Inhibition of proliferation of HT-29 colon adenocarcinoma cells by carboxylate NSAIDs and their acyl glucuronides.

Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1-3000 microM with HT-29 cells for 24 hr, with [3H]-thymidine added for an additional 2 hr incubation. IC50 values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC50 values about 200-500 microM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present.

Apparent autoinduction of valproate beta-oxidation in humans.

AIMS: The study aimed to show whether autoinduction of valproate (VPA) along its beta-oxidation pathway occurred upon chronic dosing in humans. METHODS: Twelve young volunteers without active illness took sodium valproate (NaVPA) 200 mg orally 12 hourly for 3 weeks. On days 7 and 21, serial blood samples and all urine passed over an interdosing interval from 08.00 to 20.00 h were collected for analysis of VPA and certain metabolites. RESULTS: Plasma AUC(0,12 h) of VPA was significantly lower on day 21 than on day 7 (2.40 vs 2.84 micromol ml-1 h, 95% CI for the difference 0.13-0.81 micromol ml-1 h). Significant differences in plasma AUC(0,12 h) of the beta-oxidation metabolites E-2-en-VPA and 3-oxo-VPA were not found. However, formation clearances of plasma VPA to urinary E-2-en-VPA and 3-oxo-VPA were significantly increased from day 7 to day 21 (0. 010 vs 0.024 and 2.57 vs 3.60 ml kg-1 h-1, respectively, 95% CI for the differences -0.025 to -0.004 and -1.72 to -0.34 ml kg-1 h-1, respectively). Formation clearances to VPA-glucuronide (0.534 vs 0. 505 ml kg-1 h-1) and 4-OH-VPA (0.112 vs 0.110 ml kg-1 h-1) were not significantly different. CONCLUSIONS: Regular low dose VPA intake in humans over a period of 3 weeks appears to be associated with a small induction of its metabolism by the beta-oxidation pathway, but not by glucuronidation or 4-hydroxylation.

Methimazole and propylthiouracil equally cross the perfused human term placental lobule.

Propylthiouracil (PTU) is widely believed to cross the placenta less freely than methimazole (MMI) and is therefore regarded as the preferred drug for treatment of hyperthyroidism in pregnancy. Clinical studies comparing the two drugs show, however, no differences in maternal or fetal thyroid function. We investigated transfer from the maternal to the fetal circuit in the isolated perfused term human placental lobule of low and high doses of PTU (4 micrograms/mL and 40 micrograms/mL) and MMI (1.5 micrograms/mL and 15 micrograms/mL) in protein-free perfusate and low doses of both drugs with addition of 40 g/L of bovine albumin. Both drugs readily crossed the placenta, reaching equilibrium in all experiments in about 2 h. Drug concentrations in the two circuits fitted a two compartmental model. Transfer kinetics for the two drugs were similar, nonsaturable, and unaffected by addition of albumin. Clearances (mL.min-1.g-1, means +/- SD) of PTU from maternal to fetal circuits were: 0.229 +/- 0.110, 0.216 +/- 0.065, and 0.170 +/- 0.032; and for transfer of MMI: 0.165 +/- 0.025, 0.232 +/- 0.153, and 0.174 +/- 0.009 (for low doses without, low doses with, and high doses without albumin, respectively). Clearances of PTU from fetal to maternal circuits were: 0.147 +/- 0.072, 0.109 +/- 0.014, and 0.116 +/- 0.028; and for transfer of MMI: 0.095 +/- 0.029, 0.122 +/- 0.088, and 0.12 +/- 0.005 (in the same experiments). There was no significant difference between drugs or drug doses and no effect of addition of albumin. We conclude that PTU and MMI have similar placental transfer kinetics.

Cortisol metabolism and its inhibition by glycyrrhetinic acid in the isolated perfused human placental lobule.

We have previously reported the placental metabolism of prednisolone to prednisone, 20alpha- and beta-dihydroprednisone and 20beta-dihydroprednisolone. In this study, the disposition of cortisol was investigated in vitro in the dual perfused, isolated human placental lobule after the addition of cortisol (1.2 micromol, n = 3 and 12 micromol, n = 4) to the maternal compartment. Analysis of 5 h maternal and fetal perfusate samples by high performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) revealed that cortisol was mainly metabolized to cortisone, but a significant production of 20alpha-dihydrocortisone, 20beta-dihydrocortisone, 20alpha-dihydrocortisol and 20beta-dihydrocortisol was also detected. Saturability of metabolism but not transfer was demonstrated. Metabolism was eliminated by co-perfusion with the potent 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzyme inhibitor 18beta-glycyrrhetinic acid (GA). The disposition of GA was analysed using HPLC-atmospheric pressure chemical ionisation-MS/MS (HPLC-APCI-MS/MS). GA was found to transfer from the maternal to the fetal circulations without detectable metabolism during 6 h of perfusion.

A high-performance liquid chromatography-electrospray-tandem mass spectrometry analysis of cortisol and metabolites in placental perfusate.

A reversed-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry assay (HPLC-ESI-MS/MS) was developed to quantitate cortisol, cortisone, 20 alpha- and beta-dihydrocortisol, 20 alpha- and beta-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyze perfusate from the isolated human placental lobule for cortisol and its metabolites. Analytes were prepared from the perfusion medium using C18 solid-phase extraction cartridges. The internal standard for the analyses was 6 alpha-methylprednisolone. Chromatography was performed on a Novapak C18 column at ambient temperature using 53% methanol and 47% 10 mM ammonium formate buffer (pH 4.0) as mobile phase, at a flow rate of 80 microL/min. A PE-SCIEX API III triple quadrupole instrument was used for mass spectrometric detection. An ionspray (pneumatically assisted electrospray) interface was used in negative and positive ionization mode. The assay was linear over the range 100-2000 micrograms/L for each analyte. The instrumental limit of detection was 50 pg. Assay imprecision at 400 and 800 micrograms/L was < or = 10% (total coefficient of variation). Accuracy ranged between 83.2% for 20 beta-dihydrocortisone to 102.6% for cortisone. Recovery of 1000 micrograms/L analyte ranged from 91.3% for cortisone to 109.7% for tetrahydrocortisol.

Maternal to fetal thyroxine transmission in the human term placenta is limited by inner ring deiodination.

Placental deiodination of T4 to rT3 has been proposed as the factor controlling materno-fetal transmission of T4. We investigated T4 transfer in the isolated perfused human placental lobule with and without addition of the deiodinase inhibitor, iopanoic acid. T4 (150 nmol/L) in protein-free medium was added to the maternal circuit. Without iopanoic acid, the appearance of T4 in the fetal circuit was very low, with fetal T4 levels reaching only 4.1 +/- 0.84 pmol/L at 6 h. Levels of rT3 rose progressively in both circuits, reaching 28.8 +/- 5.5 nmol/L in the maternal and 12.4 +/- 3.2 nmol/L in the fetal circuit by 6 h. No T3 could be measured in either circuit. Addition of 0.5 nmol/L iopanoic acid to maternal perfusate, however, resulted in significant reduction in the appearance of rT3 [maternal levels, 0.58 +/- 0.06 nmol/L (2% of control values); fetal levels, 0.33 +/- 0.03 nmol/L (2.7% of control values)] and a major (approximately 2700-fold) increase in T4 appearance in the fetal circuit, with fetal T4 levels reaching 10.1 +/- 3.4 nmol/L at 6 h. These results support the hypothesis that placental inner ring (type III) deiodination is a major factor controlling placental transmission of maternal T4.

High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions.

A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 micrograms ml-1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8 - 56.8 micrograms ml-1, for DOH in the concentration range 0.5 - 30.0 micrograms ml-1 and for MOH in the range 1.4-25.1 micrograms ml-1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb.

Pressor effect of ethanol in the isolated perfused human placental lobule.

Pharmacologically relevant concentrations of ethanol (0.01-0.3 g/%) were perfused via the fetal circulation in isolated human placental lobules. This resulted in a dose-related rise in fetal arterial pressure, which at 0.3 g/% ethanol, was 10.1 +/- 1.1 mmHg above the pre-drug baseline. The pressor responses to ethanol were (i) rapid in onset, reaching a stable plateau within 5-10 min following administration, (ii) readily reversible by perfusion with drug-free media, (iii) non-tachyphylactic and (iv) largely inhibited by the cyclo-oxygenase inhibitor, inhibitor, indomethacin (5 microM). This pressor action of ethanol in the fetal circulation may contribute to the pathogenesis of the fetal alcohol syndrome as well as represent an underlying mechanism of ethanol-induced hypertension.

Pathway and kinetics of prednisolone metabolism in the human placenta.

Prednisolone is metabolized in the perfused human placental lobule to prednisone, 20 alpha-dihydroprednisone, 20 beta-dihydroprednisone and 20 beta-dihydroprednisolone. The pathway of metabolite formation was defined in perfusions of placental lobules using prednisone and 20 beta-dihydroprednisone separately as substrates and with prednisolone co-perfused with glycyrrhetinic acid, a potent inhibitor of the 11-oxidase component of the 11 beta-hydroxysteroid dehydrogenase enzyme system. The pattern of metabolites identified from 6 h samples indicated a reversible formation of prednisone from prednisolone, the production of the 20 alpha- and 20 beta-dihydro metabolites of prednisone from prednisone, the formation of 20 beta-dihydroprednisolone from 20 beta-dihydroprednisone only and no direct formation of 20 beta-dihydroprednisolone from prednisolone. Kinetic analysis at two substrate concentrations confirmed that the formation of three of the four steroid metabolites followed first order kinetics. In perfusions with an initial prednisolone concentration of 1 microgram/ml (n = 4) or 100 ng/ml (n = 3), the rate constants obtained were (mean +/- SD, maternal compartment, h-1): prednisone, 1.97 +/- 0.49 and 2.25 +/- 0.15, P > 0.1; 20 alpha-dihydroprednisone, 0.0006 +/- 0.0004 and 0.0017 +/- 0.0006, P < 0.1; 20 beta-dihydroprednisone, 0.15 +/- 0.022 and 0.15 +/- 0.0077, P > 0.1. In contrast, the rate constant for formation of 20 beta-dihydroprednisolone at an initial prednisolone concentration of 100 ng/ml (0.083 +/- 0.0095 h-1) was significantly (P < 0.01) greater than the corresponding rate constant at the higher initial prednisolone concentration (0.039 +/- 0.015 h-1). A significant increase (P < 0.05) was observed for the formation of 20 beta-dihydroprednisolone at the end of 6 h perfusions at the lower initial substrate concentration (11.2 +/- 1.9%) compared with the 1 microgram/ml concentration (6.0 +/- 2.5%).

The effect of intravenous immunoglobulin on placental transfer of a platelet-specific antibody: anti-P1A1.

The isolated perfused lobule of human placenta was used as an in-vitro model to study the effect of intravenous immunoglobulin (IVGG) on the placental transfer of a human platelet-specific antibody (anti-P1A1). Normal human IgG was shown to transfer from the maternal to the fetal circulation of the placental model after a lag period of 2-3 h. IVGG also transferred across the placenta but only after a longer lag period (3-4 h) than normal human IgG at the same concentration, which suggests that IVGG may contain a factor that inhibits the transfer of its own component IgG. The sensitive Western immunoblotting technique was used to demonstrate progressive transfer of anti-P1A1 antibody to the fetal circulation after a 2-3 h lag period. When IVGG and anti-P1A1 antibody were added simultaneously to the maternal circulation, the transfer of platelet-specific antibody was strongly inhibited by IVGG. The inhibitory effect of IVGG on anti-P1A1 antibody transfer was consistent for three different batches of the same IVGG product (Sandoglobulin). These studies provide the first scientific data to support the use of IVGG to inhibit antiplatelet antibody transfer as part of the antenatal management of neonatal alloimmune thrombocytopenia.

Ketoconazole and plasma and urine steroid levels in Cushing's disease.

1. Plasma and urine steroid and plasma ACTH levels were measured for 2 weeks in eight subjects (six female, two male) with Cushing's disease with each given 200 mg ketoconazole orally four times daily. 2. Treatment was associated with major falls in excretion of free cortisol and the cortisol metabolites tetrahydrocortisol, 5 alpha-tetrahydrocortisol and tetrahydrocortisone. 3. Plasma cortisol and ACTH levels did not change significantly. 4. Excretion of the androgen metabolites, androsterone, 11 beta-hydroxyandrosterone and aetiocholanolone was also reduced but there was no significant fall in plasma testosterone. Plasma levels of progesterone, 17 alpha-hydroxyprogesterone and 11 beta-deoxycortisol and excretion of pregnanediol, pregnanetriol and tetrahydrodeoxycortisol rose with treatment. 5. Analysis of steroid precursor/product ratios indicated that ketoconazole significantly inhibited 11 beta-hydroxylase and 17,20-lyase but not 17 alpha-hydroxylase in these patients with Cushing's disease.

Metabolism of prednisolone by the isolated perfused human placental lobule.

Previous studies of the metabolism of 11 beta-hydroxy corticosteroids by placental tissue have indicated that the only product is the C11-oxidized metabolite. In the present study we have re-examined the metabolism of prednisolone in the isolated, perfused, dual recirculating human placental lobule, using a perfusate based on tissue culture medium 199. Four metabolites were identified in both the maternal and fetal compartments in 6 h perfusions by comparison of relative retention times measured by HPLC and capillary gas chromatography (GC) and of mass spectra recorded by capillary gas chromatography-mass spectrometry (GC-MS) with those of authentic reference standards. The steroids were derivatized as the MO-TMS ethers for mass spectral measurements. Analysis of samples from five perfusion experiments resulted in the following percentage conversions after 6 h perfusion (mean +/- SD, maternal and fetal perfusate, respectively): prednisone (49.1 +/- 7.8, 49.1 +/- 6.6), 20 alpha-dihydroprednisone (0.84 +/- 0.29, 0.81 +/- 0.35), 20 beta-dihydroprednisone (39.1 +/- 6.7, 39.2 +/- 5.9), 20 beta-dihydroprednisolone (6.8 +/- 2.7, 6.3 +/- 1.6) and unmetabolized prednisolone (4.1 +/- 1.8, 4.6 +/- 2.1). No evidence was found for metabolites formed by 6 beta-hydroxylation or cleavage of the C17-C20 bond.

Selective liquid chromatographic assay for propylthiouracil in plasma.

A liquid chromatographic assay was developed to quantitate propylthiouracil in plasma using an internal standard, 5-propyl-2-thiouracil, of similar structure and physical properties. Caffeine, which coelutes with propylthiouracil, was removed by extraction from serum treated with base. No other compounds were found to interfere in the assay. The drug was extracted from plasma with chloroform with a recovery of 59.4% and the intra- and inter-assay coefficients of variation were 5.7 and 3.3%, respectively. The assay was linear to 3 micrograms/ml with a lower detection limit of 40 ng/ml for a sample volume of 1 ml.

Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media.

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20 alpha-dihydro and 20 beta-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6 alpha-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0-1 microgram/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20 alpha-dihydroprednisone) to 8.8% (20 beta-dihydroprednisolone) and 4.1% (20 beta-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20 alpha-dihydroprednisone) to 9.1% (20 beta-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C18 columns which were linked together and maintained at 40 degrees C.

Disposition of phenytoin and phenobarbitone in the isolated perfused human placenta.

1. The disposition of the anti-epileptic agents phenytoin (PHT) and phenobarbitone (PB) was investigated in lobules of term human placentae perfused using separate maternal and fetal circulations for 6 h periods. 2. No evidence for metabolism of PHT or PB to their p-hydroxylated or other derivatives was found either in perfused lobules or by incubation with placental microsomes. 3. Both PHT and PB were readily transferred across the placenta after administration to either the maternal or fetal perfusates. 4. PHT, unlike PB, showed considerable accumulation in placental tissue.