10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof.

Chair

Prof. Dr.

Substrate specificity and transport kinetics of MATE1 and MATE2K and high functional overlap with OCT1 and OCT2

Ion channels and membrane transporters

BR05

Introduction: The substrate spectrum of the multidrug and toxin extrusion proteins 1 and 2 (MATE1 and -2K) was studied earlier but is still incompletely understood. The functional synergy between organic cation transporter 1 (OCT1) and MATE1 in the liver as well as OCT2 and MATE2K in the kidneys is well known but no comprehensive data exists on the corresponding substrate overlap. Therefore, we screened a very broad spectrum of mostly positively charged low molecular weight substances as potential substrates of MATE1, MATE2K, OCT1, and OCT2.Methods: We studied the uptake of substances in HEK293 cells stably overexpressing each of the transporters. Intracellular drug concentrations were analysed using high-performance liquid chromatography coupled with tandem mass spectrometry.Results: In total, 590 substances were tested as substrates of MATE1 and MATE2K from which 164 (27.8%) and 114 (19.3%) were substrates based on an uptake ratio of >2, respectively. Detailed transport kinetics revealed berberine, coptisine, pentamidine and amisulpride as substrates with highest affinity (KM) and epinephrine, methacholine, atenolol and neostigmine as substrates with highest Vmax. About 57 (34.8%) of MATE1 substrates were no MATE2K substrates but almost all MATE2K substrates were transported by MATE1. Both MATEs showed only moderate stereoselectivity with chiral drugs. Although there is only about 15% sequence homology between the MATE and OCT transporters, there was a surprisingly high overlap in the substrate spectrum between the 4 transporters with a particularly high correlation between MATE1 and OCT1. Substrates of the four transporters did not differ in typical molecular features like molecular weight, basic pKA, lipophilicity and polar surface area.Conclusion: In conclusion, the broad spectrum of MATE substrates encompasses numerous endobiotics and xenobiotics, including 120 drugs from various therapeutic classes. This diversity of MATE substrates in size, structure, and charge means that we still cannot predict the substrates by molecular features. The large number of substrates highlights the significance of MATE1 and MATE2K in pharmacokinetics and drug-drug interactions. The surprisingly high substrate overlap may be a good starting for comparison of the structure of the MATE and OCT transporters by methods of structural biology like cryogenic electron microscopy.

Basic Research

Short talk

Transdermal drug delivery: fine-tuned drug loading and release from novel mesoporous SiO2-based membranes with defined pore architectures and chemical surface modifications

Drug transport/delivery and metabolism

BR06

Transdermal drug delivery systems (TDDSs) play important roles in therapy due to distinct advantages over other forms and types of drug application. Despite being in clinical use since decades, TDDS have yet to reach their full potential. While polymer matrices are most commonly used as transdermal patches so far, inorganic carriers like mesoporous silica membranes offer several advantages, including high loading capacities, favorable physical, technical and biological properties as well as chemical and mechanical stability, allowing possible re-use / drug re-loading and a broad chemical compatibility with drugs and chemical enhancers. Mesoporous glass membranes can be prepared with a particularly wide range of possible pore architectures and chemical modifications.Here, we explored for the first time monolithic SiO2-based carriers as sustained release systems of therapeutics. By systematic variation, pore volumes and drug concentrations, but not pore diameter or pore surface area were identified as important parameters of drug loading and release kinetics. Additionally, drug loading capacities and release profiles were substantially affected by chemical surface modifications with strongly acidic SO3H groups or hydrophobic methyl groups, while introducing weakly acidic COOH groups or nitrile groups showed little effects. SO3H modification of low porosity (LP) membranes led to markedly increased loading capacity and a more sustained release profile of anastrozole. Biocompatibility studies showed full biocompatibility of SO3H functionalized membranes as indicated by efficient cell attachment and viability.In Franz cell experiments on porcine skin, we also demonstrated surprisingly substantial drug loss during skin penetration. Drug passage through porcine skin was found to be dependent on the age and pre-treatment of the skin. pH and temperature were major determinants of drug recovery rates as well, indicating drug loss in the skin by enzymatic metabolization. High loss rates in the skin were determined for different drugs, including anastrozole, xylazine and imiquimod.Conclusions: 1) SiO2-based membranes are attractive novel TDDS. 2) Their physical and chemical structure-property relationships will allow for further fine-tuning towards optimized systems for use in transdermal drug applications. 3) Drug loss is a major bottleneck in achieving high skin penetration and, thus, the systemic availability of drugs.

Prof

Hepatoproective activity of ethyl acetate fraction of Telosma pallida flower against carbon tetrachloride induced hepatotoxicity in rats

Disease models, drug development

BR07

Background: In the present work, hepatoprotective power of ethyl acetate fraction of Telosma pallida flower (TPFEAF) was evaluated against carbon tetrachloride (CCl4) in rats. T. pallida is a climber plant having flowers in the monsoon season only. The flowers are nutritious and edible.Materials and Methods: Ethyl acetate fraction was prepared from hydro-alcoholic extract. A total of thirty rats were equally divided into the five groups comprise of 6 rats in each. Group C1 did not receive any treatment. Group C2 was treated with CCl4 (1 ml kg-1). Group C3 received silymarin (200 mg kg-1) along with CCl4 (1 ml kg-1). Group T1 and T2 received TPFEAF at the dose of 100 and 200 mg kg-1 along with CCl4 (1 ml kg-1). The treatment period was seven days. On the 8th day, all the rats were killed, serum, plasma and liver tissues were collected to perform serum biochemical parameters like Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and Alkaline Phosphatase (ALP) level. Oxidative stress parameters like superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH Px), and malondialdehyde (MDA).Results and Discussion: The ALT and AST levels were significantly elevated (p<0.05) in the carbon tetrachloride-treated group compared to other treatment groups. ALT levels in the silymarin and TPFEAF-treated groups were not significantly different (p>0.05). ALP levels were significantly higher (p<0.05) in the carbon tetrachloride group compared to control and other treatment groups. The control group had significantly lower levels (p<0.05) of ALT and ALP compared to treatment groups, while AST levels in the standard control group approached normal values. SOD activity was significantly higher in the carbon tetrachloride (CCl4) group compared to all other groups, while GSH levels were significantly lower. The MDA levels were significantly elevated in the CCl4 group but showed a dose-dependent reduction with TPFEAF treatment. GSH-Px activity was significantly decreased in the CCl4 group but increased with silymarin and TPFEAF treatments, with the 200 mg/kg TPFEAF group showing non-significant differences from the control group.Conclusion: TPFEAF showed the strong protection against carbon tetrachloride induced hepatoxocity in rats. The probable mechanism via protecting liver parameters like AST, ALT, and ALP via scavenging CCl4 induced free radicals by boosting antioxidant biomarkers.

HDAC2 inactivation in CREM-IbΔC-X transgenic mice resulted in proteomic changes related to structural remodelling of atrial fibrillation

Objective: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with extensive functional and structural atrial remodeling. We have previously shown that genetic inactivation of histone deacetylase 2 (HDAC2) reduces thrombus formation, atrial fibrosis and left atrial weight in CREM-IbΔC-X transgenic (TG) mice, a well-characterized model with extensive atrial remodeling and spontaneous AF development. Here, we investigated the impact of cardiomyocyte-specific inactivation of HDAC2 (KO) in atrial cardiomyocytes from TG mice on the atrial proteome to identify regulated pathways.Material & Methods: Proteins were extracted from atrial tissue samples of TG, KO, TGxKO and control (CTR) mice. The proteome was analyzed using an ESI-LC-MS/MS system. Data analysis was performed using R and Ingenuity® pathway analysis (IPA). ShinyGO 0.81 was used for gene ontology (GO) analysis.Results: Of the 3949 proteins identified 342 were significantly regulated in both TG and TGxKO, and these were almost exclusively counterregulated. In contrast, only 120 proteins were significantly regulated in KO compared to CTR, documenting the enhanced effect of HDAC2 inactivation under disease conditions. In males, 198 proteins were regulated in TG and 196 of them were counterregulated in TGxKO. In females, only 48 proteins were regulated in TG and consequently counterregulated in TGxKO. Furthermore, only 17 proteins were exclusively regulated in TGxKO females (e.g. Vcl, Vcam1, Tppp), whereas 178 proteins were exclusively regulated in TGxKO males, such as Tnni1, various collagen or myosin chain isoforms. IPA revealed reduced activation of pathways addressing connective tissue formation and extracellular matrix organization, as well as reduced activation of biological functions related to cytoskeleton reorganization, fibroblast movement and inflammatory response in TGxKO compared to TG. (analyzed atria, male/female, CTR: 5/6, KO: 6/5, TG: 4/5, TGxKO: 5/4)Conclusion: The KO of HDAC2 in TG mice resulted in proteomic changes related to the structural remodeling of AF. Fewer proteins are affected by KO in female mice than in male mice, but both showed a variety of enriched and downregulated pathways related to cytoskeletal and contractile filament reorganization, inflammation and fibrosis. Our results emphasize the therapeutic potential of inactivating or inhibiting HDAC2 for the treatment of structural remodeling in atrial fibrillation.Supported by the DFG

Dose-dependent kinetics of thiamine in healthy volunteers with and without functional OCT1 hepatic transporter

Pharmacokinetics and PK/PD modelling

BR08

Thiamine, also known as Vitamin B1, is an essential water-soluble nutrient available in approved OTC preparations in pharmacies but is also sold as a dietary supplement in drugstores. Studies in humans have reported conflicting results on the role of the organic cation transporter 1 (OCT1) for thiamine kinetics. In this study, we aimed to determine whether the effects of OCT1 on thiamine pharmacokinetics may dependent on thiamine dose.We recruited 12 healthy volunteers with functional OCT1 (homozygous for OCT1*1, "reference") and 12 healthy volunteers with deficient OCT1 (homozygous or compound heterozygous for OCT1*3, *4, *5, *6; "poor transporter") into a 4-arm cross-over trial testing single oral doses of 5 mg, 10 mg, 50 mg, and 200 mg thiamine in each volunteer in the fasting condition. Blood and urine samples were collected and analyzed by LC-MS/MS to determine standard kinetic parameters, e.g., AUC0-24h, Cmax, Tmax, oral and renal clearance.Mean AUC0-24h for 5 mg, 10 mg, 50 mg, and 200 mg in the reference group were 1758 min*ng/ml, 2022 min*ng/ml, 2797 min*ng/ml, and 7605 min*ng/ml, resp. Mean AUC0-24h for 5 mg, 10 mg, 50 mg, and 200 mg in the poor transporter group were 1759 min*ng/ml, 2129 min*ng/ml, 2851 min*ng/ml, and 7370 min*ng/ml, resp. (genotype comparison at any dose level: p = 0.695, p = 0.833, p = 0.608, p = 0.887; Mann-Whitney-U-Test for independent samples).Mean Cmax for 5 mg, 10 mg, 50 mg, and 200 mg in the reference group were 3.3 ng/ml, 3.8 ng/ml, 7.3 ng/ml, and 29.7 ng/ml, resp. Mean Cmax for 5 mg, 10 mg, 50 mg, and 200 mg in the poor transporter group were 3.7 ng/ml, 4.5 ng/ml, 8.2 ng/ml, and 23.5 ng/ml, resp. (genotype comparison at any dose level: p=0.316, p=0.235, p=0.88, p = 1.000; Mann-Whitney-U-Test for independent samples).In conclusion, we observed a linear relationship between dose and AUC0-24h in our study, but oral thiamine kinetics was not dependent on OCT1 genotypes at any dose tested. Our study findings do not indicate that OCT1 saturation caused the lack of thiamine dependence on OCT1 genotypes. This result suggests that thiamine uptake in the liver is more complex than previously thought. OCT1 may not be the major thiamine transporter in humans as opposed to findings in mice models. Other transporters might compensate for OCT1 deficiency.

Short talk 20
Transport, drug delivery and disease models

Appointment

Chair

Programme

Short talk 20Transport, drug delivery and disease models

Appointment

Chair

Programme

Short talk 20
Transport, drug delivery and disease models