Valentina M. Fokina

Expression patterns of Wnt genes during development of an anterior part of the chicken eye

To address the roles of Wnts in the development of the anterior eye, we used a chicken model to perform comprehensive expression analysis of all Wnt genes during anterior eye development. In analyzing the available genomic sequences, we found that the chicken genome encodes 18 Wnt proteins that are homologous to corresponding human and mouse proteins. The mRNA sequences for 12 chicken Wnt genes are available in GenBank, and mRNAs for six other Wnt genes (Wnt2, Wnt5b, Wnt7b, Wnt8b, Wnt9b, and Wnt16) were identified and cloned based on the homology to the genes from other species. In addition, we found that chicken Wnt3a and Wnt7b genes encode two alternative mRNA isoforms containing different first exons. Following in situ hybridization, we found that out of 18 Wnt genes, 11 genes were expressed in the anterior eye, exhibiting distinct temporal‐spatial patterns. Several Wnts were expressed in the lens, including Wnt2 and Wnt2b in the anterior epithelium and Wnt5a, Wnt5b, Wnt7a, and Wnt7b in the differentiating lens fiber cells. In the cornea, we detected Wnt3a, Wnt6, and Wnt9b in the ocular surface ectoderm, including the corneal epithelium, and Wnt9a in the corneal endothelium from the onset of its differentiation. In the optic cup, Wnt2, Wnt2b, and Wnt9a were localized in the rim of the optic cup (presumptive iris), while Wnt5a and Wnt16 were detected in the ciliary epithelium/iris zone of the differentiated optic cup, and Wnt6 was expressed in the iridial mesenchyme. These data suggest that Wnt signaling might play important roles in anterior eye development. Developmental Dynamics 235:496–505, 2006.

Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

Effect of albumin on transplacental transfer and distribution of rosiglitazone and glyburide

Objective. The aims of this investigation were (i) to determine the rate and extent of rosiglitazone transfer across term human placenta, and (ii) to determine the effect of human serum albumin (HSA) on rosiglitazone and glyburide transfer and distribution. Methods. These aims were achieved by utilizing the technique of dual perfusion of placental lobule (DPPL). Each hypoglycemic drug was coperfused with the marker compound antipyrine (AP). In each experiment, the [3H]-isotope of the hypoglycemic drug and the [14C]-isotope of AP were added to enhance the detection limits of each drug. Human serum albumin (HSA) was added to both the maternal and fetal circuits in the experiments in which it was investigated. Results. Transplacental transfer of rosiglitazone and glyburide from the maternal to fetal circuits in media devoid of HSA was similar. However, the addition of HSA to the maternal and fetal circuits had different effects on the transfer and distribution of the two drugs, though their binding to HSA (99.8%) was almost identical. HSA increased the maternal (M) to fetal (F) transfer of rosiglitazone, as revealed by an increase in its F/M concentration ratio from 0.17 ± 0.01 (in the absence of albumin) to 0.33 ± 0.07 (p < 0.001). Moreover, the addition of albumin decreased the amount of rosiglitazone retained by placental tissue from 539 ± 148 to 60 ± 8 ng/g (p < 0.001). Conversely, the addition of HSA to the perfusion media resulted in a decrease in glyburide transfer, as revealed by the change of its F/M concentration ratio from 0.09 ± 0.02 (in the absence of albumin) to 0.03 ± 0.01 (p < 0.01). However, similar to rosiglitazone, glyburide retention by the tissue decreased from 103 ± 26 to 19 ± 6 ng/g (p < 0.001). Conclusions. These data indicate that the binding of the two drugs to albumin, though similar, is only one of the factors that could affect their placental transfer and distribution.

Transplacental transfer and metabolism of buprenorphine in preterm human placenta.

We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine.

Transplacental transfer of vancomycin and telavancin

OBJECTIVE We sought to determine the bidirectional transfer and distribution of vancomycin and telavancin across the dually perfused term human placental lobule. STUDY DESIGN The technique of dually perfused placental lobule was used in its recirculating mode to determine the maternal to fetal (M→F) (n = 20) and fetal to maternal (n = 18) transfer of each antibiotic, which were coperfused with their radioactive isotopes. The concentrations of drugs were determined by liquid scintillation spectrometry. RESULTS In the M→F direction, the transfer of vancomycin (9.6 ± 4%) and telavancin (6.5 ± 2%) were low; however, telavancin retention by the perfused lobule was greater than that of vancomycin (P < .01). The normalized transplacental transfer of telavancin across the placental lobule in the fetal to maternal direction was higher than in the M→F direction (P < .01), suggesting the involvement of placental efflux transporters. CONCLUSION The ex vivo perfusion experiments revealed low transfer of vancomycin and telavancin to the fetal circuit.

Metabolism of bupropion by baboon hepatic and placental microsomes

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36±6 μM, Vmax 258±32 pmol mg protein(-1) min(-1)), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.

Metabolism of 17-Alpha-Hydroxyprogesterone Caproate by Human Placental Mitochondria

Perfusion of 17-alpha-hydroxyprogesterone caproate (17HPC) via the maternal circuit of a dually perfused human placental lobule resulted in the extensive formation of 2 metabolites. On the other hand, human placental microsomes biotransformed 17HPC into 5 monohydroxylated metabolites, which did not correspond to those formed during perfusion. The goal of this investigation was to determine the subcellular localization of the enzymes responsible for the biotransformation of 17HPC during its perfusion in human placenta. Crude subcellular fractions of the human placental tissue were utilized. Six 17HPC metabolites were formed by the placental mitochondrial fraction, of which 4 were identical to those formed by the microsomes; whereas the other 2, namely MM and M19, were formed by the mitochondrial fraction only. The latter metabolites were identical to those formed during 17HPC perfusion, as determined by liquid chromatography–mass spectrometry (LC-MS) analysis. Therefore, these data strongly suggest that the enzymes responsible for the biotransformation of 17HPC during its perfusion are predominantly localized in human placental mitochondria.

Human adipose-derived mesenchymal stromal cell pigment epithelium-derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells.

BACKGROUND AIMS Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. METHODS ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium-derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture-conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer-focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. RESULTS ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. CONCLUSIONS These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects.