James W. Paxton

Drug transfer and metabolism by the human placenta

The major function of the placenta is to transfer nutrients and oxygen from the mother to the foetus and to assist in the removal of waste products from the foetus to the mother. In addition, it plays an important role in the synthesis of hormones, peptides and steroids that are vital for a successful pregnancy. The placenta provides a link between the circulations of two distinct individuals but also acts as a barrier to protect the foetus from xenobiotics in the maternal blood.However, the impression that the placenta forms an impenetrable obstacle against most drugs is now widely regarded as false. It has been shown that that nearly all drugs that are administered during pregnancy will enter, to some degree, the circulation of the foetus via passive diffusion. In addition, some drugs are pumped across the placenta by various active transporters located on both the fetal and maternal side of the trophoblast layer. It is only in recent years that the impact of active transporters such as P-glycoprotein on the disposition of drugs has been demonstrated. Facilitated diffusion appears to be a minor transfer mechanism for some drugs, and pinocytosis and phagocytosis are considered too slow to have any significant effect on fetal drug concentrations.The extent to which drugs cross the placenta is also modulated by the actions of placental phase I and II drug-metabolising enzymes, which are present at levels that fluctuate throughout gestation. Cytochrome P450 (CYP) enzymes in particular have been well characterised in the placenta at the level of mRNA, protein, and enzyme activity. CYP1A1, 2E1, 3A4, 3A5, 3A7 and 4B1 have been detected in the term placenta. While much less is known about phase II enzymes in the placenta, some enzymes, in particular uridine diphosphate glucuronosyltransferases, have been detected and shown to have specific activity towards marker substrates, suggesting a significant role of this enzyme in placental drug detoxification.The increasing experimental data on placental drug transfer has enabled clinicians to make better informed decisions about which drugs significantly cross the placenta and develop dosage regimens that minimise fetal exposure to potentially toxic concentrations. Indeed, the foetus has now become the object of intended drug treatment. Extensive research on the placental transfer of drugs such as digoxin and zidovudine has assisted with the safe treatment of the foetus with these drugs in utero. Improved knowledge regarding transplacental drug transfer and metabolism will result in further expansion of pharmacological treatment of fetal conditions.

Interactions of Herbs with Cytochrome P450

A resurgence in the use of medical herbs in the Western world, and the co-use of modern and traditional therapies is becoming more common. Thus there is the potential for both pharmacokinetic and pharmacodynamic herb–drug interactions. For example, systems such as the cytochrome P450 (CYP) may be particularly vulnerable to modulation by the multiple active constituents of herbs, as it is well known that the CYPs are subject to induction and inhibition by exposure to a wide variety of xenobiotics. Using in vitro, in silico, and in vivo approaches, many herbs and natural compounds isolated from herbs have been identified as substrates, inhibitors, and/or inducers of various CYP enzymes. For example, St. Johns wort is a potent inducer of CYP3A4, which is mediated by activating the orphan pregnane X receptor. It also contains ingredients that inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Many other common medicinal herbs also exhibited inducing or inhibiting effects on the CYP system, with the latter being competitive, noncompetitive, or mechanism-based. It appears that the regulation of CYPs by herbal products is complex, depending on the herb type, their administration dose and route, the target organ and species. Due to the difficulties in identifying the active constituents responsible for the modulation of CYP enzymes, prediction of herb–drug metabolic interactions is difficult. However, herb–CYP interactions may have important clinical and toxicological consequences. For example, induction of CYP3A4 by St. Johns wort may partly provide an explanation for the enhanced plasma clearance of a number of drugs, such as cyclosporine and innadivir, which are known substrates of CYP3A4, although other mechanisms including modulation of gastric absorption and drug transporters cannot be ruled out. In contrast, many organosulfur compounds, such as diallyl sulfide from garlic, are potent inhibitors of CYP2E1; this may provide an explanation for garlics chemoproventive effects, as many mutagens require activation by CYP2E1. Therefore, known or potential herb–CYP interactions exist, and further studies on their clinical and toxicological roles are warranted. Given that increasing numbers of people are exposed to a number of herbal preparations that contain many constituents with potential of CYP modulation, high-throughput screening assays should be developed to explore herb–CYP interactions.

Independent regulation of apical and basolateral drug transporter expression and function in placental trophoblasts by cytokines, steroids, and growth factors.

Placental ATP binding cassette (ABC) transporters protect placental and fetal tissues by effluxing xenobiotics and endogenous metabolites. We have investigated the effects of cytokines and survival/growth factors, implicated in various placental pathologies, on ABC transporter expression and function in primary placental trophoblast cells. Treatment of primary term trophoblasts in vitro with tumor necrosis factor-α (TNF-α) or interleukin (IL)-1β decreased mRNA and protein expression of apical transporters ABCB1/multidrug resistance gene product 1 (MDR1) and ABCG2/breast cancer resistance protein (BCRP) protein by 40 to 50% (P < 0.05). In contrast, IL-6 increased mRNA and protein expression of the basolateral transporter ABCB4/MDR3 (P < 0.05), whereas ABCC1/MRP1 expression was unaltered. Pretreatment of trophoblasts with TNF-α over 48 h resulted in significantly decreased BCRP efflux activity (increased mitoxantrone accumulation) with minimal changes in MDR1/3 activity. Epidermal growth factor (EGF) and insulin-like growth factor II, on the other hand, significantly increased BCRP expression at the mRNA and protein level (P < 0.05); EGF treatment also increased BCRP functional activity. Estradiol stimulated BCRP, MDR1, and MDR3 mRNA and protein expression by 40 to 60% and increased MDR1/3 functional activity (P < 0.05). Progesterone had modest positive effects on MRP1 mRNA and MDR1 protein expression (P < 0.05). In conclusion, this study shows that proinflammatory cytokines, sex steroids, and growth factors exert independent effects on expression of apical and basolateral placental ABC transporters in primary trophoblast. Such changes could alter placental drug disposition, increase fetal susceptibility to toxic xenobiotics, and impact on placental viability and function.

Improving the oral bioavailability of beneficial polyphenols through designed synergies.

A substantial and growing consumer demand exists for plant-based functional foods that improve general health and wellbeing. Amongst consumed phytochemicals, the polyphenolic compounds tend to be the most bioactive. Many commonly consumed polyphenols have been shown to have specific and potent health-promoting activities when assessed by high-throughput in vitro assays and when administered to experimental animals by injection. However, very few have been shown to have any beneficial effects in animals or man when orally consumed, because of the poor bioavailability exhibited by most polyphenols following the ingestion. Consumed polyphenols, like most pharmaceuticals, are regarded as xenobiotics by the body and must overcome many barriers, including extensive enzymatic and chemical modification during digestion and absorption, to reach their site(s) of action. This is especially true for polyphenols targeting the brain, which is protected by the tightly regulated blood–brain barrier. Interestingly, many polyphenols are also known to specifically modify some of the metabolic and transport processes that govern bioavailability. Therefore, the opportunity exists to increase the bioactivity of beneficial polyphenols by designing specific synergistic interactions with polyphenols that improve their oral bioavailability. This hypothesis and review paper will discuss some of the endogenous systems that limit the bioavailability of ingested polyphenols to the body and the brain, and the means by which bioavailability may be improved by specifically designing synergies between orally consumed polyphenols.

UDP-glucuronosyltransferase activity, expression and cellular localization in human placenta at term

The activity, expression and localization of the UDP-glucuronosyltransferases (UGTs) were investigated in human placenta at term. UGT activity (measured with the substrate 4-methylumbelliferone (4-MU)) was observed in all 25 placentas sampled and maximum velocity (V(max)) ranged 13-fold from 5.1+/-0.9 to 66.9+/-17.5 nmol/min/mg protein (mean+/-SD). Substrate affinity (K(m)) ranged 5-fold from 246+/-24 to 1124+/-422 microM. Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of the isoforms UGT2B4, 2B7, 2B10, 2B11 and 2B15 was observed in all (12/12) placentas sampled and expression of UGT2B17 was noted in 8/12 placentas. Northern analysis of the UGT2B7 isoform in 12 placentas revealed a 10-fold difference in expression with RT-PCR variability and the 13-fold variation observed in UGT activity. The presence of UGT2B4 and 2B7 proteins (52 and 56kDa, respectively) was demonstrated by Western blotting. The sites of placental UGT2B transcription (in situ hybridization) and protein expression (immunohistochemistry) were located in the syncytium of the placental trophoblasts bordering the placental villi. UGT1A proteins could not be observed with immunohistochemistry or Western blotting and expression could not be observed with RT-PCR. Our discovery of UGT expression and activity at the site of maternal-fetal exchange is consistent with a role for UGTs in detoxification of exogenous and endogenous ligands and the maintenance of placental function through clearance and regulation of steroid hormones.

Formulation and pharmacokinetic evaluation of an asulacrine nanocrystalline suspension for intravenous delivery.

Asulacrine (ASL) is an inhibitor of topoisomerase II, which has shown potential against breast and lung cancer. It is a poorly water soluble drug. To allow intravenous (i.v.) administration, ASL was formulated as a nanocrystalline suspension by high pressure homogenization. The nanosuspension was lyophilized to obtain the dry ASL nanoparticles (average size, 133+/-20nm), which enhanced both the physical and chemical stability of the ASL nanoparticles. ASL dissolution and saturation solubility were enhanced by the nanosuspension. Differential scanning calorimetry and X-ray diffraction analysis showed that the crystallinity of the ASL was preserved during the high pressure homogenization process. The pharmacokinetics and tissue distribution of ASL administered either as a nanosuspension or as a solution were compared after i.v. administration to mice. In plasma, ASL nanosuspension exhibited a significantly (P<0.01) reduced C(max) (12.2+/-1.3microg ml(-1)vs 18.3+/-1.0microg ml(-1)) and AUC(0-infinity) (18.7+/-0.5microg ml(-1)h vs 46.4+/-2.6microg ml(-1)h), and a significantly (P<0.01) greater volume of distribution (15.5+/-0.6lkg(-1)vs 2.5+/-0.1lkg(-1)), clearance (1.6+/-0.04lh(-1)kg(-1)vs 0.6+/-0.04lh(-1)kg(-1)) and elimination half-life (6.1+/-0.1h vs 2.7+/-0.2h) compared to the ASL solution. In contrast, the ASL nanosuspension resulted in a significantly greater AUC(0-infinity) in liver, lung and kidney (all P<0.01), but not in heart.

The ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction

The efflux pump ATP binding cassette superfamily member G2 (ABCG2)/breast cancer resistance protein (BCRP) is highly expressed in human placenta. We have investigated the role of BCRP in the protection of the human placental trophoblasts from apoptosis and its expression in idiopathic fetal growth restriction, a condition associated with abnormal pla‐cental apoptosis. Inhibition of BCRP activity with the selective inhibitor Ko143 augmented cytokine (tumor necrosis factor‐α/interferon‐γ)‐induced apoptosis and phosphatidylserine externalization in primary tropho‐blast and trophoblast‐like BeWo cells. Silencing of BCRP expression in BeWo cells significantly increased their sensitivity to apoptotic injury in response to cytokines and exogenous C6 and C8 ceramides. BCRP silencing also increased intracellular ceramide levels after cytokine exposure but did not affect cellular protoporphyrin IX concentrations or sensitivity to activators of the intrinsic apoptotic pathway. BCRP expression in placentas from pregnancies complicated by idiopathic fetal growth restriction was decreased compared with controls, suggesting reduced transport of its substrates from the placenta. We conclude that BCRP may play a hitherto unrecognized survival role in the placenta, protecting the trophoblast against cytokine‐induced apoptosis and possibly other extrinsic activators via modulation of ceramide signaling. Decreased placental BCRP expression may result in reduced viability and hence functional deficit, contributing to the fetal growth restriction phenotype.—Evseenko, D. A., Murthi, P., Paxton, J. W., Reid, G., Emerald, B. S., Mohankumar, K. M., Lobie, P. E., Brennecke, S. P., Kalionis, B. Keelan, J. A. The ABC transporter BCRP/ ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction. FASEB J. 21, 3592–3605 (2007)

Effects of Carbamazepine on Psychomotor Performance in Children as a Function of Drug Concentration, Seizure Type, and Time of Medication

Summary: Fifty children with well‐controlled seizures who were receiving carbamazepine (CBZ) monotherapy were tested on a battery of cognitive and motor tests. Time of CBZ was varied so that the children were tested both shortly after CBZ (when daily CBZ concentrations approached their peak) and before CBZ (when CBZ concentrations approached daily low or trough levels). Testing was blind so that the examiner was unaware of the time of CBZ or the childs type of seizure. Results showed only one group difference related to seizure type and no differences linked to overall CBZ concentration as measured in saliva. Peak and trough concentrations were associated with changes in several variables, however. The children performed significantly better shortly after CBZ (peak concentration day) on measures of seat activity, attention span, and motor steadiness, whereas response times appeared to be influenced in a task‐specific manner.

Active transport across the human placenta: impact on drug efficacy and toxicity

The human placenta expresses a large number of transport proteins. The ATP-binding cassette (ABC) family of active efflux pumps, predominantly localised to the maternal-facing syncytial membrane of placental microvilli, comprise the major placental drug efflux transporters. A variety of other transporters are also expressed in the placenta that can facilitate xenobiotic transfer in both the maternal and fetal directions. Many drugs administered in pregnancy are ABC transporter substrates, and many are either teratogenic or fetotoxic. The invitro, invivo and clinical evidence reviewed in this article argues that active efflux of drugs by placental transporters helps to maintain its barrier function, reducing the incidence of adverse fetal effects. ABC transporter polymorphisms may explain the wide variability observed in fetal drug concentrations, incidence of teratogenesis or drug failure in pregnancies exposed to therapeutic agents. Although our understanding of the molecular mechanics and dynamics of placental drug transfer is advancing, much work is needed to fully appreciate the significance of placental drug transporters in the face of increasing drug administration in pregnancy.

Effect of Sodium Valproate on Psychomotor Performance in Children as a Function of Dose, Fluctuations in Concentration, and Diagnosis

Summary: Forty‐six children receiving sodium valproate monotherapy, and with well‐controlled seizures, were tested three times, at weekly intervals in the morning, on an extensive battery of psychomotor tests. The first session was a practice session to minimize subsequent practice effects. On one of the remaining 2 test days, morning medication was delayed until after testing (low concentration condition), whereas sodium valproate was given before testing on the alternate session (high concentration day). The data were analyzed for the effects of diagnosis (partial vs. generalized epilepsy), dose (above or below 20 mg/kg/day), and time of medication (low and high drug concentration days). Four variables discriminated between the diagnostic groups, all favoring children with generalized epilepsy. Another four measures were significantly associated with dose, with those on lower doses performing at a superior level. Time of medication had virtually no effect on performance, however. The findings were related to previous diagnostic and dosage research. Although a deterioration in performance at higher doses appears to be consistent with other dosage research, the absence of an effect due to time of medication suggests that performance does not fluctuate between drug administrations.