Effect of Lactobacillus casei on the Pharmacokinetics of Amiodarone in Male Wistar Rats.

BACKGROUND AND OBJECTIVES: The probiotic bacterium Escherichia coli strain Nissle 1917 has previously been shown to alter the pharmacokinetics of amiodarone. The aim of this study was to determine whether the probiotic bacterium Lactobacillus casei produces similar alterations in amiodarone disposition. METHODS: A suspension of live probiotic bacteria L. casei strain DN-114 001 (1.5 × 109 CFU/dose; probiotic pre-treated group) or a saline solution (control group) was administered directly into the stomach of male Wistar rats (N = 30 in each group) by oral gavage daily for 7 consecutive days. On the eighth day, all rats (N = 60) were given a single oral dose of an amiodarone hydrochloride suspension (model drug; 50 mg/kg). The concentrations of amiodarone and of its main metabolite N-desethylamiodarone were determined in rat plasma by high-performance liquid chromatography. RESULTS: Comparison of the pharmacokinetics of amiodarone in the control group and probiotic pre-treated group revealed that the peak plasma concentration of amiodarone was delayed by >2 h in the probiotic pre-treated group. The plasma level of N-desethylamiodarone was unchanged in the probiotic pre-medicated group and its pharmacokinetic parameters were not altered. CONCLUSIONS: The slower absorption of amiodarone in the probiotic pre-treated rats compared to the control ones and the unchanged pharmacokinetics of its main metabolite suggest that the probiotic strain of L. casei DN-114 001 has probably no clinical consequences as the difference was not statistically significant.

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action.

7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro.

1. The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450. 2. The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro. 3. Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (Ki 2.88 ± 0.12 µM) and S-TAM (Ki 14.22 ± 0.53 µM) as well as for S-TOL (Ki 1.71 ± 0.03 µM) and R-TOL (Ki 4.78 ± 0.17 µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC50 23.64 ± 6.25 µM) was more potent than (-)-KET (IC50 66.12 ± 12.6 µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC50 = 51.79 ± 8.58 µM, S-MOD IC50 = 48.62 ± 9.74 µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC50 = 68.17 ± 5.70 µM, S-CIT IC50 = 62.63 ± 7.89 µM) was not enantiospecific. 4. Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).

Pharmacokinetics of imipenem in critically ill patients during empirical treatment of nosocomial pneumonia: a comparison of 0.5-h and 3-h infusions.

In critically ill patients, pathophysiological changes alter the pharmacokinetics of antibiotics. Imipenem exhibits primarily time-dependent killing. Its administration by prolonged infusion may increase the time for which its plasma concentration exceeds the minimum inhibitory concentrations (MICs) of suspected pathogens. The objectives of this study were to compare the pharmacokinetic parameters of imipenem administered by standard short infusion (1g imipenem/1g cilastatin over 30min three times daily) and by extended infusion with a reduced total dose (0.5g imipenem/0.5g cilastatin over 3h four times daily) and to compare the target pharmacokinetic/pharmacodynamic indices, namely percentage of the dosing interval for which the free plasma concentration of imipenem exceeds the MIC and 4× MIC (%fT>MIC and %fT>4×MIC) of 0.5, 1, 2 and 4mg/L, for these two regimens in critically ill adult patients with nosocomial pneumonia on Day 2 of empirical antibiotic therapy. The study included 22 patients. Whilst no significant differences were found between both groups for %fT>MIC, %fT>4×MIC was 87.4±12.19%, 68.6±15.08%, 47.31±6.64% and 27.81±9.52% of the 8-h interval in the short infusion group for MICs of 0.5, 1, 2 and 4mg/L, respectively, and 85.15±17.57%, 53.14±27.27%, 13.55±24.47% and 0±0% of the 6-h interval for the extended infusion group. In conclusion, administration of 0.5g of imipenem by a 3-h infusion every 6h does not provide sufficient drug concentrations to treat infections caused by pathogens with a MIC of ≥2mg/L.

Oxidation of endogenous N-arachidonoylserotonin by human cytochrome P450 2U1.

Cytochrome P450 (P450) 2U1 has been shown to be expressed, at the mRNA level, in human thymus, brain, and several other tissues. Recombinant P450 2U1 was purified and used as a reagent in a metabolomic search for substrates in bovine brain. In addition to fatty acid oxidation reactions, an oxidation of endogenous N-arachidonoylserotonin was characterized. Subsequent NMR and mass spectrometry and chemical synthesis showed that the main product was the result of C-2 oxidation of the indole ring, in contrast to other human P450s that generated different products. N-Arachidonoylserotonin, first synthesized chemically and described as an inhibitor of fatty acid amide hydrolase, had previously been found in porcine and mouse intestine; we demonstrated its presence in bovine and human brain samples. The product (2-oxo) was 4-fold less active than N-arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The rate of oxidation of N-arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The demonstration of the oxidation of N-arachidonoylserotonin by P450 2U1 suggests a possible role in human brain and possibly other sites.

Rosuvastatin suppresses the liver microsomal CYP2C11 and CYP2C6 expression in male Wistar rats.

The aim was to investigate whether rosuvastatin affect rat cytochrome P450 (CYP) 2C11 and CYP2C6. CYP2C11 and CYP2C6 are considered as counterparts of human CYP2C9, which metabolizes many drugs including S-warfarin, diclofenac or ibuprofen. The male Wistar rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD, 1% of cholesterol, 10% of lard fat) for 21 days. Rosuvastatin administration in STD (0.03% w/w) resulted in decreased mRNA expression of CYP2C11 as well as of CYP2C6 (here significant) and in a significant decrease of the respective protein expression as well as of the enzyme activity of both CYP2C forms. When rosuvastatin was administered in the HCD, the mRNA expression of both CYP2C forms was significantly lowered; the protein and activity parameters did not show significant changes. These results suggest that CYP2C11 as well as CYP2C6 expression and activity are negatively affected by rosuvastatin and may be modulated by high cholesterol high fat diet. Therefore, it should be taken into consideration that drugs metabolized by CYP2C9 in human could interact with rosuvastatin, as it has been already suggested for warfarin (rosuvastatin has increased its anticoagulant effect in human), and for telmisartan, sildenafil and glimepiride.

The influence of rosuvastatin on liver microsomal CYP2C6 in hereditary hypertriglyceridemic rat.

OBJECTIVES: The aim of this study was to investigate whether rosuvastatin affects expression and activity of rat CYP2C6. This cytochrome P450 is considered to be a counterpart of human CYP2C9, which metabolizes many drugs, including diclofenac, ibuprofen or warfarin. DESIGN: Male hereditary hypertriglyceridemic (HHTg) rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD: STD + 1% of cholesterol w/w + 10% of lard fat w/w) for 21 days. A third group of rats were fed high a cholesterol diet with rosuvastatin added (0.03% w/w). Expression of CYP2C6 was measured in liver samples using real-time PCR (mRNA level) and Western blotting (protein level). Formation of diclofenac metabolites (typical enzyme activity of CYP2C6) was analyzed using HPLC with UV detection. RESULTS: Administration of rosuvastatin to HHTg rats resulted in significantly increased mRNA expression and enzyme activity in HCD-fed animals; changes of CYP2C6 protein were non-significant. These results suggest that CYP2C6 expression and activity are positively affected by rosuvastatin in hereditary hypertriglyceridemic rats after intake of HCD. CONCLUSION: The results presented open the possibility that in humans, rosuvastatin may affect the metabolism of many drugs by influencing expression and activity of CYP2C6 (counterpart of human CYP2C9). Further studies are needed to elucidate the effects of this statin on CYP2C9 in humans.

Effects of Lactobacillus casei on the expression and the activity of cytochromes P450 and on the CYP mRNA level in the intestine and the liver of male rats.

OBJECTIVES: The aim of the study was to find whether probiotic Lactobacillus casei influences the expression or the activity of cytochromes P450 (CYP) and whether it has an influence on the level of CYP mRNA in male rats. DESIGN: Live bacterial suspension of L. casei was administered orally (gavage) to healthy male Wistar rats daily for 7 days. Control group of rats was treated with the saline solution. Sections of the duodenum, jejunum, ileum, caecum and colon were dissected from each experimental animal. In all individual samples, the expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using the real-time PCR method. RESULTS: There were changes observed in the expression of CYP enzymes and in the CYP mRNA levels along the intestine after application of L. casei. The expression of CYP1A1 enzyme was found to be decreased in the proximal part of the jejunum and colon, CYP1A1 mRNA level was decreased in the distal part of the jejunum, ileum and caecum. Thus, the changes in CYP1A1 protein or mRNA were observed along the intestine of male rats. Similarly, a decreased expression of the caecal CYP2E1 mRNA and of the duodenal CYP3A9 mRNA after treatment of rats with L. casei was found. CONCLUSION: Probiotic L. casei might be able to contribute to prevention against colorectal cancer by decreasing levels of certain forms of xenobiotic-metabolizing enzymes; moreover, in general, there is a possibility of interactions with concomitantly taken pharmacotherapeutic agents.

In vitro interaction of a novel neutrophil growth factor with human liver microsomal cytochromes P450 and the contribution of UDP-glucuronosyltransferases to its metabolism.

Neutrophil growth factors (NGFs) stimulate neutrophil growth and survival. The first synthetic cytokinin-derived NGF was recently discovered and is a prospective drug owing to its potential use in anti-inflammatory therapy. The metabolism of some cytokinin-derived drugs (e.g. R-roscovitine, olomoucine II) has already been studied and it has been shown that they may give rise to drug-drug interactions. In this in vitro study, the interactions of the novel neutrophil growth factor NGF1568 with two of the main classes of human drug-metabolizing enzymes, cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), were tested. Of the CYPs evaluated, NGF1568 was found to inhibit only CYP2C9, by an uncompetitive mechanism and with a K(i) value of 349 µM. Formation of a glucuronide of NGF1568 was detected by LC/MS/MS analysis after it was incubated with human liver microsomes and UDP-glucuronic acid. The human recombinant UGT1A9 enzyme (major liver expression) and UGT1A7, UGT1A8, UGT1A10 enzymes (expressed in gastrointestinal tract instead of liver) were found to be responsible for NGF1568 glucuronidation. These results show that interaction of NGF1568 with CYPs is not as important as it is in the case of the cytokinin CDK inhibitors R-roscovitine and olomoucine II, but the conjugation enzymes (UGTs) play a major role in its metabolism. Thus, possible interference of NGF1568 with metabolism of other coadministered drugs at least on level of liver, kidney or intestinal UGTs should be thoroughly considered.

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases.

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-ß-D-glucuronides, B-7-O-ß-D-glucuronides, A-20-O-ß-D-glucuronides and B-20-O-ß-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-ß-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

Effects of probiotic Escherichia coli Nissle 1917 on expression of cytochromes P450 along the gastrointestinal tract of male rats.

OBJECTIVES: The aim of the study was to find whether probiotic Escherichia coli Nissle 1917 O6:K5:H1 (EcN) influences the expression of cytochromes P450 (CYP) in the rat intestine. DESIGN: Live bacterial suspension of EcN was administered to healthy male Wistar rats daily for 7 days. Control group of rats was stressed by oral application of the saline solution daily for 7 days as well. Sections of the duodenum, jejunum, ileum, caecum and colon have been taken from each experimental animal. With all individual samples, microsomal fraction has been prepared and expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using real-time PCR. RESULTS: It was found that there are changes in expression of CYP enzymes studied along the intestine. CYP1A1, 2B1/2 and 2E1 are present mainly in the duodenum and jejunum; on the other hand, CYP2C6 is expressed mainly in the caecum and colon. CYP3A was found all over the rat intestine. The results show that there are no prominent differences between control samples and samples with EcN, only the expression of CYP3A protein in the duodenum appears to exhibit a clear tendency to decrease. In the case of the colon, a significant increase in the expression of CYP3A (most likely CYP3A1) after treatment of rats with EcN was found. CONCLUSION: This in vivo study revealed that the levels of colon CYP3A could be significantly increased in rats treated with probiotic EcN. On the contrary, the expression of CYP3A in the duodenum decreased. However, the changes in the expression of CYP enzymes are probably not as extensive to be clinically important in man; hence, most likely the probiotic EcN has little influence on the intestinal drug metabolism by CYP enzymes.

Effect of acetylcholinesterase oxime-type reactivators K-48 and HI-6 on human liver microsomal cytochromes P450 in vitro.

Substances K-48 and HI-6, oxime-type acetylcholinesterase (AChE) reactivators, were tested for their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The compounds were shown to bind to microsomal cytochromes P450 with spectral binding constants of 0.25+/-0.05 microM (K-48) and 0.54+/-0.15 microM (HI-6). To find which cytochrome P450 from the human liver microsomal fraction interacts with these compounds, an inhibition of enzyme activities specific for nine individual CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) was studied. The results have shown no prominent inhibition of individual CYP activities with both compounds except the CYP2E1 activity and the HI-6 reactivator. However, the inhibition of this activity was less than 50% which makes the possible drug interactions highly unlikely. Hence, the interaction of K-48 and HI-6 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be clinically significant and both compounds could be taken in this respect as antidotal drugs with low risk of drug interactions.

Interactions of olomoucine II with human liver microsomal cytochromes P450.

Olomoucine II is a cyclin-dependent kinase inhibitor and a potential antineoplastic agent because it can arrest animal cell cycles. This study examines its interactions with human liver microsomal cytochrome P450 (P450) enzymes. Spectroscopic and high-performance liquid chromatography (HPLC) methods were used to estimate the degree of olomoucine II-mediated inhibition of enzymatic activities of eight drug-metabolizing P450s in vitro. In addition, mass spectrometry coupled with HPLC was used to identify an olomoucine II metabolite (2,5-dihydroxyroscovitine) formed in the reaction mixtures, and CYP3A4 was found to be responsible for the hydroxylation of the N(6)-benzyl ring at position 5, leading to this compound. Olomoucine II significantly inhibited the enzymatic activities of CYP1A2, CYP2C9, and (to a lesser degree) CYP3A4. The results indicate that use of olomoucine II as a drug could affect the activities of CYP3A4, CYP1A2, and CYP2C9 in vivo. Hence, the clinical relevance of these interactions should be carefully evaluated.

Interspecies comparison of the glucuronidation processes in the man, monkey, pig, dog and rat.

OBJECTIVES: The study of interspecies differences in glucuronidation processes in the man, monkey, pig, dog and rat using liver microsomal fraction. The study is focused on determination of the enzyme activity of UGT1A6 (having also a toxicological importance) in microsomes of different species. METHODS: For determination of glucuronides formed, an HPLC method with UV detection and LC-MS characterization was used. p-Nitrophenol and 4-methylumbelliferon and silybin were chosen as model substrates. RESULTS: The data presented in this paper show an overall similarity in kinetic parameters of the UGT1A6 with p-nitrophenol and 4-methylumbelliferon for man, pig and monkey. The pattern of silybin glucuronides formed in monkey and dog samples are relatively close to this of the man. CONCLUSIONS: For studies of glucuronidation of xenobiotics where the role UGT1A6 is expected, the use of pig and monkey microsomes should be considered. As an optimal model for study of silybin glucuronidation, both the rhesus monkey and dog (Beagle) seem to be the best models. To elucidate the role of the UGT forms involved in metabolism of silybin, the experiments with recombinant UGT enzymes are needed.