Jana Malakova

Lack of Interactions between Breast Cancer Resistance Protein (BCRP/ABCG2) and Selected Antiepileptic Agents

Summary:  Purpose: Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood–brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined.

New high-performance liquid chromatography method for the determination of (R)-warfarin and (S)-warfarin using chiral separation on a glycopeptide-based stationary phase.

Warfarin is a well-known anticoagulant agent that occurs in two enantiomers, (R)-(+)-warfarin and (S)-(-)-warfarin. A new liquid chromatography method for the determination of both enantiomers was developed, validated and applied in in vitro studies with the aim of evaluating the accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line. OptiMEM cell cultivation medium samples and cellular lysates were purified using Waters Oasis MAX extraction cartridges. The chiral separation of warfarin and the internal standard p-chlorowarfarin enantiomers was performed on an Astec Chirobiotic V2 column at a flow rate of 1.2mL/min. The mobile phase was composed of 31% acetonitrile, 5% of methanol and 64% of ammonium acetate buffer (10mmol/L, pH 4.1). The enantiomers were quantified using a fluorescence detector (lambda(excit)=320nm, lambda(emiss)=415nm). The limit of detection was found to be 0.121micromol/L of (S)-warfarin and 0.109micromol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100micromol/L. The precision ranged from 1.3% to 12.2% of the relative standard deviation, and the accuracy reached acceptable values from 95.5% to 108.4%. The new bioanalytical method confirmed the same accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line.

Tissue and plasma concentrations of cephuroxime during cardiac surgery in cardiopulmonary bypass — a microdialysis study

Aim: Wound and mediastinal infections are still very serious complications of open-heart surgery, in spite of the use of prophylactic antibiotics. The use of cardiopulmonary bypass (CPB) is associated with profound physiological changes affecting the pharmacokinetic behaviour of antibiotics. The aim of this pilot study was to monitor the tissue concentrations of cephuroxime (prophylactic antibiotic) in skeletal muscle during cardiac surgery using CPB by interstitial microdialysis. These concentrations were compared with plasma concentrations of cephuroxime. Material and methods: Nine adult patients operated on using CPB were enrolled in this study. Cephuroxime was used as a prophylactic antibiotic (1st dose — 3 g of cefuroxime i.v. with anesthesia induction, 2nd dose — 1.5 g i.v. after CPB with protamine sulphate, 3rd dose — 1.5 g i.v. 8 hours after the surgery). Interstitial microdialysis was performed by probe CMA 60 (CMA Microdialysis AB, Sweden) inserted into the patients deltoid muscle. Concentrations of cephuroxime in dialysates and in plasma were determined by the modified fluid chromatography method. The unbound cephuroxime fraction in plasma was obtained by using an ultrafiltration method. Samples of dialysates were collected at the following intervals: before CPB, each 30 minutes of CPB, at the end of CPB. Samples of blood were collected at these intervals: incision, start of CPB, each 30 minutes of CPB, at the end of CPB, at the end of surgery. Concentrations of cephuroxime in tissue were corrected by in vivo recoveries of the microdialysis probes. Results: Plasma concentrations of cephuroxime were 163.5 ± 40.1, 79.3 ± 17.4, 73.7 ± 16.8, 66.1 ± 18.3, 57.0 ± 10.9, 120.7 ± 29.9 (mg . L — 1) and concentrations of free plasma fraction of cephuroxime were 119.5 ± 35.2, 67.8 ± 15.5, 66.0 ± 12.5, 54.8 ± 12.2, 49.6 ± 9.8, 102.6 ± 26.0 (mg . L — 1). The concentrations of cephuroxime in dialysates were 44.3 ± 15.7, 36.1 ± 11.6, 31.9 ± 9.3, 34.6 ± 12.3, 27.6 ± 12.9, 56.7 ± 17.6 (mg . L — 1). The mean in vivo recovery of cephuroxime in this study was 30%. Corrected concentrations (calculated by in vivo recovery) of cephuroxime in skeletal muscle were 148, 120, 106, 115, 92, 189 (mg . L — 1). Conclusion: Our preliminary results show that CPB can modify the time course of cephuroxime plasma and tissue concentrations. A decrease in plasma drug concentrations occurred at the start of CPB and lasted until CPB ended. An increase in plasma concentrations corresponds to the second drug dose after CPB. The concentrations of cephuroxime in skeletal muscle (corrected by recovery) during CPB are higher than plasma concentrations. It is influenced by important changes during CPB; closely associated with hemodilution, a shift of intravascular volume, solutes and albumin to the extravascular space and inconstant protein binding of cephuroxime during operation. Perfusion (2007) 22, 129—136.

The effect of levetiracetam on rat bone mass, structure and metabolism.

OBJECTIVE To determine the effect of levetiracetam (LEV) Lon bone mineral density (BMD), mineral content (BMC), bone markers, body composition and bone mechanical strength in the orchidectomised (ORX) rat model. METHOD 16 orchidectomised Wistar rats were divided into control and test groups, 8 rats in each group. The control rats received standard laboratory diet (SLD) while rats in the test group were fed with SLD enriched with LEV for 12 weeks. BMD was measured by dual energy X-ray absorptiometry at the whole body, lumbar spine and femur. Bone marker concentrations were examined of osteoprotegerin (OPG) and insulin-like growth factor 1 (IGF-1) in serum, and amino-terminal propeptide of procollagen type I (PINP), carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), bone alkaline phosphatase (ALPL), and bone morphogenetic protein 2 (BMP-2) in bone homogenate. The femurs were used for biomechanical testing. RESULTS Compared to the control group we found lower fat mass, lower BMD in the area of the left femur, lower BMC in both femurs, a reduced concentration of OPG, and an increased concentration of CTX-I of borderline statistical significance (p=0.0661). Biomechanical parameters did not differ between groups. CONCLUSIONS Significant loss of BMD or BMC was seen at the left and right femur area in the LEV group. Administration of LEV in the ORX-rat model significantly decreased levels of OPG (marker of bone formation) in serum and increased levels of CTX-I (marker of bone resorption) in bone homogenate, but results in this study did not reveal any change in biomechanical bone strength. Administration of LEV in the ORX-rat model may reduce adipose tissue. Further studies in animals and humans will be needed to confirm these findings.

Up-regulation of renal Mdr1 and Mrp2 transporters during amiodarone pretreatment in rats

Although amiodarone (AMD) is known to produce drug-drug interactions through inhibition of transporter-mediated excretion of drugs, its impact on these mechanisms during chronic treatment has not been described yet. Therefore, the aim of this study was to investigate the influence of AMD pretreatment on the main multidrug transporting proteins, Mdr1 and Mrp2, in the liver and kidney. The expression of the transporters and pharmacokinetics of their substrates, rhodamine-123 (Rho123) and endogenous conjugated bilirubin (CB), were evaluated in rats after either AMD oral pretreatments (4-14 days) or single intravenous bolus. AMD pretreatment of all durations up-regulated renal Mdr1 and Mrp2 protein expression to 155-190% and 152-223% of the control values, respectively. In agreement, we observed a corresponding increase in renal clearance of both substrates. Hepatic expression was increased only for Mdr1 to 234-270% of controls, which was associated with increased biliary elimination of amiodarone without change in Rho123 biliary clearance. Interestingly, hepatic expression of another Mdr transporter, Mdr2, was progressively decreased by amiodarone administration. Acute administration of AMD reduced Rho123 biliary clearance by 64%. Our results indicate that repeated administration of AMD to rats is associated with significant increase in hepatic and renal expression of Mdr1 and Mrp2 transporters, which may contribute to variability in pharmacokinetics of AMD and simultaneously applied drugs.

Amiodarone modulates pharmacokinetics of low‐dose methotrexate in rats

Clinical studies of low‐dose methotrexate (LDMTX) pharmacokinetics document increased plasma concentrations of MTX after co‐administration of the drug with amiodarone or macrolide antibiotics. As drug–drug interactions may increase the toxicity of LDMTX, a rat model was used to follow renal and biliary elimination of MTX during its constant‐rate i.v. infusion and concomitant single bolus i.v. injections of amiodarone or azithromycin. The mean steady‐state plasma concentration of 1.7±0.1 µmol/l was reached and the total clearance achieved 17.7±1.0 ml/min/kg. Administration of amiodarone decreased the biliary clearance of MTX to 73% of the control values (p<0.05). Correspondingly, the total clearance decreased to 72% and plasma MTX concentrations were augmented to 2.5±0.4 µmol/l (p<0.05). Amiodarone‐treated rats exhibited a 3.3‐fold decrease in the renal clearance (p<0.05) of conjugated bilirubin, which was associated with its increased plasma concentration. In contrast, azithromycin did not alter any of the MTX pharmacokinetic parameters. In conclusion, this is the first report describing the impairment of MTX hepatic elimination during co‐administration with amiodarone. This study also provides new insight into acute amiodarone‐induced hyperbilirubinaemia, where increased bilirubin production and decreased renal clearance may contribute to this effect. Importantly, azithromycin seems to be a safe co‐medication during LDMTX therapy. Copyright

Behavioral effects of antiepileptic drugs in rats: Are the effects on mood and behavior detectable in open-field test?

PURPOSE Behavioral side effects of antiepileptic drugs (AEDs) are common including both positive and negative effects on mood, anxiety, depression, and psychosis. We aimed to evaluate behavioral patterns in rats after administration of lamotrigine, levetiracetam, phenytoin, topiramate, carbamazepine, gabapentin, pregabalin, and zonisamide. METHODS The open-field test was performed and locomotion, rearing, grooming, central latency and defecation were recorded over a 5min interval for each rat (8 rats in each group receiving AED and 16 controls). Kruskal-Wallis nonparametric test or ANOVA were used to assess differences among the groups. RESULTS The experimental groups did not differ in latency to enter the center compartment, neither in the decline of locomotor activity in the 1st and the 5th minute of the observation, nor in number of rears. Significant differences among groups were observed in the total number of lines crossed, grooming, as well in the number of fecal pellets. Locomotor activity was significantly increased in lamotrigine, if compared with gabapentin and pregabalin (ANOVA; p <0.05). Rats exposed to topiramate displayed a significantly increased number of grooming (when compared to pregabalin: p<0.01). Defecation (the number of fecal pellets) significantly increased in the gabapentin and carbamazepine group. CONCLUSION There are significant differences between AEDs in terms of their behavioral profile. It is of great importance to evaluate these effects in clinical practice to bring more clear insight into these positive or negative side effects of AEDs.

The effect of topiramate and lamotrigine on rat bone mass, structure and metabolism

There is only limited data concerning the effect of the newer antiepileptic drugs on bone. The objective of this study was to determine the effect of topiramate (TPM) and lamotrigine (LTG) monotherapy on bone mineral density (BMD), mineral content (BMC), bone markers, body composition and bone mechanical strength in the orchidectomized (ORX) rat model. 24 orchidectomized Wistar rats were divided into control and test groups, 8 rats in each group. The control rats received standard laboratory diet (SLD) while rats in the test group were fed with SLD enriched with LTG or TPM for 12 weeks. Dual energy X-ray absorptiometry was used to measure bone mineral density. The concentrations of bone metabolism markers were assayed in bone homogenate. In addition, both femurs were measured and used for biomechanical testing. Compared to the control group, both test groups had significantly lower weight, fat mass, whole body and femur BMD, BMC and reduced mechanical strength of bone. All of these changes were more pronounced in rats exposed to LTG. In conclusion, both LTG and TPM significantly reduce BMD and body weight and impair mechanical strength of bone. A question arises as to the degree of dependence of the effect on the dose. Further studies are warranted to establish whether LTG and TPM may have a clinically significant effect on BMD exclusively in the model of gonadectomized rats, or whether the effect applies also in the model of gonadally intact animals, and in the respective human models.

No clinical evidence for performing trough plasma and intracellular imatinib concentrations monitoring in patients with chronic myelogenous leukaemia

This multicentre study focused on monitoring imatinib mesylate (IMA) trough plasma (Ctrough) and intracellular (IMA Cintrac) concentrations in 228 chronic myelogenous leukaemia patients. The median of measured IMA Ctrough in our patient group was 905.8 ng ml (range: 27.7–4628.1 ng/ml). We found a correlation between IMA Ctrough and alpha 1‐acid glycoprotein plasma concentrations (rS = 0.42; p < 0.001). All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or body mass index) impact on measured IMA Ctrough. The IMA Ctrough decreased during the first 6 months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. There were no significant differences in medians of IMA Ctrough between both groups measured during the first year of treatment. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p = 0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in chronic myelogenous leukaemia patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1‐acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results. Copyright

The effect of lamotrigine and phenytoin on bone turnover and bone strength: A prospective study in Wistar rats

OBJECTIVE Some data suggest that exposure to lamotrigine (LTG) might be associated with impaired bone health in an orchidectomized rat model. The aim of this study was to determine if LTG poses any significant risk for bone in a gonadally intact animals and to compare the effect of LTG with that of phenytoin (PHT). METHOD Twenty-four rats were divided into control and test groups, (n=8 per group). Control rats received a standard laboratory diet (SDL), while rats in the test groups were fed a SLD enriched with LTG or PHT for 12 weeks. Dual energy X-ray absorptiometry was used to measure bone mineral density (BMD). The concentrations of bone turnover markers (BTM) were assayed in bone homogenates. The femurs were measured and biomechanically tested. RESULTS Treatment with either LTG or PHT had no significant effect on BMD or on the biomechanical strength of the bones. In contrast to the effect of LTG, we did find significant changes in BTM in the PHT group: a highly significant decrease in the osteoprotegerin/receptor activator of nuclear factor kappa B ratio (p<0.01) and highly significant increases in bone alkaline phosphatase and amino-terminal propeptide of procollagen type I (p<0.001, p˂0.01, respectively). In the LTG group, the only significant change was a decrease in sclerostin (p˂0.05). The PHT level was 19.0 (15.6-19.5) μmol/l, which represents the lower end of the therapeutic range used in humans. The level of LTG was 60.7 (58.5-61.8) μmol/l. CONCLUSIONS LTG has no effect on the BMD, BTM or mechanical strength in gonadally intact animals. Although a low dose of PHT was associated with enhanced BTM, it did not affect BMD or the biomechanical properties of the bones, similar to the results observed for LTG.