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Probiotics are recommended for maintaining the good general condition and health. Current researches show that probiotics have an important role in treating diarrhoea, serious bacterial infections and inflammatory disorders, colon cancer (only in researches performed on animals, support the immune system functions, healthy urino-genital tract, play the role in treating allergies caused by nutritive allergens, hypertension, in lactose digestion.

Effects of bile acids and their salts on absorption of other substances Bile acids and their salts increase intestinal absorption of lipids and transmembrane and paracellular transfer of small and endogenous and exogenous polar molecules. It has been established that they are good promotores of insulin absorption through skin and nasal mucose, and of blood-brain barrier transfer of salycilates and quinine. Effects of bile acids and their salts on absorption of other substances and their potential action It has been established that combination of bile acids with amphotericin B has potential Leishmanicideal effect and combination with ciprofloxacine has improved its antibacterial activity against Pseudomonas aeruginosa in vitro. Bile acids pharmacodynamic effects Bile acids have analgesic and hypoglycemic effect They also have anti-HIV effect probably suppressing virus transmission from cell to cell. Conclusion New studies of natural bile acids and new synthetic bile acids have revealed that they are not only adjuvants to existing active principles in pharmaceutical forms, but they can act as new therapeutic agents. However, it is necessary to study their possible mechanisms, but they are not crucial for their therapeutic application. Toxicological and pharmacological studies will determine the role of newly synthetized bile acids and their salts in current therapy.

The present study investigated the transcellular and placental permeation of cefuroxime, an antibiotic used in cesarean sections, in pregnant women with diabetes and hypertension.Fifty-three women scheduled for cesarean section were divided into three groups: healthy women (n = 18), women with arterial hypertension (n = 21), and women with gestational diabetes (n = 14). All women received 1.5 g, i.v., cefuroxime. Cefuroxime concentrations were measured in maternal venous plasma before, during, and after delivery, as well as in fetal umbilical cord vein and artery plasma during delivery. The effects of diabetes and hypertension on cefuroxime placental-permeation were assessed by the fetomaternal plasma concentration ratios. Pharmacokinetic non-compartmental model analyses were performed and results were compared using anova.Fetomaternal drug concentration ratios were lower in the diabetic group than in the hypertensive and control groups. There were no significant differences in umbilical arterial : venous plasma drug concentration ratios in the diabetic and hypertensive groups compared with the control group. Apparent volume of distribution and clearance were significantly lower in the diabetic group compared with the control and hypertensive groups.Diabetes led to decreased placental transfer of cefuroxime, as well as volume of distribution and clearance, but did not affect other pharmacokinetic parameters. Hypertension had no significant effect on the permeation of cefuroxime or on its pharmacokinetics. Prophylactic concentrations of cefuroxime were reached in all groups, but the dosing time of cefuroxime should not be less than 30 min or greater than 2 h prior to delivery.

Background/Aim. Stevioside is a glycoside that supposedly possesses a number of pharmacodynamic effects such as anti-infective, hypoglycemic, along with the beneficial influence on the cardiovascular system. The aim of this study was to determine the effect of rats pretreatment with aqueous solution of stevioside on pharmacological actions of adrenaline, metoprolol and verapamil. Methods. Rats were administered (intraperitoneally 200 mg/kg/day) stevioside as aqueous solution or physiological saline in the course of 5 days, then anaesthetized with urethane and the first ECG recording was made. The prepared jugular vein was connected to an infusion pump with adrenaline (0.1 mg/mL), verapamil (2.5 mg/mL) or metoprolol (1 mg/mL). Control animals, pretreated with saline, in addition to the mentioned drugs, were also infused with the solution of stevioside (200 mg/mL) in the course of recording ECG. Results. The infusion of stevioside produced no significant changes in ECG, even at a dose exceeding 1,600 mg/kg. In the control group, a dose of adrenaline of 0.07 ± 0.02 mg/kg decreased the heart rate, whereas in the steviosidepretreated rats this occurred at a significantly higher dose (0.13 ± 0.03 mg/kg). In stevioside-pretreated rats, the amount of verapamil needed to produce the decrease in heart rate was significantly lower compared to the control. The pretreatment with stevioside caused no significant changes in the parameters registered on ECG during infusion of metoprolol. Conclusion. The results suggest that pretreatment with stevioside may change the effect of adrenaline and verapamile on the heart rate. [Projekat Ministarstva nauke Republike Srbije, br. 41012]

Armin Mooranian,1 Rebecca Negrulj,1 Nigel Chen-Tan,2 Hesham S Al-Sallami,3 Zhongxiang Fang,4 Trilochan Mukkur,5 Momir Mikov,6,7 Svetlana Golocorbin-Kon,6,7 Marc Fakhoury,8 Frank Arfuso,5 Hani Al-Salami1 1Biotechnology and Drug Development Research Laboratory, School of Pharmacy, Curtin Health Innovation Research Institute, Biosciences Research Precinct, Curtin University, Perth, WA, Australia; 2Faculty of Science and Engineering, Curtin University, Perth, WA, Australia; 3School of Pharmacy, University of Otago, Dunedin, New Zealand; 4School of Public Health, Curtin University, Perth, WA, Australia; 5Curtin Health Innovation Research Institute, Biosciences Research Precinct, School of Biomedical Science, Curtin University, Perth, WA, Australia; 6Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia; 7Department of Pharmacy, Faculty of Medicine, University of Montenegro, Podgorica, Montenegro; 8Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada Abstract: Gliclazide (G) is an antidiabetic drug commonly used in type 2 diabetes. It has extrapancreatic hypoglycemic effects, which makes it a good candidate in type 1 diabetes (T1D). In previous studies, we have shown that a gliclazide-bile acid mixture exerted a hypoglycemic effect in a rat model of T1D. We have also shown that a gliclazide-deoxycholic acid (G-DCA) mixture resulted in better G permeation in vivo, but did not produce a hypoglycemic effect. In this study, we aimed to develop a novel microencapsulated formulation of G-DCA with uniform structure, which has the potential to enhance G pharmacokinetic and pharmacodynamic effects in our rat model of T1D. We also aimed to examine the effect that DCA will have when formulated with our new G microcapsules, in terms of morphology, structure, and excipients' compatibility. Microencapsulation was carried out using the Büchi-based microencapsulating system developed in our laboratory. Using sodium alginate (SA) polymer, both formulations were prepared: G-SA (control) at a ratio of 1:30, and G-DCA-SA (test) at a ratio of 1:3:30. Complete characterization of microcapsules was carried out. The new G-DCA-SA formulation was further optimized by the addition of DCA, exhibiting pseudoplastic-thixotropic rheological characteristics. The size of microcapsules remained similar after DCA addition, and these microcapsules showed no chemical interactions between the excipients. This was supported further by the spectral and microscopy studies, suggesting microcapsule stability. The new microencapsulated formulation has good structural properties and may be useful for the oral delivery of G in T1D. Keywords: type 2 diabetes, bile acids, gliclazide, polymer

Introduction In previous studies carried out in our laboratory, a bile acid (BA) formulation exerted a hypoglycaemic effect in a rat model of type-1 diabetes (T1D). When the antidiabetic drug gliclazide (G) was added to the bile acid, it augmented the hypoglycaemic effect. In a recent study, we designed a new formulation of gliclazide-cholic acid (G-CA), with good structural properties, excipient compatibility and exhibits pseudoplastic-thixotropic characteristics. The aim of this study is to test the slow release and pH-controlled properties of this new formulation. The aim is also to examine the effect of CA on G release kinetics at various pH values and different temperatures. Method Microencapsulation was carried out using our Buchi-based microencapsulating system developed in our laboratory. Using sodium alginate (SA) polymer, both formulations were prepared: G-SA (control) and G-CA-SA (test) at a constant ratio (1:3:30), respectively. Microcapsules were examined for efficiency, size, release kinetics, stability and swelling studies at pH 1.5, pH 3, pH 7.4 and pH 7.8 and temperatures of 20 and 30 °C. Results The new formulation is further optimised by the addition of CA. CA reduced microcapsule swelling of the microcapsules at pH 7.8 and pH 3 at 30 °C and pH 3 at 20 °C, and, even though microcapsule size remains similar after CA addition, percent G release was enhanced at high pH values (pH 7.4 and pH 7.8, p

Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico-chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal-phase thin-layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure-activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α-hydroxy-12-oxo-5β-cholanoic and 3α-hydroxy-7-oxo-5β-cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α-hydroxy-12-oxo-5β-cholanoic, 3α-hydroxy-7-oxo-5β-cholanoic, 3,7,12-trioxo-5β-cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry.

Background: The field of bile acid research has become tremendously active. Bile acids have been shown to act as signaling molecules that are involved in many metabolic processes, but their role in carcinogenesis is also emerging. Methods: The aim of this review was to summarize the present knowledge in the innovative field of bile acids pharmacology, to reveal the novel mechanisms of their action, particularly focusing on clinically relevant aspects, and to evaluate the role of both genetic and epigenetic variation in genes encoding bile acid-activated receptors in determining the therapy outcome. Results: Most effects of bile acids are mediated by both nuclear and G protein-coupled receptors. Three natural bile acids have already been registered for the use in humans, but various semi-synthetic bile acid analogues with improved pharmacokinetic and pharmacodynamic properties have been developed, which opens up new avenues in pharmacotherapy. Many efforts have been made to evaluate the impact of nuclear receptors on inter-individual variation in responses to drugs, since nuclear receptors are significant mediators between environmental stimuli and pharmacokinetics. Genetic variation of bile acid-activated receptors is associated with both benign and malignant diseases, in terms of disease risk and severity, but also with pharmacokinetics and therapy outcome. Furthermore, the activity of these receptors may be masked or amplified by epigenetic modifications. Conclusion: Both genetic and epigenetic factors may alter complex and intricate network of bile acid signaling pathways, contributing to the development of several metabolic and non-metabolic diseases and altered activities of drug-metabolizing enzymes and transporters. These polymorphisms and epigenetic modifications may also impact the effectiveness and pharmacokinetics of bile acid analogues, which must be taken into account during the development of these compounds as novel therapeutic agents.

Background Natural antioxidant products are increasingly being used to treat various pathological liver conditions considering the role of oxidative stress in their pathogenesis. Rosemary essential oil has already being used as a preservative in food industry due to its antioxidant and antimicrobial activities, but it was shown to possess additional health benefits. The aim of our study was to evaluate the protective effect of rosemary essential oil on carbon tetrachloride - induced liver injury in rats and to explore whether its mechanism of action is associated with modulation of hepatic oxidative status. Methods Chemical composition of isolated rosemary essential oil was determined by gas chromatography and mass spectrometry. Antioxidant activity was determined in vitro using DPPH assay. Activities of enzyme markers of hepatocellular damage in serum and antioxidant enzymes in the liver homogenates were measured using the kinetic spectrophotometric methods. Results In this research, we identified 29 chemical compounds of the studied rosemary essential oil, and the main constituents were 1,8-cineole (43.77%), camphor (12.53%), and α-pinene (11.51%). Investigated essential oil was found to exert hepatoprotective effects in the doses of 5 mg/kg and 10 mg/kg by diminishing AST and ALT activities up to 2-fold in serum of rats with carbon tetrachloride - induced acute liver damage. Rosemary essential oil prevented carbon tetrachloride - induced increase of lipid peroxidation in liver homogenates. Furthermore, pre-treatment with studied essential oil during 7 days significantly reversed the activities of antioxidant enzymes catalase, peroxidase, glutathione peroxidase and glutathione reductase in liver homogenates, especially in the dose of 10 mg/kg. Conclusions Our results demonstrate that rosemary essential oil, beside exhibiting free radical scavenging activity determined by DPPH assay, mediates its hepatoprotective effects also through activation of physiological defense mechanisms.

Tacrolimus (Tac) is an immunosuppressive drug with a narrow therapeutic width and highly variable pharmacokinetics. Therefore, monitoring of Tac blood concentrations is of utmost importance in the management of renal transplant recipients. The occurrence and intensity of adverse effects depend on blood concentration and total exposure of the organism to this drug. This implies finding a new gender-dependent predictable method for Tac exposure monitoring based on determination of the area under the time concentration curve (AUC). The primary aim of this study was to investigate gender differences in systemic body exposure to Tac in renal transplant patients after the first oral dose and in a steady state by determining 12-h AUC (AUC(0-12)). The secondary objective was to find the best sampling time in which measured Tac concentration best predicts AUC value with respect to gender. Tac pharmacokinetic study was conducted in 20 kidney transplant recipients (10 men/10 women) on quaternary immunosuppressive therapy. The first oral Tac dose (0.05 mg/kg) was given on the fifth day post-transplant. After reaching steady state, regimen stabilized and dosage was adjusted in accordance with the level of Tac. Blood concentrations were measured by microparticle enzyme immunoassay method. AUC(0-12) for each patient was calculated after the first oral Tac dose and in the steady state from a plot of Tac concentration versus time from 0 to 12 h using the trapezoid rule. Associations between each sampling time point of concentrations within 12 h after the administration and AUC(0-12) were evaluated by Pearson correlation coefficients. Abbreviated sampling equations were derived by multiple stepwise regression analyses. Statistically significant difference was found in AUC(0-12) between male and female patients after the first oral dose (p0.01), but this difference was lost in a steady state. In female recipients C(2) seemed to be good indicator of total body exposure to Tac after the first oral dose and this was also confirmed in a steady state. The three-point sampling method was required for calculating AUC after the first oral dose in male patients, whereas in the steady state, concentration of C(8) seemed to be a good indicator of abbreviated AUC for a Tac monitoring strategy in male patients. Non-compartment Tac pharmacokinetic and regression analysis showed gender difference in total Tac exposure and determined the best predictable Tac concentrations after the first oral dose. Our study confirmed gender-dependent pharmacokinetics in a steady state in terms of best sampling time in which measured Tac concentration best predicts AUC value.

Background Previous studies carried out in our laboratories have demonstrated impaired drug permeation in diabetic animals. In this study the permeation of diazepam (after a single dose of 5 mg/day, administered intramuscularly) will be investigated in diabetic and hypertensive pregnant women. Methods A total 75 pregnant women were divided into three groups: group 1 (healthy control, n = 31), group 2 (diabetic, n = 14) and group 3 (hypertensive, n = 30). Two sets of diazepam plasma concentrations were collected and measured (after the administration of the same dose of diazepam), before, during and after delivery. The first set of blood samples was taken from the mother (maternal venous plasma). The second set of samples was taken from the fetus (fetal umbilical venous and arterial plasma). In order to assess the effect of diabetes and hypertension on diazepam placental-permeation, the ratios of fetal to maternal blood concentrations were determined. Differences were considered statistically significant if p ≤ 0.05. Results The diabetes and hypertension groups have 2-fold increase in the fetal umbilical-venous concentrations, compared to the maternal venous concentrations. Feto: maternal plasma-concentrations ratios were higher in diabetes (2.01 ± 1.10) and hypertension (2.26 ± 1.23) groups compared with control (1.30 ± 0.48) while, there was no difference in ratios between the diabetes and hypertension groups. Umbilical-cord arterial: venous ratios (within each group) were similar among all groups (control: 0.97 ± 0.32; hypertension: 1.08 ± 0.60 and diabetes: 1.02 ± 0.77). Conclusions On line with our previous findings which demonstrate disturbed transcellular trafficking of lipophilic drugs in diabetes, this study shows significant increase in diazepam placental-permeation in diabetic and hypertensive pregnant women suggesting poor transcellular control of drug permeation and flux, and bigger exposure of the fetus to drug-placental transport.

An autoimmune disorder (AD) is a condition in which the immune system mistakenly attacks its own body cells through the production of antibodies that target certain tissues. Such attack triggers further inflammation that result in more attacks and a significant inflammatory response leading to tissue destruction and cessation of functionality [1]. ADs include diabetes, rheumatoid arthritis, Graves' disease, systemic lupus and inflammatory bowel disease (IBD) [2]. ADs are on the rise worldwide and have major health implications from the diseases themselves as well as complications. Even though the causes of AD have been postulated to be genetic and environmental, the actual triggers remain poorly defined [3]. Genetic predisposition contribute to about 30% of AD while 70% to environmental factors such as infections (e.g., virus, bacteria) and lifestyle-associated factors such as food.

The pharmacokinetics of a new tablet formulation of carbamazepine, an antiepileptic agent, have been investigated in 9 normal healthy subjects. The drug was given as a single oral dose of 400 mg. Ten blood samples were collected after administration. Plasma carbamazepine concentrations were determined by a sensitive method (HPLC). Areas under the plasma level-time curves for each subject were evaluated by means of the trapezoidal rule. The peak plasma concentration of 3.96-8.25 mg/l was reached 4-24 h after drug administration. The terminal phase half-life was 22.19-39.61 h and a systemic clearance was 1.05-2.06 l/h. The new tablet formulation of carbamazepine seems to be bioequivalent when compared to the one used so far. For the prediction of systemic availability and estimation of the first-pass metabolism, from plasma level data, a hepatic blood flow rate limited model were used. The systemic availability was 97.8-98.9% and the elimination of the drug on its first-pass through the liver was 1.13-2.20%.

Background: Multiple resistances to antibiotics are an emergent problem worldwide. Scientists intensively search for new substances with the antimicrobial potential or the mode to restore the activity of old-generation antibiotics. Ampicillin is the antibiotic with the expanded range of antimicrobial activity, but its use has decreased due to the poor absorption and highly developed resistance. In vivo studies showed that ampicillin has better absorption and bioavailability if combined with bile acid salts. The aim of this study was to examine antimicrobial effects of ampicillin alone and its combination with semisynthetic monoketocholic acid salt (MKH) in vitro.Materials, Methods & Results: In this study, commercial preparation of ampicillin and sodium salt of 3α,7α-dihydroxy-12oxo-5β-cholanate were used. Their effects were evaluated on Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium), obtained from urine specimens of dogs with clinically manifested cystitis. The first two investigated strains were ampicillin-sensitive, while E. faecium was resistant to ampicillin. Modified macrodilution method according to Clinical and Laboratory Standards Institute Guidelines (M7-A8) was performed. Bacterial suspension equivalent to 0.5 McFarland was prepared in saline, compared to the standard (Biomerieux) ad oculi. The density was checked spectrophotometrically at a wavelength of 625 nm and adjusted if necessary to the desired absorbance from 0.08 to 0.1. The resultant suspension was diluted 1:100 and inoculated in test tubes. Number of bacteria was counted on Petri plates using dilutions from 10-3 to 10-7 in order to obtain valid and countable plates. One hundred microliters of appropriate dilutions were aseptically plated in triplicate onto nutrient agar. Plates were incubated on 37°C for 72 h, under aerobic conditions. The number of colony forming units (CFU) was determined by direct counting. As a valid for enumeration, we took plates with 30 to 300 CFU. Percentage of killed bacteria for ampicillin was from 69.33-95.19% for E. coli, 87.1296.92% for E. faecalis and 7.20-33.30% for E. faecium. Ampicillin applied in the combination with MKH killed 99.99% to 100% of E. coli, 94.59% to 99.91% of E. faecalis and 31.73% to 64.76% of E. faecium. Mean percentage of killed bacteria for ampicillin was 81.93% for E. coli, 91.64% for E. faecalis, and 18.13% for E. faecium, while in combination with MKH percentage was 99.96% for E. coli, 98.23% for E. faecalis and 47.54% for E. faecium.Discussion: Results are presented as pharmacological minimal inhibitory concentration (MIC) values. Ampicillin was applied at the concentration higher than the therapeutic one, which could explain high MIC values for E. coli and E. faecalis. The combination of ampicillin with MKH showed the best improvement of antimicrobial effect on E. faecium (Δ = 29.41%), isolate that was resistant to ampicillin when applied alone. In all the investigated isolates, the combinations with MKH were more effective than ampicillin administered alone. It seems that MKH demonstrates a synergistic antimicrobial activity with ampicillin in vitro, which considerably decreases MIC values for all investigated isolates. These results implicate that MKH could restore the previous activity of ampicillin against some bacteria, which could be a significant benefit for clinical practice.

Methods This study examined the influence of acute pretreatment with different H. perforatum extracts on pentobarbitalinduced sleeping time impairment of motor coordination caused by diazepam and paracetamol pharmacokinetics in mice. The preparations profile of St. John’s wort was determined using RP-HPLC analysis. Ethanolic extract, aqueous extract, infusion, tablet and capsule of H. perforatum were used in the experiment.

Background Diabetes mellitus is a major public health concern with devastating human, social and economic impact. It is increasing globally, affecting more than 180 million people worldwide. The objective of our study was to analyse the overall volume of use of antidiabetics in Serbia compared to Scandinavian countries (Sweden, Norway, Denmark), chosen for their rational and conservative prescription practice. Methods Data on consumption of antidiabetics (ATC group A10) in 2010 were extracted from the databases of the representative national authorities. Utilisation of these medicines was measured through the defined daily dose (DDD) unit and the results were expressed as DDD per 1000 inhabitants per day (DID). Results In 2010, antidiabetics were used at a similar rate in Serbia (47.3 DID) and Scandinavian countries (from 46.5 DID in Sweden to 47.67 DID in Norway), but the share of use of insulins (A10A) and oral antidiabetics (A10B) differed among the observed countries. The proportion of insulin in Serbia was 22.0% of all antidiabetics which is relatively low in comparison with Scandinavian countries (from 36.2% in Denmark to 50.8% in Sweden). Utilisation of long-acting insulins (A10AE) was much lower in Serbia (1.3 DID) compared to Scandinavian countries (range: 2.6–4.6 DID). The share of oral antidiabetics use also differed among these countries. In Serbia, sulfonylureas (A10BB), as a second-line treatment for type 2 diabetes, were used predominantly (55.6%) compared to metformin (44.1%). In Scandinavian countries, metformin, as preffered oral agent for type 2 diabetes and the only medicine from the biguanide class (A10BA), was used at a higher rate than in Serbia (from 51.2% in Denmark to 60.4% in Sweden). New medicinal products with effect on the incretin system (A10BH and A10BX) were also used at a higher rate in Scandinavian countries (range: 0.5–2.5 DID) in comparison to Serbia (0.002 DID). Conclusions

Background Metabolism interaction between corticosteroids and tacrolimus (Tac) exists and can be an important factor in providing rational pharmacotherapy in kidney transplantation patients. Both Tac and corticosteroids can induce adverse metabolic effects, such as hyperglycemia, post-transplantation diabetes mellitus, and dyslipidemia. Objective The main goal of this study was to detect corticosteroid dose influence on Tac level within the first 6 months of immunosuppressive therapy. The secondary goal of this research was to investigate sex differences on Tac−corticosteroid interaction. We also monitored biochemical-parameter changes, which are related to immunosuppressive treatment. Methods This retrospective pharmacokinetic study included 30 Serbian patients after kidney transplantation. Patients received a quaternary immunosuppressive regimen including Tac, mycophenolate, mofetil, basiliximab, and corticosteroids. To compare dose-normalized level and dose of Tac in different days after transplantation, we performed the Friedman test and Wilcoxon matched-pairs signed rank sum test. Mann-Whitney test was performed to compare differences in dose of Tac, level of Tac, and dose-normalized level of Tac between male and female patient groups. We used the Friedman test to compare biological and clinical data. Results Obtained results show statistical significance between dose of Tac on day 180 post transplantation and dose on days 7, 14, 21, and 60 post transplantation. There was a statistical difference in dose-normalized level of Tac between days 7 and 21 post transplantation (P < 0.01), days 7 and 60 (P < 0.01), and between days 7 and 180 (P < 0.05). There is a statistical significance between male and female levels of Tac on day 21 after transplantation (P < 0.01). Significance also exists on day 60 after transplantation between male and female dose-normalized levels (P < 0.05). There is also a statistical difference in glucose, cholesterol, triglyceride, serum creatinine, and urea level and activity of alanine aminotransferase and alkaline phosphatase before and after operation. Conclusion Our study shows that dose of corticosteroid affects Tac level in kidney transplantation patients. Tac dose and level changes showed that corticosteroid−Tac interaction has more influence on male than female patients. According to biochemical monitoring, the immunosuppressive therapy used at present is quite well tolerated.

The use of probiotics, alone or in interaction with bile acids, is a modern strategy in the prevention and treatment of hypercholesterolemia. Numerous mechanisms for hypocholesterolemic effect of probiotics have been hypothesized, based mostly on in vitro evidence. Interaction with bile acids through reaction of deconjugation catalyzed by bile salt hydrolase enzymes (BSH) is considered as the main mechanism of cholesterol-lowering effects of probiotic bacteria, but it has been reported that microbial BSH activity could be potentially detrimental to the human host. There are several approaches for prevention of possible side effects associated with BSH activity, which at the same time increase the viability of probiotics in the intestines and also in food matrices. The aim of our study was to summarize present knowledge of probiotics—bile acids interactions, with special reference to cholesterol-lowering mechanisms of probiotics, and to report novel biotechnological approaches for increasing the pharmacological benefits of probiotics.

The pharmacokinetics of a new tablet formulation of acenocoumarol racemate, an oral anticoagulant agent, has been investigated in 8 normal healthy subjects. The drug was given as a single oral dose of 12 mg. 12 blood samples were collected after administration Plasma acenocoumarol concentrations were determined by a sensitive HPLC method. Areas under the plasma level-time curves for each subject were evaluated by means of the trapezoidal rule. The peak plasma concentration of 244.19-644.23 micrograms/l was reached 1-4 h after drug administration. The terminal phase half-life was 6.29-14.22 h and a systemic clearance was 1.86-5.62 l/h. The new table formulation of acenocoumarol seems to be bioequivalent when compared to the one used so far. For the prediction of systemic availability and estimation of the first-pass metabolism, from plasma level data, a hepatic blood flow rate limited model were used. The systemic availability was 94.22-98.01% and the elimination of the drug on its first-pass through the liver was 1.99-5.78%.

Background/Aim. Probiotics help to provide an optimum balance in the intestines. Probiotics species competitive block toxic substances and growth of unwanted bacteria and yeast species while they compete for the space and food. Lactogyn® is the first oral probiotics on Serbian market dedicated to maintaining a normal vaginal flora. Lactogyn® contains two well studied probiotics strains - Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Both of them are considered as probiotic agents with therapeutic properties increase the population of beneficial lactobacillus organisms within the vagina. The aim of this study was to exam an influence of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on results of cervical smear cytological testing including detection of atypical cells, detection of false positive and false negative findings as well as on vaginal microflora content in patients with vaginal infection signs and symptoms. Methods. Totally 250 women with signs of vaginal infection were selected to participate in the study. The study group comprised 125 patients taking studied probiotic strains along with specific anti-infective therapy. The control group comprised, also, 125 patients taking anti-infective agents, only. Probiotic preparation (Lactogyn ® capsules) was administered orally (one capsule daily) during 4 weeks. Before and six weeks after beginning of the therapy a cervical smear cytological test (the Papanicolaou test), as well as microbiological examination of the vaginal smear were performed. Results. Number of cases of inflammation and atypical squamous cells of undeterminated significance (ASCUS) in the study group were significantly higher before administration of the probiotic preparation. The number of lactobacilli was significantly higher, and the number of pathogenic microorganisms lower in the group treated with this preparation. Conclusion. The application of probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 concomitantly with specific antiinfective agents provides more reliable cytological diagnostics, reduces the number of false positive and false negative findings on cervical malignancy and normalizes vaginal microflora in higher percentage of patients with vaginal infections comapred with therapy including anti-infective agents only.

Diabetes mellitus is a metabolic disorder classified as Type 1 (T1D) or Type 2 (T2D). T1D is an autoimmune disorder characterized by the destruction of the -cells of the pancreas resulting in a partial or complete lack of insulin production and the inability of the body to control glucose homeostasis (Akerblom et al. 2002). T1D is also known as juvenile-onset diabetes because it manifests at a young age (Bruno et al. 2005). As it requires the patient to inject insulin to supplement the partial or complete lack of insulin production by the pancreas, it is also called insulin-dependent diabetes mellitus (IDDM). T2D, formerly known as noninsulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, is a metabolic disorder with onset most common in middle age and later life (Campbell 1991). T2D may be controlled by diet and exercise and, unlike T1D, does not always require the use of insulin (Campbell 2004). However, the term “noninsulin-dependent” is a misnomer since many patients require insulin therapy at some time in the course of their disease. T2D is often associated with obesity, hypertension and insulin resistance and can result in the complete destruction of beta-cells of the pancreas leading to T1D (Campbell 2004; Weiss & Caprio 2006). The prevalence of T1D and T2D are on the rise worldwide, which has generated a strong drive towards developing preventative measures as well as cure. Recent data published by the International Diabetes Federation highlighted the severity of diabetes epidemic. Data show that the disease is currently affecting 246 million people worldwide, with 46% of all those affected in the 40-59 age group. Previous figures underestimated the scope of the problem, while even the most pessimistic predictions fell short of the current figure. It is predicted that the total number of people living with diabetes will increase to 380 million within twenty years if no new and substantially more effective drugs are produced (Moore et al. 2003a; Rosenbloom et al. 1999). On 2007, the health costs of diabetes have exceeded 200 billion dollars only in the US. This adds to the cost generated from higher rate of hospitalization, higher mortality rate, and impaired performance of workers with diabetes. This has generated a strong drive towards developing preventative measures as

According to the World Health Organization, counterfeit medicines are medicines that are mislabeled deliberately and fraudulently regarding their identity and/or source. All kinds of medicines have been counterfeited, both branded and generic ones. Counterfeit medicines may include products containing correct or wrong ingredients; without active or with insufficiently or over-active ingredients, or with fake packaging. Many sources of information have been explored, including reports from the national medicine regulatory authorities, pharmaceutical companies and literature data. Since the time counterfeit drugs first appeared, they have become more sophisticated and more difficult to be detected. The World Health Organization estimate is that up to 1% of medicines available in the developed world are likely to be counterfeit. This figure rises to 10% globally, although in some developing countries it is 50%. The World Health Organization estimate is that 50% of medicines available via the internet are counterfeit. The knowledge about counterfeit drugs should be used to educate students of pharmacy and medicine, health professionals and patients. The most important players in campaign against counterfeit medicines are health professionals. Pharmacists and doctors should stay vigilant and report suspicious products, and consider counterfeits as a possible cause of adverse reactions or therapeutic failure. Patients should inform their pharmacists and doctors if they suspect any irregularity concerning their medication, if they experience side effects or a decrease in beneficial effect. The crucial step in the prevention of counterfeit medicines is to get supplied from reliable sources, i.e. licensed pharmacies.

Influence of two newly synthesized bile acids derivates, namely sodium salt of monoketocholic acid MKH-Na and methyl ester of monoketocholic acid MKH-Me on tramadol (12.5 mg/kg oral and intramuscular) analgesic effect was examined in this research. Analgesic effect was measured by antinociceptive hot plate method. Interaction was estimated by detection of changes in analgesic effect of tramadol combined with bile acids (subcutaneous administration of 4 mg/kg 20 min before tramadol) compared to analgesic effect of the same dose of tramadol given alone. Hydrosoluble sodium salt of monoketocholic acid did not show interaction with tramadol, regardless of the route of administration of tramadol. However, methyl ester of monoketocholic acid increased the analgesic effect of tramadol when it was given intramuscularly. After oral administration of tramadol, methyl ester of monoketocholic acid decreased the analgesic effect of tramadol. According to the time point when interaction reached statistically significant difference, it can be presumed that after intramuscular administration of tramadol, methyl ester of monoketocholic acid increases tramadol absorption and transport to brain and in that way increases its analgesic effect. The analgesic effect of tramadol after oral administration was decreased, which could be explained by the induction of tramadol metabolism in the liver, but should be examined in more details.

Introduction. In the recent years, herbal preparations have been more used to treat diabetes. Dietetic supplement based on barley and beer yeast enriched with chromium (BBCr) is registered in Serbia as a supplement in the treatment of type 2 diabetes. Objective. To investigate the effect of the preparation based on barley and brewer’s yeast with chromium (BBCr), rosiglitazone (R) and their combination (BBCr+R) on fasting glycaemia and glycaemia in mice after glucose, adrenalin and alloxan application. Methods. The animals were divided into three groups: glucose 500 mg/kg (I); adrenalin 0.2 mg/kg (II); and alloxan 100 mg/kg (III) and into subgroups according to the substance they received (BBCr: 750 mg/kg, R: 0.75 mg/kg and BBCr+R). Each animal was its own control in respect of glycaemia before and after the treatment with test substances, except for group III which contained a placebo subgroup. Results. BBCr caused a significant decrease of fasting glycaemia and significant reduction of glycaemia after glucose load compared to the values before treatment (7.4±0.6 mmol/l vs 9.2±0.6 mmol/l; p=0.01). R and BBCr+R significantly decreased glycaemia after adrenalin load (R: 8.6±1.8 mmol/l vs 15.4±3.2 mmol/l; p=0.004; BBCr+R: 9.6±2.4 mmol/l vs 15.0±4.4 mmol/l; p=0.04). After alloxan application the glycaemia was significantly lower in the subgroups treated with BBCr, R and BBCr+R compared to placebo subgroup (10.1±8.0 mmol/l vs 6.8±2.7 mmol/l vs 13.5±9.7 mmol/l vs 24.5±4.7 mmol/l; p=0.001). Conclusion. Pretreatment with BBCr caused a significant reduction of fasting glycaemia and glycaemia after glucose load. Rosiglitazone and BBCr+R caused a significant reduction of glycaemia after adrenalin load. Pretreatment with BBCr, R and BBCr+R prevented the onset of experimental diabetes caused by alloxan, which was confirmed by histological analysis of pancreas tissue.

Purpose The aim of this study was to measure the consumption of serum lipid reducing drugs in Serbia from 2004 to 2008, to compare this data with that from Scandinavian countries, and to compare the consumption of lipid lowering drugs and the rate of mortality from cardiovascular diseases in these countries. Methods A population-based study was undertaken to analyse lipid lowering drug consumption using the Anatomical Therapeutic Chemical/Defined Daily Dose methodology. Cause-specific mortality rates were obtained from the WHOSIS annual report for the year 2009. Results In 2008, a total of 1207.44 DDD/1000 inh/day of all drugs, was used in Serbia, of which 38.89% belonged to drugs for cardiovascular diseases. While in Scandinavian countries 17.03–24.80% of drugs for cardiovascular diseases belonged to lipid-lowering drugs, in Serbia it was substantially lower (3%). In 2004 in Serbia, 1.50 DDD/1000 inh/day of statins were used. In 2008, this value was 14.24 DDD/1000 inh/day. In every investigated country, simvastatin made up more than 50% of the consumption of statins. After simvastatin, the next most frequently used statin was atorvastatin, with 5.52, 11.00, 11.17 and 24.82 DDD/1000 inh/day, in Serbia, Denmark, Finland and Norway, respectively. In 2004 Serbia has the highest mortality rate for cardiovascular diseases among investigated countries with 762/100.000 inhabitants and Norway has the lowest rate with 158/100.000 inhabitants. Conclusion The use of lipid lowering drugs is 6–8 times lower in Serbia than in Scandinavian countries but there is an evident rise in lipid lowering drugs consumption in Serbia during years. Copyright © 2010 John Wiley & Sons, Ltd.

Bile salts are known to enhance the permeability of biological barriers but little is known about their effects on drug permeability across the blood-brain barrier (BBB). In this paper, the rat brain endothelial 4 (RBE4) cell monolayer incubated with astrocyte-conditioned medium was used as an in vitro model of the BBB to investigate the effects of cholate (C), 12-monoketocholate (MKC), deoxycholate (DC), and taurocholate (TC) on the transport of the hydrophilic drug, morphine-6-glucuronide (M6G). C, MKC, and TC at a concentration of 5 mM each and DC at 1 mM increased the permeability of M6G through the paracellular pathway based on a similar permeability pattern to that of sucrose. RBE4 cell uptake of M6G was unaffected by 5 mM C and TC, whereas 1 mM DC dramatically increased it due to an effect shown to be cytotoxicity as measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Surprisingly, 1 mM MKC significantly increased M6G uptake without any cytotoxicity. In summary, all bile salts increased paracellular permeation of M6G but MKC also enhanced transcellular transport with little cytotoxicity. MKC appears to have the potential to modulate biophysical properties of the cell membrane or membrane-bound transporters and may therefore enhance drug delivery to the brain.

AIM: To investigate the influence of probiotic pre-treatment on the permeation of the antidiabetic drug gliclazide in healthy and diabetic rats. METHODS: Wistar rats (age 2-3 months, weight 350 +/- 50 g) were randomly allocated into one of 4 groups (N = 16 each group): healthy control, healthy probiotic, diabetic control, and diabetic probiotic. Probiotics (75 mg/kg, equal quantities of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus) were administered twice a day for three days to the appropriate groups after diabetes had been induced with alloxan i.v. 30 mg/kg. Rats were sacrificed, ileal tissues mounted in Ussing chambers and gliclazide (200 microg/mL) was administered for the measurement of the mucosal to serosal absorption Jss((MtoS)) and serosal to mucosal secretion Jss((StoM)) of gliclazide. RESULTS: Treatment of healthy rats with probiotics reduced Jss((MtoS)) of gliclazide from 1.2 +/- 0.3 to 0.3 +/- 0.1 microg/min/cm(2) (P0.01) and increased Jss((StoM))from 0.6 +/- 0.1 to 1.4 +/- 0.3 (P0.01) resulting in net secretion while, in diabetic tissues, treatment with probiotics increased both Jss((MtoS)) and Jss((StoM))fluxes of gliclazide to the comparable levels of healthy tissues resulting in net absorption. DISCUSSION: In healthy rats, the reduction in Jss((MtoS)) after probiotics administration could be explained by the production of bacterial metabolites that upregulate the mucosal efflux drug transporters Mrp2 that control gliclazide transport. In diabetic rats, the restored fluxes of gliclazide after probiotic treatment, suggests the normalization of the functionality of the drug transporters resulting in a net absorption. CONCLUSION: Probiotics may alter gliclazide transport across rat ileal tissue studied ex vivo.

l-Selenomethionine (SeMet) and sodium selenite are widely used selenium nutritional supplements with potential benefit in preventing cancer. However, supplementation is not without risks of toxicity if intake is too high. The aim of the present study was to investigate SeMet and selenite metabolism in the gastrointestinal tract with particular focus on the formation of the volatile selenium excretion products, dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). Adult male Wistar rats (n = 5) were euthanized, their intestinal tracts removed and the contents of jejunum, ileum, caecum and colon used to prepare 10% suspensions in saline. SeMet and selenite (0.5–0.6 mM) were then incubated with these suspensions at 37°C for 3 h. Caecum and colon contents were the most metabolically active towards SeMet with 30% and 15% metabolized over 3 h. DMDSe was the only volatile selenium metabolite detected accounting for 8.7 ± 1.3% of the selenium lost in caecum contents. Selenite was completely metabolized by caecum contents and 73% by colon contents under the same conditions forming DMSe (5.7 ± 0.9% of the selenium lost in caecum) and a precipitate of red amorphous elemental selenium. Based on previous literature and these results, we conclude that the gut microbiota contributes to the excretion of excess selenium through the production of methylated selenium compounds and elemental selenium.

Lamotrigine (LTG, 3,5-diamino-6- (2,3-dichlorphenyl)-l,2,4-triazine) is an antiepileptic drug used mainly for partial and generalized seizures. The efficacy of LTG in treating resistant partial seizures was optimized when it was combined with valproate (VPA). The aim of this study was to investigate the influence of VPA on LTG pharmacokinetics in epileptic patients. Methods: 38 patients were randomly divided into two groups, one given LTG (n=18) and the other given LTG+VPA(n=20). The first group consisted of 10 females (32.50 ± 12.46 years old, 67.80 ± 15.18 kg) and 8 males (24.88 ± 8.92 years old, 69.88 ± 11.41 kg) and the second group consisted of 9 females (28.33 ± 6.52 years old, 62.89 ± 13.28 kg) and 11 males (37.64 ± 10.43 years old, 85.64±15.4 kg). Patients were either administered an oral dose of LTG (157 ± 74 mg/day) or LTG+VPA (150 ± 83.11 mg/day & 774 ± 330 mg/day respectively). LTG steady state serum concentrations were determined 1.5–8 h post dose. Analyses were performed by a validated HPLC method. Results: LTG serum concentrations were increased significantly from 4.67 ± 3.66 and 9.56 ± 5.27 μg/ml by concomitant administration of VPA. Discussion: The inhibition of LTG metabolism by VPA was shown to have a marked effect on LTG kinetics. This inhibitory effect was complicated further by inter-patients variation in body weight and gender. This emphasizes the importance of continuous monitoring of LTG serum concentrations on an individual basis. Accordingly, if the use of potentially interacting drugs cannot be avoided, adverse reactions can be minimized by dose adjustments guided by careful monitoring of clinical response and measurement of LTG serum concentrations.

This study presents application of statistical power function for the t-test and ANOVA F-test on the evaluation of diclofenac bioequivalence in trials with the wide variations in sample sizes (N = 12, 18 and 24). The power function, together with appropriate equations tables and figures, is used to calculate the power of the ANOVA for crossover design, the number of subjects for a given value of power and the minimum detectable difference in treatment means for different pharmacokinetic parameters of the formulations. The power of the trial with a small, sample size (N = 12) to detect 20% differences between diclofenac formulations is shown to be more than 0.9 and almost the same as the power of the trial with a large sample size (N = 24). In all trials for all pharmacokinetic parameters the power to detect 20% difference is shown to be more than 0.8. For the power of 0.8, the needed subject number to detect 20% difference in treatment means is the same or smaller than used and the minimum detectable difference is smaller than 20% in all our trials. This investigation shows that bioequivalence studies with small number of subjects (N = 12) may be quite adequate for valid conclusions.

Bile salts have been shown to decrease the absorption of methotrexate in the rat intestine by an unknown mechanism. We aimed to examine this effect.We assessed apical-to-basolateral (AP-BL) permeation of methotrexate (5 muM) across Caco-2 cell monolayers pretreated with various concentrations (0, 0.25, 0.5, 1, 3 and 5 mM) of sodium cholate or its semisynthetic analogue, sodium 12-monoketocholate. We also determined the effect of orally administered 12-monoketocholate on the intestinal absorption of methotrexate in rats to evaluate a possible in-vitro-in-vivo correlation.It was found that sodium cholate and sodium 12-monoketocholate decreased the AP-BL permeation of methotrexate at low concentrations (maximal inhibition at 0.25 and 1 mM, respectively) and increased it at higher concentrations. Determination of [(14)C] mannitol permeation and electrical resistance of monolayers during experiments showed that membrane integrity was not compromised at low concentrations of bile salts but was disrupted at higher concentrations. Subsequently, we examined the effect of the simultaneous oral administration of sodium 12-monoketocholate (4, 20, 40 and 80 mg/kg) on the intestinal absorption of methotrexate in rats after an oral dose (5 mg/kg). The pharmacokinetic study showed that 12-monoketocholate at 4 and 20 mg/kg did not change the methotrexate area under the serum concentration-time curve whereas sodium 12-monoketocholate at 40 and 80 mg/kg significantly reduced it.Sodium 12-monoketocholate appears to decrease the intestinal absorption of methotrexate in rats by inhibition of transcellular active transport.

The aim of ths study was to investigate the pharmacokinetics of cefotaxime sodium (CEF) pharmacokinetics after oral application in the form of sodium 3alpha,7alpha-dihydroxy-12-keto-5beta-cholanate (MKC) microvesicles (MV) in rat. Thirty Male Wister rats were divided into six groups (n=5 per group). Groups were treated orally with: (i) CEF (15 mg/kg) saline solution (15 mg/kg); (ii) CEF (15 mg/kg) saline solution with MKC (2 mg/kg); (iii) CEF saline solution mixed with blank microvesicles; (iv) CEF (15 mg/kg) encapsulated in microvesicles with saline solution; (v) CEF saline solution (15 mg/kg) mixed with blank MKC microvesicules; (vi) CEF (15 mg/kg) encapsulated in MKC microvesicules with saline solution. Data were analyzed using noncompartmental model. CEF oral bioavailability was increased twofold when coadministered with MKC and when encapsulated in microvesicles and ninefold when encapsulated in MKC microvesicles compared to the same CEF dose administered orally as saline solution. The increased bioavailability of CEF resulting from CEF encapsulation in microvesicules with MKC suggests that this formulation can extend the application of CEF from parenteral only to oral application.

Selenomethionine (SeMet) is a widely used nutritional supplement that has potential benefit for people living in selenium-deficient areas. Previous research has shown that selenium administered as SeMet undergoes significant enterohepatic recycling which may involve the gut microflora. In order to investigate this we have developed a simple method for the quantitation of l-SeMet in rat gut content suspensions prepared from jejunum, ileum, caecum and colon. After incubation of l-SeMet with gut content suspensions, samples were deproteinized with sulfosalicylic acid and derivatized with o-phthaldialdehyde (OPA) and N-acetyl-l-cysteine (NAC). Mass spectrometry confirmed the formation of a 1:1:1 derivative of l-SeMet with OPA and NAC. Samples were analysed by reversed-phase high-performance liquid chromatography with fluorescence detection. The assay was linear in the concentration range 0.5–100 µg/mL (r2 = 0.9992) with a limit of detection of 0.025 µg/mL (signal-to-noise ratio of 5). Intra-day and inter-day accuracies were 91.1–92.8 and 91.7–95.5%, respectively with corresponding precisions as relative standard deviation of

In the last years there appeared many articles about the adverse influence of non-steroidal anti-inflammatory drugs on the liver and heart. This study is concerned with the influence of the duration of treatment with diclofenac and ketoprofen on the macroscopic and microscopic changes in the liver, lungs, heart, and kidneys in rats. Experiments were carried out on mature Wistar strain rats. Animals of test groups received diclofenac and ketoprofen in a dose of 8 mg/kg/day (equivalent to the therapeutic dose for man) during 7 per os (p.o.) or 28 days intraperitoneally (i.p.), whereas controls received physiological solution p.o. A high morbidity was observed in the animals receiving diclofenac p.o. and somewhat lower in those treated with ketoprofen. On the other hand, the rats got through the 28-day i.p. treatment with both drugs mainly without significant complications. Macroscopic examinations revealed some changes in treated rats: distension of the stomach, ascites, fibrin deposits on the internal organs, lung effusion and the changes in color and structure of the liver. These changes were more frequent in the group of rats receiving diclofenac for the 7 days compared with those that received ketoprofen for the same time. It may be thought that the high mortality and macroscopic changes in the internal organs of experimental animals are a consequence of the microscopic changes in the liver and its lowered function.

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu(n), n=2-5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu(n) in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 x 10(6) cells) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H](+) ions of MTX and MTXGlu(n) to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3-->294.2. Calibration curves for all analytes were linear in the range 2-250 nM (r(2)>0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4-15.1% and 4.3-18.4%, respectively with corresponding accuracies (as relative error) of -3.6 to +6.6% and -5.5 to +7.5%, respectively. Recoveries were in the range 60+/-4 to 108+/-13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.