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Abomasal emptying rate of diarrhoeic and healthy suckling calves fed with oral rehydration solutions
(2020)
Abstract
The aim of the study was to determine the abomasal emptying rate (AER) of calves suffering from naturally occurring diarrhoea compared with that of healthy calves. Furthermore, the effects of an oral rehydration solution (ORS) mixed into milk replacer on the AER were determined. Acetaminophen absorption test (APAT) was performed to estimate the AER. Sixty Holstein–Frisian calves (age < 14 days) were included in the study and divided into groups as follows: healthy calves (H; n = 16), healthy calves fed with ORS (HORS; n = 14), diarrhoeic calves (D; n = 15) and diarrhoeic calves fed with ORS (DORS; n = 15). For the APAT, the calves were fed 2 L of milk replacer containing 50 mg acetaminophen (AP)/kg body weight. Venous blood samples were collected before and after milk replacer and AP intake in 30–60 min intervals for 12 hr. During the APAT, no significant differences in median maximum acetaminophen concentration (Cmax) were observed among all groups. Time to reach maximum acetaminophen concentration (Tmax) in DORS (median 390 min, 25/75 quartiles: 300/480 min) was significantly higher compared with that in H (median: 270 min 25/75 quartiles: 210/315 min) and HORS (median: 300 min (25/75 quartiles: 240/360 min). Non‐linear regression revealed that the calculated abomasal half‐life (AP t1/2) tended to be delayed in DORS (median: 652 min, 25/75 quartiles: 445/795 min, p = .10). The area under the AP curve values (AUC) from 0 to 120 min and 0 to 240 min of the observation period were significantly higher in H than D and DORS. In conclusion, significant differences in the AER indices reflected delayed abomasal emptying in diarrhoeic calves. Furthermore, the hypertonic ORS tended to have an additive delaying impact on the AER, which needs attention for the feeding management of diarrhoeic calves.
Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the β2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human–mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar β2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure–function data of OCT1 is not directly transferrable between substrates or species.
OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.
Pentathiepins are polysulfur-containing compounds that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and inhibit glutathione peroxidase (GPx1). This renders them promising candidates for anticancer drug development. However, the biological effects and how they intertwine have not yet been systematically assessed in diverse cancer cell lines. In this study, six novel pentathiepins were synthesized to suit particular requirements such as fluorescent properties or improved water solubility. Structural elucidation by X-ray crystallography was successful for three derivatives. All six underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigating the inhibition of GPx1 and cell proliferation, cytotoxicity, and the induction of ROS and DNA strand breaks. Furthermore, selected hallmarks of apoptosis and the impact on cell cycle progression were studied. All six pentathiepins exerted high cytotoxic and antiproliferative activity, while five also strongly inhibited GPx1. There is a clear connection between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks. Additionally, these studies support apoptosis but not ferroptosis as the mechanism of cell death in some of the cell lines. As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for an optimization of the anticancer activity of pentathiepins in the future.
Course of disease and risk factors for hospitalization in outpatients with a SARS-CoV-2 infection
(2022)
We analyzed symptoms and comorbidities as predictors of hospitalization in 710 outpatients in North-East Germany with PCR-confirmed SARS-CoV-2 infection. During the first 3 days of infection, commonly reported symptoms were fatigue (71.8%), arthralgia/myalgia (56.8%), headache (55.1%), and dry cough (51.8%). Loss of smell (anosmia), loss of taste (ageusia), dyspnea, and productive cough were reported with an onset of 4 days. Anosmia or ageusia were reported by only 18% of the participants at day one, but up to 49% between days 7 and 9. Not all participants who reported ageusia also reported anosmia. Individuals suffering from ageusia without anosmia were at highest risk of hospitalization (OR 6.8, 95% CI 2.5–18.1). They also experienced more commonly dyspnea and nausea (OR of 3.0, 2.9, respectively) suggesting pathophysiological connections between these symptoms. Other symptoms significantly associated with increased risk of hospitalization were dyspnea, vomiting, and fever. Among basic parameters and comorbidities, age > 60 years, COPD, prior stroke, diabetes, kidney and cardiac diseases were also associated with increased risk of hospitalization. In conclusion, due to the delayed onset, ageusia and anosmia may be of limited use in differential diagnosis of SARS-CoV-2. However, differentiation between ageusia and anosmia may be useful for evaluating risk for hospitalization.
Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery
(2021)
Introduction
Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery.
Materials and Methods
In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus.
Results
Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P.
Discussion
In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.
(1) Background: Sepsis is a leading cause of death and a global public health problem. Accordingly, deciphering the underlying molecular mechanisms of this disease and the determinants of its morbidity and mortality is pivotal. This study examined the effect of the rs951818 SNP of the negative costimulatory lymphocyte-activation gene 3 (LAG-3) on sepsis mortality and disease severity. (2) Methods: 707 consecutive patients with sepsis were prospectively enrolled into the present study from three surgical ICUs at University Medical Center Goettingen. Both 28- and 90-day mortality were analyzed as the primary outcome, while parameters of disease severity served as secondary endpoints. (3) Results: In the Kaplan–Meier analysis LAG-3 rs951818 AA-homozygote patients showed a significantly lower 28-day mortality (17.3%) compared to carriers of the C-allele (23.7%, p = 0.0476). In addition, these patients more often received invasive mechanical ventilation (96%) during the course of disease than C-allele carriers (92%, p = 0.0466). (4) Conclusions: Genetic profiling of LAG-3 genetic variants alone or in combination with other genetic biomarkers may represent a promising approach for risk stratification of patients with sepsis. Patient-individual therapeutic targeting of immune checkpoints, such as LAG-3, may be a future component of sepsis therapy. Further detailed investigations in clinically relevant sepsis models are necessary.
Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.
The tricyclic antidepressant amitriptyline is frequently prescribed but its use is limited by its narrow therapeutic range and large variation in pharmacokinetics. Apart from interindividual differences in the activity of the metabolising enzymes cytochrome P450 (CYP) 2D6 and 2C19, genetic polymorphism of the hepatic influx transporter organic cation transporter 1 (OCT1) could be contributing to interindividual variation in pharmacokinetics. Here, the impact of OCT1 genetic variation on the pharmacokinetics of amitriptyline and its active metabolite nortriptyline was studied in vitro as well as in healthy volunteers and in depressive disorder patients. Amitriptyline and nortriptyline were found to inhibit OCT1 in recombinant cells with IC50 values of 28.6 and 40.4 µM. Thirty other antidepressant and neuroleptic drugs were also found to be moderate to strong OCT1 inhibitors with IC50 values in the micromolar range. However, in 35 healthy volunteers, preselected for their OCT1 genotypes, who received a single dose of 25 mg amitriptyline, no significant effects on amitriptyline and nortriptyline pharmacokinetics could be attributed to OCT1 genetic polymorphism. In contrast, the strong impact of the CYP2D6 genotype on amitriptyline and nortriptyline pharmacokinetics and of the CYP2C19 genotype on nortriptyline was confirmed. In addition, acylcarnitine derivatives were measured as endogenous biomarkers for OCT1 activity. The mean plasma concentrations of isobutyrylcarnitine and 2-methylbutyrylcarnitine were higher in participants with two active OCT1 alleles compared to those with zero OCT1 activity, further supporting their role as endogenous in vivo biomarkers for OCT1 activity. A moderate reduction in plasma isobutyrylcarnitine concentrations occurred at the time points at which amitriptyline plasma concentrations were the highest. In a second, independent study sample of 50 patients who underwent amitriptyline therapy of 75 mg twice daily, a significant trend of increasing amitriptyline plasma concentrations with decreasing OCT1 activity was observed (p = 0.018), while nortriptyline plasma concentrations were unaffected by the OCT1 genotype. Altogether, this comprehensive study showed that OCT1 activity does not appear to be a major factor determining amitriptyline and nortriptyline pharmacokinetics and that hepatic uptake occurs mainly through other mechanisms.
Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.
Organic cation transporter OCT1 is strongly expressed in the sinusoidal membrane of hepatocytes. OCT1 mediates the uptake of weakly basic and cationic compounds from the blood into the liver and may thereby facilitate the first step in hepatic metabolism or excretion of many cationic drugs. OCT1 is a polyspecific transporter and has a very broad spectrum of structurally highly diverse ligands (substrates and inhibitors). The exact transport mechanism and the amino acids involved in polyspecific ligand binding of OCT1 are poorly understood.
The aim of this work was to utilize the polyspecificity to better understand the structure-function relationships of OCT1 and to gain first insights into potential mechanisms conferring the polyspecificity. We followed two strategies, analyzing the effects of variability in both ligand and transporter structure on OCT1 function. The effects of ligand structure were analyzed by comparing uptake and inhibitory potencies of structurally similar drugs of the group of opioids. The effects of transporter structure were analyzed by comparing the effects of variability caused by naturally occurring genetic variants or artificial mutations on OCT1 uptake and inhibition of several substrates. Most importantly, the effects of interspecies variability in transporter structure were analyzed by comparing uptake kinetics between human and mouse OCT1 orthologs. To this end, we used stably or transiently transfected HEK293 cells overexpressing OCT1 and different chimeric and mutant variants thereof.
Focusing on OCT1 ligands, we compared the uptake and inhibitory potencies of structurally similar opioids. Only minor changes of the ligand structure strongly affected the interaction with OCT1. The presence of the ether linkage between C4 and C5 of the morphinan ring was associated with reduced OCT1 inhibitory potencies, while passive membrane permeability was the major negative determinant of OCT1-mediated uptake among structurally highly similar morphinan opioids. Only minor structural changes strongly increased the inhibitory potency by 28-fold from the lowest IC50 of 2004 µM for oxycodone to 72 µM for morphine. Additional removal of the ether linkage between C4-C5 increased the inhibitory potency by a total of 313-fold to the lowest IC50 of 6 µM for dextrorphan. Consequently, our data demonstrates that despite its polyspecificity, OCT1-mediated uptake and inhibition of this uptake is still somewhat very specific.
Focusing on OCT1 protein structure, we first analyzed the effects of variability caused by naturally occurring genetic variants on OCT1 uptake and inhibition. OCT1 transport was strongly affected by OCT1 genetic variants and these effects were often substrate-specific. Correlation of these effects revealed several substrates that were similarly affected by the variants and may therefore be suggested to share similar or overlapping binding sites in OCT1. In addition, the effects of the genetic variants OCT1*2 and OCT1*3 on different substrates correlated well which may suggest that the structural variability caused by these two variants similarly affects substrate uptake. OCT1 genetic variants also affected the inhibition of OCT1, with both substrate and genotype-specific differences. Ranitidine inhibited the uptake of several substrates, among them the clinically relevant drugs metformin and morphine. Moreover, the inhibition was more potent (about 2-fold) on the uptake mediated by the common genetic variant OCT1*2 than on the uptake mediated by the reference OCT1*1.
Second, we analyzed the effects of artificial mutations of key amino acids. Tyr222 and Asp475 in rat OCT1 had strongly substrate-specific and also species-specific effects on both OCT1-mediated uptake and inhibition. Mutation of these amino acids strongly decreased OCT1-mediated uptake, which further underscored an important role especially of Asp475. Interestingly, despite a proposed essential role of this amino acid, we observed Asp475-independent transport. This transport was observed in mouse, but not in human OCT1 and was substrate-specific. TMH10 was identified to be involved in determining the Asp475-independent uptake of mouse OCT1.
Finally and most importantly, we analyzed the effects of sequence differences between human and mouse OCT1 on the transport kinetics of several OCT1 substrates. The transport kinetics differed strongly between human and mouse OCT1 orthologs. These differences were substrate-specific and affected both the affinity (KM) and capacity (vmax) of transport. Human OCT1 had an 8-fold higher capacity of trospium transport, while mouse OCT1 had an 8-fold higher capacity of fenoterol transport. Furthermore, mouse OCT1 had a 5-fold higher affinity for metformin transport compared to human OCT1. The difference between Phe32 in human and Leu32 in mouse OCT1 in TMH1 was identified to confer a higher capacity of transport by human compared to mouse OCT1, while the difference between Cys36 in human and Tyr36 in mouse OCT1 in TMH1 was identified to confer a higher capacity of transport by mouse compared to human OCT1. Furthermore, Leu155 in human OCT1, corresponding to Val156 in mouse OCT1 in TMH2, in concert with TMH3 were identified to confer the differences in affinity for metformin transport between the species.
It may be speculated that ligand binding in OCT1 involves a core binding region that includes Asp474/475 and that polyspecific ligand binding is enabled by providing further binding partners (different amino acids) in more peripheral regions that different ligands can selectively interact with. This mechanism may also be a first step in explaining the substrate-specific effects of genetic variants with clinical relevance. Based on our findings, these “polyspecificity regions” may include TMH1, TMH2, and TMH3. Further analyses are warranted to characterize and narrow down these regions to unravel the structure-function relationships and with that the polyspecificity of OCT1.
To summarize, variability in both ligand and transporter structure strongly affected OCT1 function and we were able to identify ligand structures that affect inhibitory potency and protein structures that confer species-specific differences in OCT1 transport. This work emphasizes again the complexity of OCT1 transport and structure-function relationships. We also showed that, in spite of the difficulties for experimental analysis and data interpretation that arise from the polyspecific nature of OCT1, polyspecificity can also be used as a tool to better understand the structure-function relationships of this transporter.
About 30 % of epileptic patients are non-responsive to multidrug antiepileptic therapy. One of non-responsiveness in epilepsy hypothesis claims that non-responsiveness occurs because of reduced access of antiepileptic drugs to their targets, as a result of increased efflux of antiepileptic drugs away from these targets. Transporters believed to be involved in non-responsiveness in epilepsy are mainly but not exclusively the members of the ABC superfamily including P-gp (MDR1, ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and others. These proteins are normally found in the blood-brain barrier and the blood-cerebrospinal fluid barrier where they function as protectors. There is emerging evidence that P-gp, MRP1 and MRP2 are up-regulated in epileptogenic brain tissue. The risk of non-responsiveness could be related also to the MDR1 or MRP2 gene polymorphisms. We hypothesised that changes in expression and function of multidrug transporters involved in non-responsiveness of epilepsy might be detectable not only in the brain but also in other tissues such as lymphocytes. Therefore we evaluated the expression of MDR1, MRP1 and MRP2 and function of P-gp in lymphocytes in patients with epilepsy and healthy subjects. Three groups of epileptic patients and 15 healthy subjects as a control group were included in the study. The patients’ group was defined as follows: Monotherapy – patients treated with carbamazepine monotherapy, without seizures - corresponded to group responders. Combined therapy – patients after monotherapy (two different medicines have been tried) and combined therapy (two trials of combined therapy), not free of seizures. Monotherapy and combined therapy groups each embraced 15 patients. Neurosurgery – patients who had undergone neurosurgery, afterwards were or were not additionally treated with carbamazepine, with or without seizures. This group comprised 24 patients. Combined therapy and neurosurgery groups composed the group of non-responders. The mRNA expression of MRP1, MRP2 and MDR1 by means of quantitative real-time PCR as well as MRP2 and P-gp protein content by Western blot in lymphocytes was measured. For P-gp functional analysis rhodamine efflux from lymphocytes and natural killer (NK) cells was performed. The influence of the polymorphisms C3435T, G2677T/A in the MDR1 gene and C24T, G1249A, C3972T in the MRP2 gene for the transporters expression, function and their association with non-responsive epilepsy phenotype was investigated. Our results showed that MRP1 expression in lymphocytes was significantly lower in epileptics than in healthy subjects. Non-responders had lower MRP1 mRNA content in lymphocytes than responders. We did not find any difference in MRP2 expression between epileptics and healthy volunteers. MRP2 mRNA levels in lymphocytes were higher in non-responders than in responders. However, at protein level epileptic patients had significantly lower MRP2 content in lymphocytes than controls. MRP2 protein content did not differ in responders and non-responders. There was no reliable correlation between MRP2 mRNA expression and MRP2 protein content in lymphocytes. Epileptics had significantly lower MDR1 expression in lymphocytes than healthy individuals. MDR1 expression was decreasing according to the consumption of antiepileptic drugs and seizures frequency: patients after neurosurgery had significantly lower MDR1 expression than patients after combined therapy and monotherapy. MDR1 expression was significantly lower in non-responders than in responders. At protein level epileptics had lower P-gp content than controls. Detected P-gp amount in lymphocytes did not differ between responders and non-responders. Rhodamine efflux from lymphocytes and NK cells did not differ significantly between epileptics and healthy subjects, but it was higher in patients after neurosurgery than in patients after monotherapy. Rhodamine efflux from NK cells, which are known to express the highest levels of P-gp, was significantly higher in non-responders than in responders. In this study, we showed that MRP1 mRNA expression in lymphocytes was significantly correlated to its expression in the brain. We detected also a significant co-correlation between MRP1 expression in the hippocampus and MDR1 expression in lymphocytes. We found no evidence regarding the impact of the MDR1 polymorphisms on mRNA expression, P-gp content and rhodamine efflux from lymphocytes. Our data showed lack of evidence regarding the impact of the MRP2 polymorphisms on mRNA expression and protein content. We did not detect any association between MDR1 or MRP2 polymorphisms and non-responsiveness in epilepsy or epilepsy in the main. In conclusion, our results suggest that lymphocytes are an appropriate surrogate for studies on changes of multidrug transporters expression in epilepsy. Lymphocytes as an easily accessible tissue might serve as a marker for responsiveness to antiepileptic drug therapy in epilepsy studies.
Doxorubicin is a frequently used anticancer drug to treat many types of tumors, such as breast cancer or bronchial carcinoma. The clinical use of doxorubicin is limited by its poorly predictable cardiotoxicity, the reasons of which are so far not fully understood. The drug is a substrate of several efflux transporters such as P-gp or BCRP and was recently reported to be a substrate of cation uptake transporters. To evaluate the potential role of transporter proteins in the accumulation of doxorubicin at its site of action (e.g., mammary carcinoma cells) or adverse effects (e.g., heart muscle cells), we studied the expression of important uptake and efflux transporters in human breast cancer and cardiac tissue, and investigated the affinity of doxorubicin to the identified transporters. The cellular uptake studies on doxorubicin were performed with OATP1A2*1, OATP1A2*2, and OATP1A2*3-overexpressing HEK293 cells, as well as OCT1-, OCT2-, and OCT3- overexpressing MDCKII cells. To assess the contribution of transporters to the cytotoxic effect of doxorubicin, we determined the cell viability in the presence and absence of transporter inhibitors in different cell lines. Several transporters, including P-gp, BCRP, OCT1, OCT3, and OATP1A2 were expressed in human heart and/or breast cancer tissue. Doxorubicin could be identified as a substrate of OCT1, OCT2, OCT3, and OATP1A2. The cellular uptake into cells expressing genetic OATP1A2 variants was markedly reduced and correlated well with the increased cellular viability. Inhibition of OATP1A2 (naringin) and OCT transporters (1-methyl-4-phenylpyridinium) resulted in a significant decrease of doxorubicin-mediated cytotoxicity in cell lines expressing the respective transporters. Similarly, the excipient Cremophor EL significantly inhibited the OCT1-3- and OATP1A2-mediated cellular uptake and attenuated the cytotoxicity of doxorubicin. In conclusion, genetic and environmental-related variability in the expression and function of these transporters may contribute to the substantial variability seen in terms of doxorubicin efficacy and toxicity.
The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).
In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.