Network-guided modeling allows tumor-type independent prediction of sensitivity to all-trans-retinoic acid.

Background: All-trans-retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia (APL) and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires the development of predictive tools allowing identification of sensitive cancer types and responsive individuals. Materials and methods: RNA-sequencing data for 10 080 patients and 33 different tumor types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was carried out using machine learning methods and network analysis. Results: We profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor types for which higher ATRA-21 predictions are associated with better outcomes. Conclusions: In summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor types that are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

In situ perfusion in the rabbit: effects of different umbilical flow rates on placental transfer of compounds.

The effects of different umbilical flow rates on the placental transfer of compounds were studied in order to optimize an in situ placental perfusion model in the rabbit for establishing the most suitable perfusion flow rate range and assessing alterations in the placental architecture related to the umbilical flow. Placental transfer of a tool compound theophylline (TH) at different umbilical flow rates was compared with that of antipyrine (AP), the commonly used indicator of placental exchange, in maternal arterial drug steady-state conditions after a two-step infusion program. Placentas of six rabbits were perfused for 250 min with Earle's enriched bicarbonate buffer at 0.5, 1, 1.5, 2, 3, and 4 mL/min flow rates. Plasma and placental perfusate effluent biochemical parameters, gas exchange, body temperature, and electrocardiogram were recorded. Umbilical arterial perfusion pressure was controlled throughout the experiments. A detailed pharmacokinetic analysis of unbound maternal plasma, perfusate TH, and AP concentrations was made. Placental clearance of TH and AP rose up to the 3 mL/min flow rate and then remained constant. Placental clearance was linear up to 2.0 mL/min for TH and 1.5 mL/min for AP. The umbilical flow rate limit was 1.76 +/- 0.29 mL/min for TH and 1.72 +/- 0.49 mL/min for AP. The clearance index was 0.71 +/- 0.04. The correlation between umbilical flow and perfusion pressure was linear, with mean values from 4 to 25 mmHg. Placental resistances did not change significantly at all flow rates with mean values between 6 and 9 mmHg/mL/min.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring of blood gas parameters and acid-base balance of pregnant and non-pregnant rabbits (Oryctolagus cuniculus) in routine experimental conditions.

Blood gas parameters and acid-base balance values were determined in adult pregnant New Zealand rabbits (Oryctolagus cuniculus) in standard laboratory housing conditions and during anaesthesia with an association of ketamine-chlorpromazine, administered before surgical procedures. All the variables were also studied in adult non-pregnant female, used as controls. No differences in pH, sO2c, O2Hb, COHb, sO2m and a-vDO2 were found between pregnant and non-pregnant rabbits in physiological conditions and during anaesthesia. Ketamine-chlorpromazine and pregnancy seemed to change the other parameters used to assess the acid-base balance and the oxygenation conditions. Anaesthesia affected only Hb, O2Ct, O2Cap, CcO2 and P50. The additive effect of pregnancy and anaesthesia modified pCO2, pO2, HCO3-, TCO2, BEb, SBC, BEecf, A-aDO2, RI, MetHb, RHb, CaO2 and CvO2. The patterns described are close to those of other species, suggesting the New Zealand rabbit might be a reliable animal model for monitoring selected variables.

Placental transfer of theophylline in an in vitro closed perfusion system of human placenta isolated lobule.

Theophylline (TH) is a methylated xanthine widely used in the treatment of asthmatic pregnant women. Because of the scant available information on the transplacental profile, the time course of TH transfer was studied by an in vitro human placental perfusion. 6 placentas were perfused with Earle's enriched bicarbonate buffer for 180 min using recirculating maternal and fetal circuits. The physiological and biochemical properties of the tissue were well maintained. TH data were compared to those of antipyrine (AP), an usual marker in placental perfusions. The disappearance of TH from the maternal circuit was studied after administration of 15 mg/l in maternal perfusate. TH appeared in the fetal circuit within 5 min. Equilibrium was achieved in both circuits. TH fetomaternal mass ratio became constant (FMM = 0.45 +/- 0.01) after 80 min of perfusion and maternal to fetal clearance was 2.59 +/- 0.24 ml/min. About 16% of TH maternal dose was recovered in the tissue, while 18% appeared in fetal circulation. TH recovery was 89 +/- 9%. On the basis of our results, similar concentrations could be predicted in mother and fetus after maternal TH intake. The TH transfer profile is consistent with in vivo values reported in humans and animals at delivery.

Pharmacokinetic profile of theophylline in isolated perfused liver of rabbits at different ages. Development of drug-metabolizing activity during ontogenesis.

The ontogeny of the biotransformation of exogenous and endogenous compounds has been mostly studied using liver cells and microsomal fractions. We have used liver perfusion for the first time to characterize the development of the total P-450 cytochrome-dependent system in the rabbit, with theophylline (TH) as tool substance. Livers of 0- to 60-day-old rabbits were perfused with TH (10 micrograms/ml) for 3 hr. Metabolizing enzymes (cytochrome P-450), ATP, glutathione, and glycogen were measured in liver tissue after perfusion. Lactate dehydrogenase, glutamic-oxalacetic transaminase, glucose, and urea were assayed in the medium throughout perfusion. The pharmacokinetic profile of TH was determined. The activity of total cytochrome P-450, as well as the intrinsic unbound clearance and TH metabolites production, increased following a similar sigmoidal pattern and reached a plateau around 30-45 days of the postnatal development of rabbit liver. The perfused tissue showed no signs of age-related hepatic damage or toxic effects of TH. Thus, the results in perfused liver predict its metabolic capacity during ontogenesis.

Theophylline transfer across human placental cotyledon during in vitro dual perfusion.

In vitro placental perfusion is widely used to investigate the placental transfer of endogenous compounds and, to a lesser extent, that of drugs. The aim of this study was to assess the suitability and reliability of such in vitro systems for application on drug placental transfer studies. We investigated the time course of theophylline (TH) transfer, a drug frequently used in the perinatal period. Eight experiments were performed with maternal and fetal circuits maintained in an open system, perfusing placentas for 160 min with Earle's enriched bicarbonate buffer containing two test substances, antipyrine (AP), (80 mg/L) and creatinine (CR), (150 mg/L), and the tool drug TH (15 mg/L). All substances equilibrated in our system with times proportional to the chemical-physical characteristics of each compound, being the time required to reach the steady state 5 to 12 min for AP, 12 to 31 min for CR and 10 to 35 min for TH. AP and CR clearances were 2.94 +/- 0.33 and 0.83 +/- 0.26 mL/min, respectively. The transfer profile of TH was similar to that of AP and its clearance was 2.39 +/- 0.37 mL/min, with a clearance index of 0.80 +/- 0.11. Transfer percentages of TH are in agreement with in vivo values for both humans and animals, and with results obtained during in situ placental perfusion in the rabbit. Physiological conditions and biochemical properties of the tissue were well maintained throughout perfusion. Glucose consumption and lactate release were, respectively, 0.65 +/- 0.16 and 0.73 +/- 0.11 mumoles/min/g. Oxygen consumption was 5.29 +/- 1.32 mL/min/kg and oxygen transfer from the maternal to fetal circuit was 0.99 +/- 0.43 mL/min/kg. The findings support the reliability of this technique to study transplacental passage of drugs, and the relevance of such a model to obtain information concerning potential therapeutic or toxicologic effects of drugs during the last trimester of pregnancy.

Placental transfer of theophylline during in situ perfusion in the rabbit.

Many physiological changes take place during pregnancy, and the disposition profile of endogenous and exogenous compounds may change, too. Thus knowledge of the disposition pattern of a compound may be useful in relation to its therapeutic effect(s) and its potential toxicity on the fetus and the newborn. Because the amount of a compound received by the fetus is a product of placental transfer rate, and available maternal amount, and because it is difficult to control and evaluate the factors that may affect such a transfer in women, we set up an in situ perfused placental model in the rabbit. The reliability of the model was borne out by comparing the placental transfer of theophylline with antipyrine, a commonly used marker of placental exchange, at steady state after a two-step infusion at mean arterial plasma concentrations of 8 and 5 mg/L, respectively for theophylline and antipyrine. The rabbit placenta was perfused in situ with a modified Earle's buffer at a 1-mL/min flow rate. During perfusion, maternal plasma, placental perfusate, biochemical parameters, gas exchange, body temperature, and electrocardiogram were carefully monitored. The maternal plasma and perfusate drug concentrations over time were fitted by appropriate models and kinetic parameters were calculated. Umbilical vein/maternal artery concentration ratios reached equilibrium soon after the loading infusion was stopped for both drugs. Placental clearance averaged 0.62 and 0.77 mL/min for theophylline and antipyrine, respectively, and the clearance index of theophylline was 0.81 +/- 0.07. Although human and rabbit placentas are structurally dissimilar, the rabbit placenta perfused in situ appears to be a useful preparation for measuring the transfer processes and the related and governing factors, of different compounds.

Pharmacokinetics of theophylline in the newborn and adult rabbit. In vivo and isolated perfused liver approaches.

The isolated perfused liver technique is the in vitro system most nearly comparable to the intact liver for experimental investigations on drug metabolism. The model currently used employs liver from different species, but only adults. For the first time, we have set up an experimental investigation involving perfusion of the liver of newborn animals. Using theophylline (TH) as tool drug, an in vivo/in vitro and adult/newborn disposition study was made in the rabbit. After a 10 mg/kg dose iv to adult rabbits and ip to rabbits at birth, the pharmacokinetic profile of TH was analyzed during liver perfusion at comparable TH concentrations in the medium. A few biochemical variables were recorded. No age-related differences were observed in the release of glutamic-oxalacetic transaminase and lactate dehydrogenase over the perfusion time. O2 consumption was higher in adults than in newborns, in accordance with the lower metabolic capacity of the neonatal liver, supported by the lower values of cytochrome P-450, cytochrome c, and glutathione. In vivo and in vitro values were close in adults and newborns for half-life (average 5.2 vs. 5.4 and 27 vs. 35 hr, respectively) and intrinsic clearance of TH (13 vs. 11 and 0.032 vs. 0.021 ml/min). The qualitative and quantitative TH metabolic patterns in the medium and in vivo also were close in adult animals. Only unchanged TH was detected in newborn perfusate. The isolated perfused liver technique appears to offer a reliable model for studying the in vitro ontogeny of drug metabolism, and for making in vitro and in vivo physiological and pharmacological comparisons.

Placental transfer and tissue distribution of vitamin E in pregnant rabbits.

alpha-Tocopherol (vitamin E) is widely prescribed in neonatal intensive care units, in large doses and by different schedules, for the prevention of retrolental fibroplasia, intraventricular haemorrhage, bronchopulmonary dysplasia, and haemolytic anaemia. Since the efficacy of the drug in premature newborns seems related to early administration, the physicochemical characteristics of the drug itself and available formulations limit the major therapeutic aim of promptly raising levels of vitamin E in premature babies during the early hours of life. It has thus been suggested that vitamin E be given to the mother before delivery to produce higher drug concentrations in the newborn. To see whether this would work, the tissue distribution and transplacental transfer of vitamin E were studied in six pregnant rabbits at steady-state after an i.v. bolus + infusion to give a mean venous blood concentration of about 325 mumol l-1 of alpha-tocopherol acetate, corresponding to about 30 mumol l-1 of alpha-tocopherol. Endogenous levels were measured in six control pregnant rabbits. In treated animals alpha-tocopherol was increased in liver, spleen, placenta, lung, mammary gland, blood, and bile but not in brain, heart, fat, muscle or adrenals probably because distribution into these tissues is very slow. Vitamin E levels in the placenta of treated mothers were 15 times those of control rabbits, but the vitamin was not detectable in amniotic fluid and only very low levels were found in fetal blood. These findings do not indicate any advantage of giving mothers alpha-tocopherol acetate before delivery.

Simultaneous determination of retinol, alpha-tocopherol and retinyl palmitate in plasma of premature newborns by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of retinol, alpha-tocopherol and retinyl palmitate in plasma. Plasma containing an internal standard (tocol) was deproteinized with ethanol, then extracted with n-hexane. The organic layer was removed and evaporated under a nitrogen stream, and chromatographed on a reversed-phase RP-18 column using a water/acetonitrile-ethyl acetate/2-propanol gradient solvent system over 15 min at 305 nm. The recovery exceeded 93%. The detection limit was 0.1 microgram/ml for retinol, 1.3 micrograms/ml for alpha-tocopherol and 0.95 micrograms/ml for retinyl palmitate. The reproducibility, precision (expressed as coefficients of variation) and accuracy were less than 8% for all analytes. The small sample requirement, the simplicity of extraction, the short run-time and the good reproducibility make this procedure particularly useful for monitoring retinol and alpha-tocopherol supplementation in premature newborns.

Molecular cloning of the cDNA coding for mouse aldehyde oxidase: tissue distribution and regulation in vivo by testosterone.

The cDNA coding for mouse aldehyde oxidase (AO), a molybdoflavoprotein, has been isolated and characterized. The cDNA is 4347 nt long and consists of an open reading frame predicting a polypeptide of 1333 amino acid residues, with 5' and 3' untranslated regions of 13 and 335 nt respectively. The apparent molecular mass of the translation product in vitro derived from the corresponding cRNA is consistent with that of the monomeric subunit of the AO holoenzyme. The cDNA codes for a catalytically active form of AO, as demonstrated by transient transfection experiments conducted in the HC11 mouse mammary epithelial cell line. The deduced primary structure of the AO protein contains consensus sequences for two distinct 2Fe-2S redox centres and a molybdopterin-binding site. The amino acid sequence of the mouse AO has a high degree of similarity with the human and bovine counterparts, and a significant degree of relatedness to AO proteins of plant origin. Northern blot and in situ hybridization analyses demonstrate that hepatocytes, cardiocytes, lung endothelial or epithelial cells and oesophagus epithelial cells express high levels of AO mRNA. In the various tissues and organs considered, the level of AO mRNA expression is not strictly correlated with the amount of the corresponding protein, suggesting that the synthesis of the AO enzyme is under translational or post-translational control. In addition, we observed sex-related regulation of AO protein synthesis. In the liver of male animals, despite similar amounts of AO mRNA, the levels of the AO enzyme and corresponding polypeptide are significantly higher than those in female animals. Treatment of female mice with testosterone increases the amounts of AO mRNA and of the relative translation product to levels similar to those in male animals.

Mefloquine transfer during in vitro human placenta perfusion.

Mefloquine (MQ) is highly effective in the treatment and prophylaxis of chloroquine-resistant Plasmodium falciparum malaria. Despite its widespread use, scant information is available on the transplacental profile and time course of MQ transfer across the human placenta. Six human placentas were perfused with human plasma for 180 min using recirculating maternal and fetal circuits. The viability of the placental preparation was validated measuring oxygen and carbon dioxide balance and the rates of glucose consumption and lactate production. MQ data were compared with antipyrine, a routine marker in placental perfusions. Disappearance of MQ from the maternal circulation after a dose of 0.8 mg/liter was biexponential, with a first, rapid distribution phase into the placental tissue. The apparent first-order distribution (lambda 1) and elimination (lambda z) rate constants were 0.043 +/- 0.014 min-1 and 0.020 +/- 0.007 min-1, respectively. The fetomaternal mass ratio became constant (0.46 +/- 0.07) after 120 min of perfusion and the time needed to achieve equal concentrations on both sides of the placenta was 178 +/- 31 min. MQ clearance was 3.36 +/- 0.38 ml/min. About 40% of the MQ maternal dose was recovered in tissue and 11% appeared in the fetal circulation. These data provide support for using MQ in pregnant women for both the treatment and prophylaxis of Plasmodium malaria, although comparison with other compounds are needed.

Placental transfer of valproic acid after liposome encapsulation during in vitro human placenta perfusion.

Valproic acid (VPA) is an antiepileptic drug that crosses the placenta freely. Because its use in pregnancy is associated with an increased incidence of fetal malformation and toxic effects, this study was designed to check whether the placental transfer of VPA entrapped in liposomes was reduced. VPA was encapsulated in dehydrated-rehydrated liposomes prepared with equimolar concentrations of phosphatidylcholine, cholesterol and alpha-tocopherol. Liposomes were analyzed for their physicochemical characteristics, their stability and percentage of encapsulation of VPA. A system of dual perfusion of an isolated lobule of term human placenta was used. Six placentas were perfused with liposome-VPA and six with free VPA for 180 min using recirculating maternal and fetal circuits. The rate of transfer and time to reach equilibrium of VPA was similar in placentas perfused with free VPA and with liposome-encapsulated VPA. Liposomes significantly reduced VPA transplacental transfer and placental uptake. This was confirmed by FMM at equilibrium, that was 0.548 +/- 0.058 in free VPA and 0.393 +/- 0.075 in liposome-VPA. The ratio of fetal to maternal concentrations at equilibrium was 0.90 +/- 0.10 in controls and 0.66 +/- 0.13 in liposome-VPA. The amount of VPA recovered in fetal circulation and in placental tissue were 28 +/- 4 and 7 +/- 3% in controls and 19 +/- 4 and 3 +/- 2% in liposome-VPA. In conclusion, our data indicate that encapsulating VPA in liposomes significantly reduces the fetal amount and exposure, and further in vitro and in vivo investigations are needed to optimize the use of liposomes, particularly in pregnancy.