2B1 with its orthologue in rats (rOatp2b1). Even if the general expression pattern was comparable, the transporters exhibited substantial differences on functional level. While bromosulfophthalein and atorvastatin were substrates of both transporters, the steroid sulfate conjugates estrone 3-sulfate (E1S), progesterone sulfate and dehydroepiandrosterone sulfate were only transported by hOATP2B1. To further elucidate these functional differences, experiments searching for the E1S substrate recognition site were conducted generating human-rat chimera as well as partly humanized variants of rOatp2b1. The rOatp2b1-329-hOATP2B1 chimera led to a significant increase in E1S uptake suggesting the C-terminal part of the human transporter is involved. However, humanization of various regions within this part, namely of the transmembrane domain (TMD)-9, TMD-10 or the extracellular loop-5 did not significantly change E1S transport function. Replacement of the intracellular loop-3, slightly enhanced cellular accumulation of sulfated steroids. Taken together, we report that OATP2B1 exhibited differences in recognition of steroid sulfate conjugates comparing the rat and human orthologues.

Genetic variants of SLCO1B7 are of relevance for the transport function of OATP1B3-1B7.

The family of Organic Anion Transporting Polypeptides are known to facilitate the transmembrane transport. OATP1B3-1B7 is a novel member of the OATP1B-subfamily, and is encoded by SLCO1B3-SLCO1B7 readthrough deriving from the genes SLCO1B3 and SLCO1B7 on chromosome 12. The resulting protein is expressed in the smooth endoplasmatic reticulum of hepatocytes, is functional, and transports dehydroepiandrosterone-sulfate (DHEAS). In the gene area encoding for the 1B7-part of the protein, there are coding polymorphisms. It was the aim of this study to test the frequency and the impact of these genetic variants on transport activity. The minor allele frequency (MAF) of the coding polymorphisms was determined in a cohort of 192 individuals. DHEAS transport function was determined by applying the vTF-7 based heterologous expression system using plasmids encoding for OATP1B3-1B7 or the respective variants. The genetic variants 641 T (MAF 0.021), 1073 G (MAF 0.169) and 1775 A (MAF 0.013) significantly reduced DHEAS accumulation in cells transfected with OATP1B3-1B7, albeit without significantly influencing expression of the transporter as determined by Western blot analysis and immunofluorescence after heterologous expression. Genotyping revealed complete linkage of the variants 884A, 1073 G and 1501C. Presence of the haplotype abolished the DHEAS-transport function of OATP1B3-1B7. Naturally and frequently occurring genetic variants located within the gene region of SLCO1B7 encoding for the 1B7-part of OATP1B3-1B7 influence the in vitro function of this member of the OATP1B-family. With their functional characterisation, we provide the basis for pharmacogenetic studies, which may help to understand the in vivo relevance of this transporter.

PDMS-PMOXA-Nanoparticles Featuring a Cathepsin B-Triggered Release Mechanism.

BACKGROUND: It was our intention to develop cathepsin B-sensitive nanoparticles for tumor-site-directed release. These nanoparticles should be able to release their payload as close to the tumor site with a decrease of off-target effects in mind. Cathepsin B, a lysosomal cysteine protease, is associated with premalignant lesions and invasive stages of cancer. Previous studies have shown cathepsin B in lysosomes and in the extracellular matrix. Therefore, this enzyme qualifies as a trigger for such an approach. METHODS: Poly(dimethylsiloxane)-b-poly(methyloxazoline) (PDMS-PMOXA) nanoparticles loaded with paclitaxel were formed by a thin-film technique and standard coupling reactions were used for surface modifications. Despite the controlled release mechanism, the physical properties of the herein created nanoparticles were described. To characterize potential in vitro model systems, quantitative polymerase chain reaction and common bioanalytical methods were employed. CONCLUSIONS: Stable paclitaxel-loaded nanoparticles with cathepsin B digestible peptide were formed and tested on the ovarian cancer cell line OVCAR-3. These nanoparticles exerted a pharmacological effect on the tumor cells suggesting a release of the payload.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Hyperforin-Induced Activation of the Pregnane X Receptor Is Influenced by the Organic Anion-Transporting Polypeptide 2B1.

The herbal remedy St. John's wort (SJW) is used in the treatment of mild depressive symptoms and is known for its drug-drug interaction potential when enhanced expression of CYP3A4 modifies clearance of concomitantly applied substrate drugs. Hyperforin is one constituent of SJW that alters CYP3A4 expression by activation of the nuclear receptor pregnane X receptor (PXR). However, little is known about the transmembrane transport of hyperforin. One membrane protein that modulates cellular entry of drugs is the organic anion-transporting polypeptide (OATP) 2B1. It was the aim of this study to test whether hyperforin interacts with this transport protein. Transport inhibition studies and competitive counterflow experiments suggested that hyperforin is a substrate of OATP2B1. This notion was validated by showing that the presence of OATP2B1 enhanced the hyperforin-induced PXR activation in cell-based luciferase assays. Moreover, in Caco-2 cells transcellular transport of the known OATP2B1 substrate atorvastatin was changed in the presence of hyperforin, resulting in an increased efflux ratio. Eleven commercially available SJW formulations were assessed for their influence on OATP2B1-mediated transport of estrone 3-sulfate and for their impact on CYP3A4 promoter transactivation. The correlation between effect size and the hyperforin content as determined by high-performance liquid chromatography with ultraviolet detection suggested that hyperforin is the major determinant. Our results indicate an interaction between hyperforin and OATP2B1, which is not only known to contribute to hepatocellular uptake but also to intestinal absorption of its substrates. These findings extend the complexity of mechanisms that should be considered when evaluating the interaction potential of SJW preparations.

Thyroid Hormones Are Transport Substrates and Transcriptional Regulators of Organic Anion Transporting Polypeptide 2B1.

Levothyroxine replacement therapy forms the cornerstone of hypothyroidism management. Variability in levothyroxine oral absorption may contribute to the well-recognized large interpatient differences in required dose. Moreover, levothyroxine-drug pharmacokinetic interactions are thought to be caused by altered oral bioavailability. Interestingly, little is known regarding the mechanisms contributing to levothyroxine absorption in the gastrointestinal tract. Here, we aimed to determine whether the intestinal drug uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1) may be involved in facilitating intestinal absorption of thyroid hormones. We also explored whether thyroid hormones regulate OATP2B1 gene expression. In cultured Madin-Darby Canine Kidney II/OATP2B1 cells and in OATP2B1-transfected Caco-2 cells, thyroid hormones were found to inhibit OATP2B1-mediated uptake of estrone-3-sulfate. Competitive counter-flow experiments evaluating the influence on the cellular accumulation of estrone-3-sulfate in the steady state indicated that thyroid hormones were substrates of OATP2B1. Additional evidence that thyroid hormones were OATP2B1 substrates was provided by OATP2B1-dependent stimulation of thyroid hormone receptor activation in cell-based reporter assays. Bidirectional transport studies in intestinal Caco-2 cells showed net absorptive flux of thyroid hormones, which was attenuated by the presence of the OATP2B1 inhibitor, atorvastatin. In intestinal Caco-2 and LS180 cells, but not in liver Huh-7 or HepG2 cells, OATP2B1 expression was induced by treatment with thyroid hormones. Reporter gene assays revealed thyroid hormone receptor α-mediated transactivation of the SLCO2B1 1b and the SLCO2B1 1e promoters. We conclude that thyroid hormones are substrates and transcriptional regulators of OATP2B1. These insights provide a potential mechanistic basis for oral levothyroxine dose variability and drug interactions.

LST-3TM12 is a member of the OATP1B family and a functional transporter.

Organic anion transporting polypeptides (OATPs) and particularly the two members of the OATP1B family are known for their role in pharmacokinetics. Both SLCO1B3 and SLCO1B1 are located on chromosome 12 encompassing the gene locus SLCO1B7. Hitherto, this particular gene has been assumed to be a pseudogene, even though there are published mRNA sequences linked to this chromosomal area. It was aim of this study to further investigate SLCO1B7 and the associated mRNA LST-3TM12. In a first step, we aligned all mRNAs linked to the chromosomal region of SLCO1B-transporters. This in silico analysis revealed that LST-3TM12 is a product of splicing of SLCO1B3 and SLCO1B7, and encodes for a protein with twelve transmembrane domains. The existence of LST-3TM12 mRNA was verified by polymerase chain reaction showing liver enriched expression. In addition, immunohistological staining showed that LST-3TM12 protein was expressed in the endoplasmic reticulum (ER) of hepatocytes. Localization in the ER was further verified by immunoblot analysis showing high amounts of LST-3TM12 in liver microsomes. Function of LST-3TM12 was assessed by transport studies after heterologous expression in HeLa cells, where the transporter was shown to be expressed not only in the ER but also in the plasma membrane. Overexpression of LST-3TM12 was associated with enhanced cellular accumulation of dehydroepiandrosterone sulfate (Vmax 300.2 pmol mg-1 min-1; Km 34.2 µm) and estradiol 17ß-glucuronide (Vmax 29.9 mol mg-1 min-1 and Km 32.8 µM). In conclusion, LST-3TM12 is a functional splice variant of SLCO1B3 and SLCO1B7 expressed in the ER of human liver.

Regulation of PDZ domain-containing 1 (PDZK1) expression by hepatocyte nuclear factor-1α (HNF1α) in human kidney.

In the renal proximal tubule the secretion and reabsorption of glomerularly filtrated compounds is realized by a functional network of uptake and efflux transporters. The activity and localization of several transporters expressed at the apical tubular membrane are regulated by the membrane-associated protein PDZ domain-containing 1 (PDZK1). We aimed to characterize the transcriptional regulation of this modulator of renal transport. Coexpression analyses of PDZK1 and putative regulators were performed using human kidney samples. Protein and mRNA expression of PDZK1 in renal proximal tubule epithelial cells after adenoviral transfer and siRNA knockdown of transcription factor hepatocyte nuclear factor-1α (HNF1α) was assessed by quantitative real-time PCR and Western blot analysis. Transactivation of the PDZK1 promoter was quantified in cell-based reporter gene assays. Subsequently, the binding of HNF1α to the PDZK1 promoter was verified by in silico analyses and chromatin immunoprecipitation assay. HNF1α positively regulated the promoter activity of PDZK1. Adenoviral overexpression of HNF1α in renal proximal tubule epithelial cells (RPTEC) increased PDZK1 mRNA and protein expression, whereas siRNA knockdown of HNF1α resulted in decreased expression of PDZK1. Our results show that HNF1α, which has previously been described as a modulator of several transporters of the renal transportosome, is also a key determinant of PDZK1 transcription.

Pimecrolimus increases the expression of interferon-inducible genes that modulate human coronary artery cells proliferation.

The pharmacodynamics of the loaded compounds defines clinical failure or success of a drug-eluting device. Various limus derivatives have entered clinics due to the observed positive outcome after stent implantation, which is explained by their antiproliferative activity resulting from inhibition of the cytosolic immunophilin FK506-binding protein 12. Although pimecrolimus also binds to this protein, pimecrolimus-eluting stents failed in clinics. However, despite its impact on T lymphocytes little is known about the pharmacodynamics of pimecrolimus in cultured human coronary artery cells. We were able to show that pimecrolimus exerts antiproliferative activity in human smooth muscle and endothelial cells. Furthermore in those cells pimecrolimus induced transcription of interferon-inducible genes which in part are known to modulate cell proliferation. Modulation of gene expression may be part of an interaction between calcineurin, the downstream target of the pimecrolimus/FK506-binding protein 12-complex, and the toll-like receptor 4. In accordance are our findings showing that silencing of toll-like receptor 4 by siRNA in A549 a lung carcinoma cell line reduced the activation of interferon-inducible genes upon pimecrolimus treatment in those cells. Based on our findings we hypothesize that calcineurin inhibition may induce the toll-like receptor 4 mediated activation of type I interferon signaling finally inducing the observed effect in endothelial and smooth muscle cells. The crosstalk of interferon and toll-like receptor signaling may be a molecular mechanism that contributed to the failure of pimecrolimus-eluting stents in humans.

Identification of anti-HIV active dicaffeoylquinic- and tricaffeoylquinic acids in Helichrysum populifolium by NMR-based metabolomic guided fractionation.

South Africa being home to more than 35% of the world's Helichrysum species (c.a. 244) of which many are used in traditional medicine, is seen potentially as a significant resource in the search of new anti-HIV chemical entities. It was established that five of the 30 Helichrysum species selected for this study had significant anti-HIV activity ranging between 12 and 21 µg/mL (IC50) by using an in-house developed DeCIPhR method on a full virus model. Subsequent toxicity tests also revealed little or no toxicity for these active extracts. With the use of NMR-based metabolomics, the search for common chemical characteristics within the plant extract was conducted, which resulted in specific chemical shift areas identified that could be linked to the anti-HIV activity of the extracts. The NMR chemical shifts associated with the activity were identified to be 2.56-3.08 ppm, 5.24-6.28 ppm, 6.44-7.04 ppm and 7.24-8.04 ppm. This activity profile was then used to guide the fractionation process by narrowing down and focusing the fractionation and purification processes to speed up the putative identification of five compounds with anti-HIV activity in the most active species, Helichrysum populifolium. The anti-HIV compounds identified for the first time from H. populifolium were three dicaffeoylquinic acid derivatives, i.e. 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid as well as two tricaffeoylquinic acid derivatives i.e. 1,3,5-tricaffeoylquinic acid and either 5-malonyl-1,3,4-tricaffeoylquinic or 3-malonyl-1,4,5-tricaffeoylquinic acid, with the latter being identified for the first time in the genus.

Ethnopharmacology in overdrive: the remarkable anti-HIV activity of Artemisia annua.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua contains the well-known antimalarial compound artemisinin, which forms the backbone of the global malaria treatment regime. In African countries a tea infusion prepared from Artemisia annua has been used for the treatment of malaria only for the past 10-20 years. Several informal claims in Africa exist that the Artemisia annua tea infusions are also able to inhibit HIV. Since HIV is a relatively newly emerged disease, the claims, if substantiated, could provide a very good example of "ethnopharmacology in overdrive". The objective of this study was to provide quantitative scientific evidence that the Artemisia annua tea infusion exhibits anti-HIV activity through in vitro studies. A second objective was to determine if artemisinin plays a direct or indirect (synergistic) role in any observed activity. This was done by the inclusion of a chemically closely related species, Artemisia afra, known not to contain any artemisinin in our studies. MATERIALS AND METHODS: Validated cellular systems were used to test Artemisia annua tea samples for anti-HIV activity. Two independent tests with different formats (an infection format and a co-cultivation format) were used. Samples were also tested for cellular toxicity against the human cells used in the assays. RESULTS: The Artemisia annua tea infusion was found to be highly active with IC(50) values as low as 2.0 µg/mL. Moreover we found that artemisinin was inactive at 25 µg/mL and that a chemically related species Artemisia afra (not containing artemisinin) showed a similar level of activity. This indicates that the role of artemisinin, directly or indirectly (synergism), in the observed activity is rather limited. Additionally, no cellular toxicity was seen for the tea infusion at the highest concentrations tested. CONCLUSION: This study provides the first in vitro evidence of anti-HIV activity of the Artemisia annua tea infusion. We also report for the first time on the anti-HIV activity of Artemisia afra although this was not an objective of this study. These results open the way to identify new active pharmaceutical ingredients in Artemisia annua and thereby potentially reduce the cost for the production of the important antimalarial compound artemisinin.