Haemoglobin adducts of acrylonitrile and ethylene oxide in acrylonitrile workers, dependent on polymorphisms of the glutathione transferases GSTT1 and GSTM1

Fifty-nine persons with industrial handling of low levels of acrylonitrile (AN) were studied. As part of a medical surveillance programme an extended haemoglobin adduct monitoring [N-(cyanoethyl)valine, CEV; N-(methyl)valine, MV; N-(hydroxyethyl)valine, HEV] was performed. Moreover, the genetic states of the polymorphic glutathione transferases GSTM1 and GSTT1 were assayed by polymerase chain reaction (PCR). Repetitive analyses of CEV and MV in subsequent years resulted in comparable values (means, 59.8 and 70.3 μg CEV/l blood; 6.7 and 6.7 μg MV/l blood). Hence, the industrial AN exposures were well below current official standards. Monitoring the haemoglobin adduct CEV appears as a suitable means of biomonitoring and medical surveillance under such exposure conditions. There was also no apparent correlation between the CEV and HEV or CEV and MV adduct levels. The MV and HEV values observed represented background levels, which apparently are not related to any occupational chemical exposure. There was no consistent effect of the genetic GSTM1 or GSTT1 state on CEV adduct levels induced by acrylonitrile exposure. Therefore, neither GSTM1 nor GSTT1 appears as a major AN metabolizing isoenzyme in humans. The low and physiological background levels of MV were also not influenced by the genetic GSTM1 state, but the MV adduct levels tended to be higher in GSTT1− individuals compared to GSTT1+ persons. With respect to the background levels of HEV adducts observed, there was no major influence of the GSTM1 state, but GST− individuals displayed adduct levels that were about 1/3 higher than those of GSTT1+ individuals. The coincidence with known differences in rates of background sister chromatid exchange between GSTT1− and GSTT1+ persons suggests that the lower ethylene oxide (EO) detoxification rate in GSTT1− persons, indicated by elevated blood protein hydroxyethyl adduct levels, leads to an increased genotoxic effect of the physiological EO background. Received: 13 January 1999 / Accepted: 15 March 1999     

The combined impact of CYP2C19 and CYP2B6 pharmacogenetics on cyclophosphamide bioactivation

AIMS

The role of CYP pharmacogenetics in the bioactivation of cyclophosphamide is still controversial. Recent clinical studies have suggested a role for either CYP2C19 or CYP2B6. The aim of this study was to clarify the role of these pharmacogenes.

METHODS

We used a combined in vitro–in vivo approach to determine the role of these pharmacogenes in the bioactivation of the prodrug to 4-hydroxy cyclophosphamide (4-OHCP). Cyclophosphamide metabolism was determined in a human liver biobank (n = 14) and in patients receiving the drug for treatment of lupus nephritis (n = 16)

RESULTS

In livers of known CYP2C19 and CYP2B6 genotype and protein expression we observed that there was a combined role for both CYP2C19 and CYP2B6 in the bioactivation of cyclophosphamide in vitro. The presence of at least one loss of function (LoF) allele at either CYP2C19 or CYP2B6 resulted in a significant decrease in both V_max (P = 0.028) and CL_int (P = 0.0017) compared with livers with no LoF alleles. This dual genotype relationship was also observed in a preliminary clinical study, with patients who had ≥1 LoF allele at either CYP2C19 or CYP2B6 also displaying significantly (P = 0.0316) lower bioactivation of cyclophosphamide. The mean 4-OHCP : CP bioactivation ratio was 0.0014 (95% CI 0.0007, 0.002) compared with 0.0071 (95% CI 0.0001, 0.014) in patients with no LoF alleles at either of these genes.

CONCLUSIONS

The presence of ≥1 LoF allele(s) at either CYP2B6 or CYP2C19 appeared to result in decreased bioactivation of cyclophosphamide both in vitro and in patients. Further clinical studies to confirm this relationship are warranted.     

Hydrolytic metabolism of pyrethroids by human and other mammalian carboxylesterases

Pyrethroid chemicals are attractive alternatives to the organophosphates (OPs) because of their selective toxicity against pests rather than mammals. The carboxylesterases (CEs) are hepatic enzymes that metabolize ester-containing xenobiotics such as pyrethroids. The primary aim of this study was to gain insight into the catalytic properties of the CE enzymes in humans that metabolize pyrethroids, while a secondary aim was to investigate pyrethroid metabolism using CEs from other mammalian species. Pure human CEs (hCE-1 and hCE-2), a rabbit CE (rCE), and two rat CEs (Hydrolases A and B) were used to study the hydrolytic metabolism of the following pyrethroids: 1Rtrans-resmethrin (bioresmethrin), 1RStrans-permethrin, and 1RScis-permethrin. hCE-1 and hCE-2 hydrolyzed trans-permethrin 8- and 28-fold more efficiently than cis-permethrin (when k(cat)/K(m) values were compared), respectively. In contrast, hydrolysis of bioresmethrin was catalyzed efficiently by hCE-1, but not by hCE-2. The kinetic parameters for the pure rat and rabbit CEs were qualitatively similar to the human CEs when hydrolysis rates of the investigated pyrethroids were evaluated. Further, a comparison of pyrethroid hydrolysis by hepatic microsomes from rats, mice, and humans indicated that the rates for each compound were similar between species, which further supports the use of rodent models for pyrethroid metabolism studies. An eight-fold range in hydrolytic rates for 11 individual human liver samples toward trans-permethrin was also found, although this variability was not related to the levels of hCE-1 protein in each sample. We also determined that the CE inhibitor 2-chloro-3,4-dimethoxybenzil blocked hCE-2-catalyzed trans-permethrin hydrolysis 36 times more potently than hCE-1. Thus, this inhibitor will be useful in future studies that examine CE-mediated metabolism of pyrethroids. While there are likely other esterases in human liver that hydrolyze pyrethroids, the results of this study clearly demonstrate that hCE-1 and hCE-2 are human pyrethroid-hydrolyzing CEs.     

N6-methyl-AMP aminohydrolase activates N6-substituted purine acyclic nucleoside phosphonates

In this study we present the identification and characterization of the enzyme involved in the N6-cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (N6-cyclopropyl-PMEDAP) conversion to biologically active 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) as well as abacavir 5'-phosphate to carbovir 5'-phosphate. This enzyme was purified from rat liver to homogeneity; it appears to be composed from six 42 kDa subunits and its native form has the molecular weight 260 kDa. This so far unknown enzyme catalyzes conversion of both N6-methyl-AMP and N6-methyl-dAMP to IMP and/or dIMP, respectively. The enzyme acts as 6-(N-substituted amino)purine 5'-nucleotide aminohydrolase with the reaction mechanism very similar to AMP deaminase. The enzyme does not deaminate AMP and dAMP, or the corresponding nucleosides. It is inhibited by deoxycoformycin 5'-phosphate but not by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).     

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant’s leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.     

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.     

Growth Inhibitory Effect of (E)-2,4-bis(p-hydroxyphenyl)-2-Butenal Diacetate through Induction of Apoptotic Cell Death by Increasing DR3 Expression in Human Lung Cancer Cells

The Maillard Reaction Products (MRPs) are chemical compounds which have been known to be effective in chemoprevention. Death receptors (DR) play a central role in directing apoptosis in several cancer cells. In our previous study, we demonstrated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP product, inhibited human colon cancer cell growth by inducing apoptosis via nuclear factor-κB (NF-κB) inactivation and G₂/M phase cell cycle arrest. In this study, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate, a new (E)-2,4-bis(p-hydroxyphenyl)-2-butenal derivative, was synthesized to improve their solubility and stability in water and then evaluated against NCI-H460 and A549 human lung cancer cells. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate reduced the viability in both cell lines in a time and dose-dependent manner. We also found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate increased apoptotic cell death through the upregulation of the expression of death receptor (DR)-3 and DR6 in both lung cancer cell lines. In addition to this, the transfection of DR3 siRNA diminished the growth inhibitory and apoptosis inducing effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate on lung cancer cells, however these effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate was not changed by DR6 siRNA. These results indicated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate inhibits human lung cancer cell growth via increasing apoptotic cell death by upregulation of the expression of DR3.     

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST ω1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST ω2 (GSTO2) Asn142Asp, GST π1 (GSTP1) Ile105Val, GST μ1 (GSTM1) wild/null, and GST θ1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As^V than the wild homo type. Higher percentage of DMA^V in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As^V to As^III. Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.     

Roles of endogenous glutathione levels on 6-hydroxydopamine-induced apoptotic neuronal cell death in human neuroblastoma SK-N-SH cells

We investigated the roles of endogenous glutathione on 6-hydroxydopamine (6-OHDA)-induced apoptosis in human neuroblastoma SK-N-SH cells using DNA fragmentation enzyme-immunoassay and the DNA dye Hoechst 33258 staining. We observed that exogenous reduced glutathione (GSH), but not oxidized glutathione (GSSG), protected 6-OHDA (25 micro M)-induced apoptosis in a dose-dependent manner. Preincubation (18 h) with the glutathione synthesis inhibitor DL-buthionine-(S,R)-sulfoximine (BSO) significantly potentiated the toxic effects of 6-OHDA (12.5 or 25 micro M). In contrast to BSO, N-acetylcysteine (NAC) blocked, and L-(-)-cystine, the glutathione precursor, significantly attenuated 6-OHDA (25 micro M)-induced apoptosis, respectively. No alterations in endogenous glutathione concentrations were detected at 5, 15, 30, 60 min, 1 hour, 3 hours, or 6 hours after 6-OHDA (25 micro M) treatment. However, we found a 3.5-fold increase of intracellular glutathione levels 24 hours later. On the contrary, higher concentration (100 micro M) of 6-OHDA treatment, which caused more severe cell death, showed no changes of glutathione levels. These results suggest that delayed induction of endogenous glutathione might play an important role in the neuroprotective mechanism against dopamine cell death. In addition, we found that NAC might work as a beneficial catecholaminergic neuron-survival factor more efficiently than exogenous glutathione or L-cystine.     

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.     

Cellular transformation of the investigational new anticancer drug NB1011, a phosphoramidate of 5-(2-bromovinyl)-2'-deoxyuridine,results in modification of cellular proteins not DNA

NB1011 [E-5-(2-bromovinyl)-2'-deoxyuridine-5'-(L-methylalaninyl)-phenylphosphoramidate], a phosphoramidate prodrug of E-5-(2-bromovinyl)-2'-deoxyuridine-5'-monophosphate (BVdUMP), is an investigational new anticancer drug. NB1011 targets thymidylate synthase (TS), which catalyzes the transformation of BVdUMP into cytotoxic reaction products. Due to the elevated levels of TS expression in tumor cells compared to normal cells, these cytotoxic products are preferentially generated inside tumor cells, and, as expected, NB1011 is more toxic to cells with higher levels of TS expression. Therefore, NB1011 therapy should kill tumor cells without severely damaging normal cells. Radiolabeled NB1011 was used to determine the intracellular fate of NB1011 reaction products and, possibly, the mechanism of action of this investigational new drug. We found significant incorporation of the radiolabel into cellular macromolecules. In contrast to our expectations that NB1011 product(s) would be incorporated into DNA, we discovered that cellular proteins were the labeled macromolecular fraction. Herein, we report that the intracellular transformation of NB1011 involves formation of the corresponding monophosphate, TS-dependent transformation into highly reactive intermediates, and subsequent incorporation into cellular proteins. TS itself appears to escape irreversible inactivation. Our data suggest that protein modification not DNA incorporation accounts for the therapeutic effect of NB1011. The proposed mechanism is rather unexpected for a nucleotide analogue and could lead to the discovery of new cellular protein targets for future drug design.     

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine and BCNU

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine being equally potent inhibitors of purified human GR (IC(50) values of 55.5 microM and 54.6 microM, respectively). Human erythrocytes, following exposure to 50 microM BCNU for 1h at 37 degrees C, had an 84% decrease in GR activity, whereas 50 microM Cloretazine caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.     

The effect of thiopurine drugs on DNA methylation in relation to TPMT expression

The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2′-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene (MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5’-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.     

Separation and Identification of Phenolic Acid and Flavonoids from Nerium indicum Flowers

Four major compounds were separated and identified from the methanol extracts of Nerium indicum flowers (Arali) using HPLC and mass spectral data. Through mass data, the chemical structures were elucidated as: trans5-O-caffeoylquinic acid (1), quercetin-3-O- rutinoside (2), luteolin-5-O-rutinoside (3) and luteolin-7-O-rutinoside (4). In addition, the cis isomers of 5-O-caffeoylquinic acid in Nerium indicum flowers were confirmed by Mass, HPLC and UV. The structures of these compounds confirmed with the help of liquid chromatography mass spectrometry.     

Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats

Saquinavir (SQV) was the first human immuno-virus-1 (HIV-1) protease inhibitor approved by FDA. However, P-glycoprotein (P-gp), an efflux pump limits its oral and brain bioavailabilities. The objective of this study is to investigate whether prodrug modification of SQV to dipeptide prodrugs Valine-Valine-Saquinavir (Val-Val-SQV) and Glycine-Valine-Saquinavir (Gly-Val-SQV) targeting intestinal peptide transporter can enhance intestinal permeability of SQV by circumventing P-gp mediated efflux. Single pass intestinal perfusion experiments in rat jejunum were performed to calculate the absorption rate constant and intestinal permeability of SQV, Val-Val-SQV and Gly-Val-SQV. Equimolar concentration (25 microM) of SQV, Val-Val-SQV and Gly-Val-SQV were employed in the perfusion studies. Perfusion experiments were also carried out in the presence of cyclosporine (10 microM) and glycyl-sarcosine (20 mM). Absorption rate constants in rat jejunum (ka) for SQV, Val-Val-SQV and Gly-Val-SQV were found to be 14.1+/-3.4x10(-3), 65.8+/-4.3x10(-3), and 25.6+/-5.7x10(-3) min(-1), respectively. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. Significant permeability enhancement of SQV across rat jejunum was observed in the presence of cyclosporine 10 microM (P-gp inhibitor). However, permeability of Val-Val-SQV was unchanged in the presence of cyclosporine suggesting lack of any interaction of the prodrug with efflux pump. Intestinal absorption of Val-Val-SQV was significantly inhibited in the presence of gly-sar indicating the involvement of peptide transporter in intestinal absorption. In conclusion, peptide transporter targeted prodrug modification of P-gp substrates could lead to shielding of these drug molecules from efflux pumps.     

Synthesis and antifungal activity of new N-(1-phenyl-4-carbetoxypyrazol-5-yl)-, N-(indazol-3-yl)- and N-(indazol-5-yl)-2-iodobenzamides

N-(1-Phenyl-4-carbetoxypyrazol-5-yl)-, N-(indazol-3-yl)- and N-(indazol-5-yl)-2-iodobenzamides 6, with a Benodanil-like structure, were synthesized by refluxing in acetic acid the corresponding benzotriazinones 5 with potassium iodide for 1 h in order to study the role on the antifungal activity of the N-substitution with an aromatic heterocyclic system on benzamide moiety. Among the tested iododerivatives, compounds 6d,f,g,h possess interesting activities toward some phytopathogenic fungal strains.     

Relation between the ability of some compounds to modulate the MRP1-mediated efflux of glutathione and to inhibit the MRPl-mediated efflux of daunorubicin

Much effort has been recently directed to identify the transport-modulating agents in order to overcome the P-gp- and MRP1-mediated drug resistance. Contrary to what is observed for P-gp, very few compounds have been shown to reverse multi-drug resistance (MDR) mediated by MRP1. On the other hand, despite of critical role of GSH in transporting the MRP1 substrates, not much is known about GSH interactions with MRP1. In this work, three compounds that were shown to inhibit the MRP1-mediated efflux of daunorubicin (DNR) have been studied. Depending on their nature the selected compounds have different effects, e.g. at 40 microM, verapamil inhibits 50% of DNR efflux whereas GSH efflux is increased about two-fold. PAK-104P has shown the same effect, i.e. the inhibition of the MRP1-mediated efflux of DNR is accompanied by a stimulation of GSH efflux. However, the PAK-104P concentration required to obtain the same effect is about 40 times smaller that in the case of verapamil. MK571 has been shown to inhibit the efflux of both DNR and GSH. Based on these observations and those reported earlier, a working model is proposed.     

Inhibition of thymidine phosphorylase (PD-ECGF) from SD-lymphoma by phosphonomethoxyalkyl thymines

A series of thymine phosphonomethoxyalkyl derivatives were evaluated for their ability to inhibit thymidine phosphorylase (dThdPase) purified from rat spontaneous T-cell lymphoma. A kinetic study of thymidine phosphorolysis catalyzed by dThdPase was performed with thymidine and/or inorganic phosphate as substrates. Data show that the substantial inhibitory effect of these acyclic nucleotide analogues is decreasing in the order of (R)-FPMPT>(S)-FPMPT>or=(R)-HPMPT>(S)-PMPT>(S)-HPMPT>PMET>or=(R)-PMPT. The inhibitory potency (K(i)/(dThd)K(m)) of the most efficient inhibitors from this series against T-cell lymphoma enzyme is 0.0026 for (R)-FPMPT and 0.0048 for (S)-FPMPT. The studied compounds do not inhibit Escherichia coli and human enzyme and possess lower inhibitory potency against rat liver thymidine phosphorylase.