Stephan Krähenbühl

The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies

The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood‐brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para‐ or transcellular permeabilities (Pe) of inulin (0.74u2003×u200310−3u2003cm/min), sucrose (1.60u2003×u200310−3u2003cm/min), lucifer yellow (1.33u2003×u200310−3u2003cm/min), morphine (5.36u2003×u200310−3u2003cm/min), propranolol (4.49u2003×u200310−3u2003cm/min) and midazolam (5.13u2003×u200310−3u2003cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1–5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1‐mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p‐glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood‐brain barrier.

Dose adjustment in patients with liver disease.

Unfortunately, there is no endogenous marker for hepatic clearance that can be used as a guide for drug dosing. In order to predict the kinetic behaviour of drugs in cirrhotic patients, agents can be grouped according to their extent of hepatic extraction. For drugs with a high hepatic extraction (low bioavailability in healthy subjects), bioavailability increases and hepatic clearance decreases in cirrhotic patients. If such drugs are administered orally to cirrhotic patients, their initial dose has to be reduced according to hepatic extraction. Furthermore, their maintenance dose has to be adapted irrespective of the route of administration, if possible, according to kinetic studies in cirrhotic patients. For drugs with a low hepatic extraction, bioavailability is not affected by liver disease, but hepatic clearance may be affected. For such drugs, only the maintenance dose has to be reduced, according to the estimated decrease in hepatic drug metabolism. For drugs with an intermediate hepatic extraction, initial oral doses should be chosen in the low range of normal in cirrhotic patients and maintenance doses should be reduced as for high extraction drugs. In cholestatic patients, the clearance of drugs with predominant biliary elimination may be impaired. Guidelines for dose reduction in cholestasis exist for many antineoplastic drugs, but are mostly lacking for other drugs with biliary elimination. Dose adaptation of such drugs in cholestatic patients is, therefore, difficult and has to be performed according to pharmacological effect and/or toxicity. Importantly, the dose of drugs with predominant renal elimination may also have to be adapted in patients with liver disease. Cirrhotic patients often have impaired renal function, despite a normal serum creatinine level. In cirrhotic patients, creatinine clearance should, therefore, be measured or estimated to gain a guideline for the dosing of drugs with predominant renal elimination. Since the creatinine clearance tends to overestimate glomerular filtration in cirrhotic patients, the dose of a given drug may still be too high after adaptation to creatinine clearance. Therefore, the clinical monitoring of pharmacological effects and toxicity of such drugs is important. Besides the mentioned kinetic changes, the dynamics of some drugs is also altered in cirrhotic patients. Examples include opiates, benzodiazepines, NSAIDs and diuretics. Such drugs may exhibit unusual adverse effects that clinicians should be aware of for their safe use. However, it is important to realise that the recommendations for dose adaptation remain general and cannot replace accurate clinical monitoring of patients with liver disease treated with critical drugs.

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.

Toxicity of amiodarone and amiodarone analogues on isolated rat liver mitochondria.

BACKGROUNDnAmiodarone is a well-known mitochondrial toxin consisting of a benzofuran ring (ring A) coupled to a p-OH-benzene structure substituted with 2 iodines and a diethyl-ethanolamine side chain (ring B).nnnAIMnTo find out which part of amiodarone is responsible for mitochondrial toxicity.nnnMETHODSnAmiodarone, ring A and B without the ethanolamine side-chain and iodines (B0), ring A and B with iodines but no ethanolamine (B2), ring B with 1 iodine and no ethanolamine (C1) and ring B with ethanolamine and 2 iodines (D2) were studied.nnnRESULTSnIn freshly isolated rat liver mitochondria, amiodarone inhibited state 3 glutamate and palmitoyl-CoA oxidation and decreased the respiratory control ratios. B0 and B2 were more potent inhibitors than amiodarone and B2 more potent than B0. C1 and D2 showed no significant mitochondrial toxicity. After disruption, mitochondrial oxidases and complexes of the electron transport chain were inhibited by amiodarone, B0 and B2, whereas C1 and D2 revealed no inhibition. Beta-oxidation showed a strong inhibition by amiodarone, B0 and B2 but not by C1 or D2. Ketogenesis was almost unaffected.nnnCONCLUSIONSnAmiodarone, B0 and B2 are uncouplers of oxidative phosphorylation, and inhibit complexes I, II and III, and beta-oxidation. The benzofuran structure is responsible for mitochondrial toxicity of amiodarone and the presence of iodine is not essential.

Prevalence of Potentially Inappropriate Medication Use in Elderly Patients Comparison between General Medical and Geriatric Wards

Background and objectiveInappropriate drug use is one of the risk factors for adverse drug reactions in the elderly. We hypothesised that, in elderly patients, geriatricians are more aware of potentially inappropriate medications (PIMs) and may replace or stop PIMs more frequently compared with internists. We therefore evaluated and compared the prevalence of PIMs as well as anticholinergic drug use throughout hospital stay in elderly patients admitted to a medical or geriatric ward.MethodsIn this retrospective cross-sectional study, 800 patients aged ≥65 years admitted to a general medical or geriatric ward of a 700-bed teaching hospital in Switzerland during 2004 were included. PIMs were identified using the Beers criteria published in 2003. The prevalence of anticholinergic drug use was assessed based on drug lists published in the literature.ResultsThe prevalence of use of PIMs that should generally be avoided was similar in medical and geriatric inpatients both at admission (16.0% vs 20.8%, respectively; p = 0.08) and at discharge (13.3% vs 15.9%, respectively; p = 0.31). In contrast to medical patients, the reduction in the prevalence of use of PIMs between admission and discharge in geriatric patients reached statistical significance (p < 0.05). Overall, the three most prevalent inappropriate drugs/drug classes were amiodarone, long-acting benzodiazepines and anticholinergic antispasmodics. At admission, the prevalence of use of PIMs related to a specific diagnosis was not significantly different between patients hospitalised to a medical or a geriatric ward (14.0% vs 17.5%, respectively; p = 0.17), as compared with the significant difference evident at hospital discharge (11.7% vs 23.7%, respectively; p < 0.001). This was largely because of ahigher prescription rate of platelet aggregation inhibitors in combination with low-molecular-weight heparins and benzodiazepines in patients with a history of falls and syncope. The proportions of patients taking anticholinergic drugs in medical and geriatric patients at admission (13.0% vs 17.5%, respectively; p = 0.08) and discharge (12.2% vs 16.5%, respectively; p = 0.10) were similar.ConclusionInappropriate drug use as defined by the Beers criteria was common in both medical and geriatric inpatients. Compared with internists, geriatricians appear to be more aware of PIMs that should generally be avoided, but less aware of PIMs related to a specific diagnosis, and of the need to avoid anticholinergic drug use. However, the results of this study should be interpreted with caution because some of the drugs identified as potentially inappropriate may in fact be beneficial when the patient’s clinical condition is taken into consideration.

Determination of carnitine and acylcarnitines in urine by high-performance liquid chromatography–electrospray ionization ion trap tandem mass spectrometry

A high-performance liquid chromatography-mass spectrometry method has been developed for the simultaneous determination of native carnitine and eight acylcarnitines in urine. The procedure uses a solid-phase extraction on a cation-exchange column and the separation is performed without derivatization within 17 min on a reversed-phase C8 column in the presence of a volatile ion-pairing reagent. The detector was an ion trap mass spectrometer and quantification was carried out in the MS-MS mode. Validation was done for aqueous standards at ranges between 0.75 and 200 micromol/l, depending on the compound. Carnitine was quantified in urine and comparison with a radioenzymatic assay gave a satisfactory correlation (R2 = 0.981). The assay could be successfully applied to the diagnostic of pathological acylcarnitines profile of metabolic disorders in urines of patients suffering from different organic acidurias.

HLA Haplotype Determines Hapten or p-i T Cell Reactivity to Flucloxacillin

Drug-induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*57:01 allele. At present, the mechanism of FLUX-DILI is not understood, but the HLA association suggests a role for activated T cells in the pathomechanism of liver damage. To understand the interaction among FLUX, HLA molecules, and T cells, we generated FLUX-reacting T cells from FLUX-naive HLA-B*57:01+ and HLA-B*57:01− healthy donors and investigated the mechanism of T cell stimulation. We found that FLUX stimulates CD8+ T cells in two distinct manners. On one hand, FLUX was stably presented on various HLA molecules, resistant to extensive washing and dependent on proteasomal processing, suggesting a hapten mechanism. On the other hand, in HLA-B*57:01+ individuals, we observed a pharmacological interaction with immune receptors (p-i)–based T cell reactivity. FLUX was presented in a labile manner that was further characterized by independence of proteasomal processing and immediate T cell clone activation upon stimulation with FLUX in solution. This p-i–based T cell stimulation was restricted to the HLA-B*57:01 allele. We conclude that the presence of HLA-B*57:01 drives CD8+ T cell responses to the penicillin-derivative FLUX toward nonhapten mechanism.

Effects of drug interactions on biotransformation and antiplatelet effect of clopidogrel in vitro

BACKGROUND AND PURPOSE The conversion of clopidogrel to its active metabolite, R‐130964, is a two‐step cytochrome P450 (CYP)‐dependent process. The current investigations were performed to characterize in vitro the effects of different CYP inhibitors on the biotransformation and on the antiplatelet effect of clopidogrel.

Analysis of carnitine and acylcarnitines in urine by capillary electrophoresis.

A capillary electrophoresis method is described for the simultaneous analysis of carnitine and short-chain acylcarnitines in aqueous standard solutions and urine samples. Samples were worked up using silica gel extraction and derivatization with 4-bromophenacyl trifluoromethanesulfonate. Separation was performed in less than 8 min using a binary buffer system containing phosphate/phosphoric acid and sodium dodecyl sulfate. 3-(2,2,2-Trimethylhydrazinium)propionate (mildronate) was used as an internal standard. The method was developed with aqueous standard solutions and then applied successfully to spiked and unspiked human urine samples. The limit of detection for both carnitine and acetylcamitine is 3 microM.

T Cells Infiltrate the Liver and Kill Hepatocytes in HLA-B∗57:01-Associated Floxacillin-Induced Liver Injury

Drug-induced liver injury is a major safety issue. It can cause severe disease and is a common cause of the withdrawal of drugs from the pharmaceutical market. Recent studies have identified the HLA-B(∗)57:01 allele as a risk factor for floxacillin (FLUX)-induced liver injury and have suggested a role for cytotoxic CD8(+) T cells in the pathomechanism of liver injury caused by FLUX. This study aimed to confirm the importance of FLUX-reacting cytotoxic lymphocytes in the pathomechanism of liver injury and to dissect the involved mechanisms of cytotoxicity. IHC staining of a liver biopsy from a patient with FLUX-induced liver injury revealed periportal inflammation and the infiltration of cytotoxic CD3(+) CD8(+) lymphocytes into the liver. The infiltration of cytotoxic lymphocytes into the liver of a patient with FLUX-induced liver injury demonstrates the importance of FLUX-reacting T cells in the underlying pathomechanism. Cytotoxicity of FLUX-reacting T cells from 10 HLA-B(∗)57:01(+) healthy donors toward autologous target cells and HLA-B(∗)57:01-transduced hepatocytes was analyzed in vitro. Cytotoxicity of FLUX-reacting T cells was concentration dependent and required concentrations in the range of peak serum levels after FLUX administration. Killing of target cells was mediated by different cytotoxic mechanisms. Our findings emphasize the role of the adaptive immune system and especially of activated drug-reacting T cells in human leukocyte antigen-associated, drug-induced liver injury.