The effect of probenecid and MK-571 on the feto-maternal transfer of saquinavir in dually perfused human term placenta.

Human placenta, particularly the blood-placenta barrier, with various transporters has crucial role to protect the fetus and, on the other hand, to facilitate movement of compounds towards the fetal circulation. This study aimed to characterize the role of basal transporters of the syncytiotrophoblast, which appear to be yet less studied, in the fetal-to-maternal transfer of saquinavir by use of dually perfused human placentas. A dual perfusion of human placenta was performed to study effect of MK-571 and probenecid, inhibitors of the MRP1 and OATP transporters, expressed in the basal trophoblast membrane, on the transfer of saquinavir. The fetal-to-maternal placental transfer of saquinavir in the control group as measured by TPT(AUC)% (absolute fraction of the dose crossing placenta) was 14.0%, which is 73% less than the transfer of the freely diffusible antipyrine. The two inhibitors, MK-571 and probenecid caused a non-significant (P = 0.34 for ANOVA) reduction of 43% and 24%, respectively, in the mean amount of saquinavir transferred from the fetal to the maternal side. MK-571 also somewhat (by 31%) reduced the TPT(AUC)% of antipyrine, but this finding did not reach statistical significance (P = 0.25). Neither of the employed inhibitors had an effect on the placental transfer index of saquinavir transfer (P = 0.77). The present results indicated lack of significant effect by MK-571 and probenecid on the fetal-to-maternal transfer of saquinavir and suggest that MRP1 and, possibly, OATP2B1 do not play a significant role in the fetal-to-maternal transfer of saquinavir.

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

Placental transfer of quetiapine in relation to P-glycoprotein activity.

Atypical antipsychotic drugs are well tolerated and thus often preferred in women of fertile age; yet the information on their placental transfer and use during the prenatal period is limited. The aim of this study was to study the placental transfer of quetiapine, a widely used atypical antipsychotic, with special reference to the role of the placental transporter protein, P-glycoprotein (P-gp). This was performed in 18 dually perfused placentas, using the well established P-gp inhibitors PSC833 (valspodar) and GG918 to inhibit the function of P-gp. We also aimed to clarify the significance of two potentially functional ABCB1 single nuclear polymorphisms (SNPs), 2677G>T/A and 3435C>T, on the transplacental transfer (TPT) of quetiapine. The placental transfer of quetiapine in the control group as measured by TPT(AUC) % (absolute fraction of the dose crossing placenta) was 3.7%, which is 29% less than the transfer of the freely diffusible antipyrine, which was 5.2%. The P-gp inhibitors had no significant effect on the transfer of quetiapine as measured by TPT(AUC) % (P = 0.77). No correlation was found between the transplacental transfer of quetiapine (TPT(AUC) %) and placental P-gp expression (P = 0.61). The 3435T allele in exon 26 was associated with significantly higher placental transfer of quetiapine (P = 0.04). We conclude that quetiapine passes the human placenta but that the blood-placental barrier partially limits the transplacental transfer of quetiapine. Administration of P-gp inhibiting drugs with quetiapine is not likely to increase fetal exposure to quetiapine, although the ABCB1 C3435T polymorphism may contribute to inter-individual variation in fetal exposure to quetiapine.

Effects of low and high plasma concentrations of dexmedetomidine on myocardial perfusion and cardiac function in healthy male subjects.

BACKGROUND: Dexmedetomidine, a selective alpha2-adrenoceptor agonist, has counteracting effects on the cardiovascular system. It mediates sympatholysis by activating alpha2 adrenoceptors in the central and peripheral nervous system, and vasoconstriction and vasorelaxation by activating postsynaptic alpha2 adrenoceptors in blood vessels. The goal of this study was to determine the effects of therapeutic and high concentrations of dexmedetomidine on myocardial perfusion and cardiac function in healthy subjects. METHODS: The authors studied 12 healthy young men. Myocardial blood flow (assessed with positron emission tomography), myocardial function (by echocardiography), and hemodynamic data were collected before and during low (measured mean plasma concentration, 0.5 ng/ml) and high (5 ng/ml) plasma concentrations of dexmedetomidine. RESULTS: The low concentration of dexmedetomidine reduced myocardial perfusion (mean difference, -27% from baseline [95% confidence interval, -31 to -23%], P < 0.001) in parallel with a reduction in myocardial oxygen demand (estimated by the rate-pressure product (-23% [-28 to -18%], P < 0.001). The high dexmedetomidine plasma concentration did not further attenuate myocardial perfusion (-3% [-12 to +6%] from low dexmedetomidine, P > 0.05; -29% [-39 to -18%] from baseline, P < 0.001) or statistically significantly affect the rate-pressure product (+5% [0 to +10%], P > 0.05). Systolic myocardial function was attenuated by sympatholysis during the low infusion rate and was further attenuated by a combination of the sustained sympatholysis and increased afterload during the high infusion rate. CONCLUSIONS: In healthy subjects, plasma concentrations of dexmedetomidine that significantly exceed the recommended therapeutic level do not seriously attenuate myocardial perfusion below the level that is observed with usual therapeutic concentrations and do not induce evident myocardial ischemia.

Determination of plant and enterolignans in human serum by high-performance liquid chromatography with tandem mass spectrometric detection.

An HPLC-MS/MS method was validated for the determination of the plant lignans 7-hydroxymatairesinol (HMR), matairesinol (Mat), secoisolariciresinol (Seco), lariciresinol (Lar), and cyclolariciresinol (CLar) and for the enterolignans 7-hydroxyenterolactone (HEL), enterodiol (ED), and enterolactone (EL) in human serum. The method included sample enzymatic hydrolysis, solid-phase extraction, and lignan analysis using a triple quadrupole mass spectrometer with electrospray ionisation in the multiple-reaction monitoring mode. The serum lignans were quantified using deuterated Mat or EL as internal standards. The method met the validation criteria for selectivity, intra- and inter-assay precision, and accuracy. The method was applied to ten serum samples collected from healthy individuals (five men and five women) consuming their habitual Finnish diet. All lignans except HMR and Seco were found in quantifiable amounts in the samples. All serums contained EL; the average concentration was 34 nM. In three individuals, the serum concentration of plant lignans was higher than that of enterolignans. Using the method, common dietary plant lignans and their major metabolites can be reliably quantified in human serum at low-nanomolar concentrations in a simple and rapid way.

Yersiniophage phiR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine.

Bacteriophage piR1-37 was isolated based on its ability to infect strain YeO3-R1, a virulence-plasmid-cured O antigen-negative derivative of Yersinia enterocolitica serotype O : 3. In this study, the phage receptor was found to be a structure in the outer core hexasaccharide of Y. enterocolitica O : 3 LPS. The phage receptor was present in the outer core of strains of many other Y. enterocolitica serotypes, but also in some Yersinia intermedia strains. Surprisingly, the receptor structure resided in the O antigen of Yersinia pseudotuberculosis O : 9. Electron microscopy demonstrated that phiR1-37 particles have an icosahedral head of 88 nm, a short neck of 10 nm, a long contractile tail of 236 nm, and tail fibres of at least 86 nm. This implies that the phage belongs to the order Caudovirales and the family Myoviridae in the ICTV (International Committee for Taxonomy of Viruses) classification. phiR1-37 was found to have a lytic life cycle, with eclipse and latent periods of 40 and 50 min, respectively, and a burst size of approximately 80 p.f.u. per infected cell. Restriction digestions and PFGE showed that the phiR1-37 genome was dsDNA and approximately 270 kb in size. Enzymically hydrolysed DNA was subjected to HPLC-MS/MS analysis, which demonstrated that the phiR1-37 genome is composed of DNA in which thymidine (T) is >99 % replaced by deoxyuridine (dU). The only organisms known to have similar DNA are the Bacillus subtilis-specific bacteriophages PBS1 and PBS2. N-terminal amino acid sequences of four major structural proteins did not show any similarity to (viral) protein sequences in databases, indicating that close relatives of phiR1-37 have not yet been characterized. Genes for two of the structural proteins, p24 and p46, were identified from the partially sequenced phiR1-37 genome.

Transfer of proinflammatory cytokines across term placenta.

OBJECTIVE: Increased concentrations of proinflammatory cytokines in amniotic fluid indicate the presence of intra-amniotic inflammation and increase the risk of preterm birth, cerebral palsy, and bronchopulmonary dysplasia. The purpose of this study was to find out if the proinflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, transfer across the placenta, and thereby determine whether intra-amniotic inflammatory response, measured from the amniotic fluid, is of maternal or fetal origin. METHODS: Nineteen placentas from healthy women undergoing elective cesarean delivery at term with intact membranes and without labor, were dually perfused ex vivo in an open circulation system for either 30 minutes or 2 hours. Tumor necrosis factor-alpha, IL-1beta, and IL-6 were added to maternal or fetal circulation in a concentration usually found in chorioamnionitis. As a reference, placentas without added cytokine were also perfused. The concentrations of cytokines were determined by enzyme immunoassays (enzyme-linked immunosorbent assay [ELISA]). RESULTS: After the addition of the cytokine to the arterial perfusate, the venous concentration on the same side of the placenta increased rapidly and reached a plateau at 10 minutes. No transfer of any cytokine in either direction was detected. Some endogenous release of IL-6 was observed in response to the perfusion. CONCLUSION: Proinflammatory cytokines do not cross normal term placenta.

Functional role of P-glycoprotein in the human blood-placental barrier.

OBJECTIVE: In vitro and animal experiments suggest that P-glycoprotein forms a functional barrier between maternal and fetal blood circulation in the placenta, thus protecting the fetus from exposure to xenobiotics during pregnancy. In this study we aimed to characterize the role of P-glycoprotein in the blood-placental barrier by use of dually perfused human placenta. METHODS: Twenty-eight human placentas were obtained after delivery, and both the maternal side and the fetal side were perfused for 2 hours. Saquinavir was used as a probe drug for P-glycoprotein-dependent active transfer, and PSC833 (valspodar) or GG918 was used as an inhibitor of P-glycoprotein function in a maternal-to-fetal and fetal-to-maternal perfusion setting. Genotyping for ABCB1 (C3435T and G2677A/T) polymorphism and quantification of P-glycoprotein expression were done for each placenta. RESULTS: The fetal-to-maternal transfer of saquinavir was 108-fold higher (P = .003) compared with transfer from the maternal to the fetal direction. Preperfusion with PSC833 increased the placental transfer of saquinavir by 7.9-fold (P < .001), and preperfusion with GG918 increased it by 6.2-fold (P < .001). The end-perfusion transfer (percentage) of saquinavir at 120 minutes was 11-fold (P < .001) and 6-fold (P < .001) higher in placentas preperfused with PSC833 and GG918, respectively, compared with control. However, PSC833 had no effect on the transfer of saquinavir from the fetal to the maternal direction (P = .79). P-glycoprotein expression was correlated with the PSC833-induced change in the saquinavir transfer (r = 0.75, P = .086). ABCB1 polymorphism did not affect the PSC833- or GG918-induced change in the saquinavir transfer. CONCLUSIONS: P-glycoprotein has a major functional role in the human blood-placental barrier but a negligible role in the removal of substances from the fetal circulation to maternal blood. Pharmacologic blockade of P-glycoprotein function can lead to disruption of the blood-placental barrier and increase the transfer of P-glycoprotein substrates to the fetal side by several-fold, which may be a noteworthy mechanism for teratogenicity.

The involvement of alpha 2A-adrenoceptors in morphine analgesia, tolerance and withdrawal in mice.

Alpha(2)-adrenoceptor agonists potentiate opioid analgesia and alleviate opioid withdrawal. The effects of two alpha(2)-adrenoceptor agonists, clonidine (2 mg/kg) and dexmedetomidine (20 and 100 microg/kg), and the alpha(1)-adrenoceptor antagonist prazosin (0.5 mg/kg) were tested on morphine analgesia, tolerance, and withdrawal in wild-type and alpha(2A)-adrenoceptor knock-out (KO) mice. Analgesia and tolerance were assessed with the tail-flick test. Withdrawal was precipitated with naloxone. Prazosin potentiated morphine analgesia equally in both genotypes. Clonidine and dexmedetomidine had no analgesic effects in alpha(2A)-adrenoceptor KO mice, but morphine analgesia and tolerance were similar in both genotypes. Alpha(2A)-Adrenoceptor KO mice exhibited 70% fewer naloxone-precipitated jumps than wild-type mice; weight loss was similar in both genotypes. The alpha(2)-adrenoceptor agonists reduced opioid withdrawal signs only in wild-type mice. We conclude that alpha(2A)-adrenoceptors are not directly involved in morphine analgesia and tolerance, and not critical for potentiation of morphine analgesia by prazosin, but that alpha(2A)-adrenoceptors modulate the expression of opioid withdrawal signs in mice.

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 2.5 mM) was allowed to react with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC and HPLC-MS technique. Although the predominant reaction observed was chlorambucil hydrolysis, 1 reacted with various heteroatoms of the nucleosides. The principal site of alkylation with all pyrimidine nucleosides was N3, as judged by 1H NMR and HPLC-MS analyses. Also, several other adducts were detected, which could be tentatively characterized by means of HPLC-MS and MS/MS. As expected, thymidine was the least reactive pyrimidine nucleoside studied, and in addition of the N3 derivative, it reacted only at the carbohydrate moiety. Overall reactivity of cytosine nucleosides with 1 was considerably higher. The N3 adducts of dCyd and 5-Me-dCyd partially deaminated under the reaction conditions employed, but the reaction was not catalyzed by the participation of the omega-hydroxy function of the alkyl substituent but presumably by the nitrogen atom of the chlorambucil moiety. In the case of cytosine nucleosides, the O2 derivatives were the second most abundant species. 5-Me-dCyd reacted more readily at O2 than dCyd. These O2 adducts were labile under acidic, neutral, and basic conditions. No N4 derivatives or cross-links were detected, but dCyd reacted also at C5, although the yield of this derivative was very low. The role of chlorambucil-pyrimidine 2'-deoxyribonucleoside adducts on the cytotoxicity and mutagenity of 1 is also discussed.

Liquid chromatographic-tandem mass spectrometric method for the plant lignan 7-hydroxymatairesinol and its potential metabolites in human plasma.

A HPLC-MS-MS method was developed for the determination of the plant lignan 7-hydroxymatairesinol and its potential metabolites matairesinol, oxomatairesinol, alpha-conidendrin, 7-hydroxyenterolactone, enterodiol, and enterolactone in human plasma. The method included sample cleanup by solid-phase extraction (SPE) and analysis using a PE Sciex API3000 triple quadrupole mass spectrometer with electrospray ionisation. The lignans were quantified using two deuterated internal standards. They showed good chromatographic linearity, analysis repeatability, and SPE recovery in the presence of plasma. In pooled plasma and in plasma samples collected from two individual subjects lignan glucuronides and sulfates were enzymatically hydrolysed to free lignans and then analysed. All the lignans could be detected in the samples.

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyadenosine.

N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 0.6 mM) was allowed to react with 2'-deoxyadenosine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC-MS and HPLC-MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 7% of 1 reacted with various heteroatoms of the nucleoside. The principal site of alkylation was N1. Several other adducts were also detected. The N1, N6, N3, and N7 derivatives were characterized by means of MS/MS, UV, and (1)H NMR. The N6 adduct is derived directly from alkylation of N6 of 2'-dAdo. Dimroth rearrangement of the N1 adduct to the N6 adduct was very slow under the reaction conditions employed. Minor adducts such as a carbohydrate derivative were tentatively characterized by MS/MS. No cross-links were detected. The role of chlorambucil-2'-deoxyadenosine adducts in the cytotoxicity and mutagenicity of 1 is also discussed.

Structural determinants of plant lignans for the formation of enterolactone in vivo.

The quantity of mammalian lignans enterolactone (ENL) and enterodiol (END) and of plant lignans secoisolariciresinol (SECO) and 7-hydroxymatairesinol (HMR) excreted in a 24-h rat urine sample was measured after a single p.o. dose of an equivalent quantity of secoisolariciresinol diglycoside (SDG), secoisolariciresinol (SECO), matairesinol (MR), 7-hydroxymatairesinol (HMR) and ENL. Plant lignans (SECO and HMR) were partially absorbed as such. The aglycone form of SECO was more efficiently converted into mammalian lignans END and ENL than the glycosylated form, SDG. Of plant lignans, MR produced the highest quantities of ENL: the quantity was over twofold compared with HMR or SDG. The majority of the animals, which had been given SECO, excreted higher quantities of END than ENL into urine, but ENL was the main lignan metabolite after SDG. The highest quantities of ENL in urine were measured after the administration of ENL as such. The (-)SECO isolated from Araucaria angustifolia was converted into (-)ENL only. The administration of (-)SDG, which was shown to produce (+)SECO, resulted in excretion of (+)ENL only and (-)HMR was converted into (-)ENL only. This confirmed that the absolute configurations at C8 and C8' are not changed during the microbial metabolism. Whether the biological effects are enantiomer-specific, remains to be resolved.