[A pharmacogenetic analysis of dopaminergic and opioidergic genes in opioid addicts treated with the combination of naltrexone and guanfacine]. / Farmakogeneticheskii analiz vliyaniya genov dofaminovoi i opioidnoi sistem na effektivnost' kombinirovannoi terapii naltreksonom i guanfatsinom bol'nykh opioidnoi zavisimost'yu.

AIM: To evaluate an effect of opioid receptor and dopamine system gene polymorphisms on the efficacy of combined treatment with oral naltrexone and guanfacine in a randomized double blinded double dummy placebo controlled clinical trial. MATERIAL AND METHODS: Three hundred and one patients with opioid dependence were randomized into 4 treatment groups: naltrexone 50 mg/day + guanfacine 1 mg/day (N+G); naltrexone + placebo guanfacine (N+GP); placebo naltrexone + guanfacine (NP+G); double placebo (NP+GP). The primary outcome was treatment retention. All enrolled participants were genotyped for polymorphisms in the following genes: mu- (OPRM1), kappa-opioid receptors (OPRK1), catechol-O-methyltransferase (COMT), dopamine receptors types 2 (DRD2) and 4 (DRD4), dopamine-beta-hydroxylase, and dopamine transporter (SLC6A3, DAT1) and alpha-2-adrenoreceptor (ADRA2A) a pharmacological target of guanfacine. RESULTS: The efficacy of the combination of naltrexone and guanfacine was comparable to naltrexone monotherapy. Regardless of treatment, several gene polymorphisms were associated with higher chance to complete the treatment program: allele Т DRD4 - 521 С/Т (rs1800955) (р=0.039; OR (95% CI)=3.7 (1.1-12.7); log-rank test: р=0.01); allele С DRD2 С957Т (rs6277) (р=0.03; HR=0.6 (0.34-0.95); genotype combination: DRD4 VNTR (LL) + OPRM1 A118G (rs1799971) (AA), р=0.051; DRD2 C957T (ТТ) + OPRM1 (rs1074287) (СС), р=0.025; DRD2 - 141С (II) + OPRM1 (rs510769) (АА), р=0.035; DBH Fau(СС) + OPRM1 (rs1074287) (СС), р=0.0497. Regardless of treatment several polymorphisms were associated with high risk of relapse: allele Т (rs510769) OPRM1 (р=0.053), allele А (rs1799971, A118G) OPRM1 (р=0.056), allele S exon III 48 bp DRD4 VNTR (р=0.001; HR=3.1 (ДИ 95% 1.57-6.18); genotype combinations: DRD4 - 521 С/Т (ТТ) + DRD2 Nco I (TT), р=0.026; DRD4 -521 С/Т (ТТ) + DRD2 -141 С (II), р=0.011; DRD4 - 521 С/Т (ТТ) + OPRM1 A118G (rs1799971) (AA), р=0.011; DRD2 Nco I(ТТ) + ADRA2A (СС), р=0.012; DRD2 Nco I(ТТ) + OPRM1 A118G (AA), р=0.02. The effects dependent on the treatment group were as follows: 1) in the N+G group, patients with the DRD4 -521 С/Т TT genotype had higher probability of completion of treatment program in comparison with other genotypes (CC and CT) (log-rank test: p=0.002); 2) in NP + GP group, patients with the OPRM1 rs510769 T allele had higher risk of relapse compared to the genotype GG (p=0.008) (FDR p<0.0125). CONCLUSION: The additive effect of opioid receptor genes and dopaminergic system genes on outcomes of treatment opioid dependence with oral naltrexone and guanfacine was shown. Pharmacological effects of naltrexone and guanfacine were associated with genetic variants of the DRD4 - 521C/T polymorphism, since its effect was shown only in the N+G group. The effect of the OPRM1 rs510769 polymorphism was demonstrated in the double placebo group that was associated with personality traits (temperament, character) and determined compliance. Genetic analysis is useful for determining potential responders to treatment of opioid dependence; genotyping can increase the efficacy of pharmacotherapy.

[Stabilization of remission in patients with opioid dependence with naltrexone implant: a pharmacogenetic approach]. / Stabilizatsiia remissii u bol'nykh opiinoi narkomaniei implantom naltreksona: farmakogeneticheskii aspekt.

AIM: To evaluate the effect of opioid receptor genes and dopamine system genes polymorphisms on treatment outcomes of opioid dependence with implantable and oral naltrexone. MATERIAL AND METHODS: Authors carried out a randomized double-blind, double-dummy, placebo-controlled clinical trial. Three hundred and six patients with opioid dependence were randomized into 3 equal treatment groups. The first group received implantation of 1000 mg naltrexone every 2 months during 6 months + oral naltrexone placebo; the second group - placebo implant every 2 months + oral naltrexone (50mg/day) and the third group - placebo implant + oral naltrexone placebo. It was genotyped polymorphisms in the following genes: mu-opioid receptor (OPRM1), kappa-opioid receptor (OPRK1), catechol-O-methyltransferase (COMT), dopamine receptors types 2 (DRD2) and 4 (DRD4), dopamine-beta-hydroxylase, and dopamine transporter (DAT1). RESULTS: Regardless of treatment several polymorphisms of these genes were associated with high risk of relapse: an allele L (2R) DRD4 120bp (p=0.05; OR (95% CI)=3.3(1.1-10.1)); an allele С DRD2 NcoI (р=0,051; OR (95% CI)=2,86 (1,09-7,52)); the genotype 9.9 DAT VNTR 40bp (р=0,04; OR (95% CI)=1,4 (1,3-1,5)); on the contrary, (СС+СТ)-(ТТ)) variants of OPRK1-DRD2Ncol increased a chance to complete treatment program (р=0,004; OR (95% CI)=7.4 (1.8-30.4)), Kaplan-Meier survival analysis (р=0,016). The probability of completing treatment program by the carriers of these variants was higher in the oral naltrexone group (p=0.016), lower in the double placebo group (p=0.015), but did not influence on treatment outcomes in the naltrexone-implant group. CONCLUSION: Naltrexone-implant is a highly effective medication for treatment of opioid dependence and its effectiveness exceeds that of oral naltrexone and placebo. The study has shown the joint influence of opioid receptor genes and genes of dopaminergic system on treatment outcomes of opioid dependence. Genetic analysis is useful for determining potential responders to naltrexone treatment of opioid dependence.

[Effect of deletion of 3'-noncoding region of the Bacillus intermedius glutamyl endopeptidase gene on the active protein production level in the culture of the B. subtilis cells].

The Bacillus intermedius glutamyl endopeptidase is a secretory serine proteinase from the subfamily of chymotrypsin. Its gene was previously cloned and sequenced. The enzyme was thoroughly characterized including 3D structure determination. The present work demonstrates that removal of 3'-noncoding region of the enzyme gene resulted in a decrease of the active glutamyl endopeptidase production level in culture of B. subtilis cells. In this 3'-noncoding region, the sequence with all typical features of transcription terminators of the Firmicutes type was found.

Modification of substrate-binding site of glutamyl endopeptidase from Bacillus intermedius.

Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family. Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear. The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system). In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213-Thr) and studied its catalytic properties. Such modification proved not to affect the primary specificity of the enzyme. The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times). The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity.

Rapid HPLC analysis of melphalan applied to hyperthermic isolation limb perfusion.

Hyperthermic isolated limb perfusion (HILP) with melphalan (MH) as a standard cytotoxic drug has been performed in 28 patients suffering from malignant melanoma. MH has been administered by HILP via extracorporeal circulation system. The drug given locoregionally reduces subsequent toxicity of organs. For all that residues can leak into the systemic circulation during HILP. Because of known carcinogenic potential and secondary cancer formation, the main interest of this work is to determine MH concentration profile in the patient plasma during and after HILP and evaluation of its potential toxicity in patients. Reversed-phase HPLC assay, which uses isocratic elution and fluorimetric detection has been shown to be sensitive, reliable and suitable for routine analyses. The assay was validated for the concentration range of 50-2500 ng.ml-1 with the limit of detection (LOD) 6.881 ng.ml-1. The samples were treated by methanol precipitation with the recovery more than 80%. The stability of standard solutions and methanolic extracts of MH were also followed. The concentration profile of MH in patient samples has been pursued in three time points during and after chemoperfusion (45 min after application of MH in extracorporeal circulation, 10 min after the joining the extremity to systemic circulation and one hour after the great vessels reconstruction). The concentrations of MH ranged 100-1500 ng.ml-1 and varied from patient-to-patient. Some complications were observed after HILP in 11 patients and are correlated with the higher con- centrations of MH (over 150 ng x ml-1) found in plasma.

Study of antioxidant effect of apigenin, luteolin and quercetin by DNA protective method.

A DNA protective capacity of three flavonoids, apigenin (AP), luteolin (LU) and quercetin (QU) against free radicals generated by H202, resp. Fe2+ is reported. This effect corresponding with scavenging of free radicals or with chelating of iron was assayed at two concentrations of flavonoids studied (1 microM and 10 microM). The quantitative analysis has shown that LU possesses the highest DNA protective effect of flavonoids investigated in the presence of H2O2. On the other hand, in the presence of 10 microM Fe2+, AP exhibited the highest DNA protective effect at the concentration of 1 microM and the following order was reached at the stoichiometric concentrations (10 microM) of Fe2+. It is believed that this discrepancy is caused by the ability of LU and QU iron-complex formation as it was separately investigated using UV-VIS spectrometry.

Determination of apigenin in rat plasma by high-performance liquid chromatography.

Apigenin (4',5,7-trihydroxyflavone, AP) belongs to a less-toxic and non-mutagenic flavone subclass of flavonoids, the biotransformation and metabolism of which have been little studied until now. Therefore, this study is focussed on the determination of AP in free form. AP was administered to rats via the i.p. route (25 mg kg(-1)) and then the blood was collected at 10, 15, 30 and 45 min after injection. Methanol was used for rat plasma deproteinization. The HPLC assay (mobile phase, 2% formic acid-acetonitrile-methanol, 40:35:25, v/v; flow-rate, 1 ml min(-1); UV detection at 349 nm) for AP determination was validated and used for the quantification of AP in rat plasma. The unknown concentration was calculated from the equation obtained by the least-squares regression analysis (y = 0.521x + 1.130, r2 = 0.998). The highest concentration of AP in plasma was found to be 30 min after injection. The concentration profile of AP obtained here may contribute to until known results about AP metabolism. They could be applied to other studies of AP or related flavonoids because of favourable effects on human health.

UV spectrometric and DC polarographic studies on apigenin and luteolin.

Remarks on polyphenolic compounds has been arisen since past few years. The flavonoids appears to be the important groups of compounds with their capability to inhibit DNA damage, lipid peroxidation, to quench free radicals and, at least, anticarcinogenic and antiproliferative effects. On the other hand, their mechanism of action is still unexplained. Apigenin and luteolin are the most wide-spread flavones and they exhibited to be useful in chemoprevention. UV spectrometric and DC polarographic studies on these two compounds have been carried out with regard to changing pH. The most significant changes were observed at basic pH. These results could aid to elucidation of their mechanism of action as pH is one of the important factors for bioprocesses passing in living organisms.

Enhanced effects of adriamycin by combination with a new ribonucleotide reductase inhibitor, trimidox, in murine leukemia.

Ribonucleotide reductase is the rate limiting enzyme of de novo DNA synthesis; its activity is significantly increased in tumor cells related to the proliferation rate. Therefore the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study we tested the in vitro and in vivo antitumor effects of a drug combination using trimidox (3,4,5-trihydroxybenzamidoxime), a novel inhibitor of ribonucleotide reductase with adriamycin, a widely used anticancer drug. This combination was selected because adriamycin generates free radicals being responsible for cardiotoxic side effects; trimidox has been shown to be a good free radical scavenger. The in vitro cytotoxic effect of the drug combination was examined in L1210 mouse leukemia cells employing a MTT chemosensitivity assay. Incubation of these cells with adriamycin and trimidox together yielded less than additive cytotoxic effects compared to either drug alone. These effects were not caused by the involvement of p-glycoprotein mediated drug efflux. However, when the effect of trimidox and adriamycin in combination was examined in L1210 leukemia bearing mice antitumor effects of adriamycin could be enhanced by the presence of trimidox. Our data indicate, that the in vivo combination of adriamycin together with trimidox might be beneficial for the treatment of malignancies.

Isolation and initial characterization of the uridine phosphorylase from Salmonella typhimurium.

The structural udp gene encoding uridine phosphorylase (UPase) was cloned from the Salmonella typhimurium chromosome and overexpressed in E. coli cells. The S. typhimurium UPase was purified to an apparently homogeneous state, and some physicochemical characteristics of the enzyme were studied. The molecular weight of one subunit of UPase is 27.5 kD, and the optimal pH for its activity is 7.2--7.4. The native S. typhimurium UPase consists of six identical subunits, and its molecular weight is about 165 kD. According to these parameters, the S. typhimurium UPase is similar to the E. coli UPase. However, these enzymes differ substantially from one another by the substrate sensitivity and sensitivity to polarity of the medium. The S. typhimurium UPase has much higher phosphorylation activity toward thymidine, deoxyuridine, and 5;-bromide- or 5;-fluoride-containing analogs of nucleosides than that of E. coli UPase.

Staurosporine enhanced benzamide riboside-induced apoptosis in human multidrug-resistant promyelocytic leukemia cells (HL-60/VCR) in vitro.

The inosine monophosphate (IMP) dehydrogenase inhibitor benzamide riboside (BR) induced apoptosis (detected with the aid of flow cytometric identification of cells with sub-G0 DNA content and increased side angle light scatter) equally or slightly more intensively in the multidrug-resistant human promyelocytic leukemia cell line (HL-60/VCR: MDR-1 gene, Pgp positive) in comparison with the parental drug sensitive HL-60 cells. Staurosporine alone induced relatively low level of apoptosis in parental HL-60 cells but higher level (approximately 35%) of apoptosis in multidrug-resistant HL-60/VCR cells after 24 hour induction. The combination of benzamide riboside and staurosporine induced in both drug-sensitive and drug-resistant HL-60 cells a marked proportion of apoptotic cells already after short (6 hour) induction (more than 30% of apoptotic cells).

DNA-protective activity of new ribonucleotide reductase inhibitors.

The DNA-protective activity of hydroxyurea (HU) and novel ribonucleotide reductase (RR) inhibitors amidox (AX), didox (DX) and trimidox (TX) was examined using hydrogen peroxide as the DNA-damaging agent. The exposure of superspiralized plasmid DNA molecules (pBR 322) to H2O2 under precisely defined in vitro conditions initiates a change in DNA topology (DNA from I relaxes to DNA form II). This electrophoretically monitored change in the plasmid DNA topology is related to the induction of ss-DNA breaks and corresponds with DNA exposition to free radicals. The inhibition of DNA relaxation (the prevention of DNA damage induced by hydrogen peroxide) depended on the free radical scavenging capacity of the drugs investigated. HU exerted DNA protective activity at a concentration of 4 mM, AX at concentration of 1 microM, TX at a concentration of 5 microM and DX at a concentration of 25 microM (the free radical scavenging activity increases from HU to AX in following manner: HU << DX < TX < AX). It can be concluded that the new synthetic RR-inhibitor AX which is being investigated at the preclinical level as a potential anti-cancer drug possess the highest capacity for scavenging of free radicals.

Simultaneous determination of the new anticancer agent amidox and its metabolites in rat bile and plasma by high-performance liquid chromatography.

A new reversed-phase ion-pair high-performance liquid chromatography method was developed to study the first-pass hepatic metabolism of the anti cancer drug amidox in bile. Separation of the metabolites was achieved on a Spherisorb C18 column after liquid-liquid extraction using a linear gradient system of heptanesulfonic acid in potassium phosphate monobasic (pH 4.0) with increasing amounts of methanol (0-40%). The method was further applied to a pharmacokinetic study of amidox in rats after 200 mg kg-1 intraperitoneal administration. Using 50 microliters of rat bile and 300 microliters of rat plasma the limit of detection for amidox was 60 ng and 85 ng, respectively, and the assay was linear from 0.1 to 150 micrograms ml-1. This method appears to be sensitive enough to be used in further pharmacokinetic studies of amidox in human volunteers.

The new inhibitors of ribonucleotide reductase--comparison of some physico-chemical properties.

Amidox (AX), didox (DX) and trimidox (TX), compounds synthetized as new ribonucleotide reductase inhibitors, have been investigated by ultraviolet (UV) spectrophotometry, polarography and high performance liquid chromatography (HPLC). The experiments have been performed at various pH values. The changes in UV absorption of the compounds studied were recorded and it was demonstrated that these changes are related to the pH and to structural features of the investigated molecules. From the compounds included in our series of experiments, only amidox and trimidox are reduced during polarographic experiments in Britton-Robinson buffer. The reduction of both compounds proceeded in two one-electron steps in acidic pH. One two-electron diffuse irreversible wave was observed at basic pH. The values of the half-wave potential became more negative in accordance with the increasing pH. HPLC assay also showed changes in the retention of compounds investigated, particularly when the pH of the mobile phase was close to the dissociation constant of the particular drug. The changes of physico-chemical properties detected by the all used methods are related to different chemical structures (the most significant changes were observed in alkaline pH).

Chromatographic properties of cytosine, cytidine and their synthetic analogues.

A direct reversed-phase high-performance liquid chromatographic (RP-HPLC) assay was used for the study of the effects of methanol concentration, pH, flow-rate of the mobile phase and column temperature on the retention of the natural nuclei acid components cytosine and cytidine and their synthetic 1-beta-D-arabinofuranosyl, 5-aza and 6-aza analogues. The pKa values were also determined. The greatest changes were observed with changes in pH. The relationship between the capacity factors and the hydrophobicity of the compounds studied was also investigated.

Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K.

Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.

Enzyme-catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation.

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Automatic solid-phase extraction and high-performance liquid chromatographic determination of quinidine in plasma.

High-performance liquid chromatography (HPLC) was used for the therapeutic drug monitoring of quinidine in clinical samples. Solid-phase extraction (SPE) was studied in both off-line and on-line modes. SPE was performed in an automatic on-line mode using a fully automated Prospekt system. Extraction recoveries were in the range 97.1-99.4% for 1-2 micrograms/ml quinidine concentrations. For HPLC separation an Ultrasep RP-8 reversed-phase column was applied with acetonitrile-water (9:1) containing 0.3% triethylamine (pH 2.5) as the mobile phase. The Prospekt system is recommended for the routine monitoring of quinidine in plasma samples. Concentrations were in therapeutic range (1.2-3.6 micrograms/ml).

Selective modification of putative uridine-binding site of uridine phosphorylase from E. coli with fluorescein 5'-isothiocyanate.

A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5'-isothiocyanate (FITC). Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit. Addition of uridine prevents the enzyme inactivation by FITC. In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme. The modified protein retains the ability to bind phosphate but not uridine. According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.