Outcomes by sex following treatment initiation with darunavir/cobicistat in a large Spanish cohort of the CODAR study (GeSIDA 9316).

BACKGROUND: Few women have been included in darunavir/cobicistat clinical development studies, and hardly any of them were antiretroviral experienced or treated with anything other than triple-based therapies. OBJECTIVES: Our aim was to increase our knowledge about women living with HIV undergoing darunavir/cobicistat-based regimens. METHODS: A multicentre (21 hospitals), retrospective study including a centrally selected random sample of HIV-1 patients starting a darunavir/cobicistat-based regimen from June 2014 to March 2017 was planned. Baseline characteristics, 24 and 48 week viral load response (<50 copies/mL), CD4+ lymphocyte count increase, time to change darunavir/cobicistat and adverse event occurrence were all compared by sex. The study was approved by each of the 21 ethics committees, and patients signed informed consent. RESULTS: Out of 761 participants, 193 were women. Similar characteristics were found for both sexes, except that the women had a longer duration of HIV infection (P = 0.001), and were less frequently pre-treated with darunavir/cobicistat in their previous regimen (P = 0.02). The main reason for using a darunavir/cobicistat-based regimen was simplification, without differences by sex, while monotherapy seems to be more frequently prescribed in women than in men (P = 0.067). The main outcomes, HIV viral load response, CD4+ lymphocyte count increase at 24 or 48 weeks, occurrence of adverse events, main reasons for changing and time to the modify darunavir/cobicistat regimen, did not show differences between the sexes. CONCLUSIONS: No sex disparities were found in the main study outcomes. These results support the use of a darunavir/cobicistat-based regimen in long-term pre-treated women. Clinical Trial.gov No. NCT03042390.

Expression profiling of the GBP1 gene as a candidate gene for porcine reproductive and respiratory syndrome resistance.

A genomic region in pig chromosome 4 has been previously associated with higher viraemia levels and lower weight gain following porcine reproduction and respiratory syndrome virus (PRRSV) infection. The region includes the marker WUR1000125, a G>A polymorphism next to a putative polyadenylation site in the 3'-untranslated region (3'-UTR) of the guanylate-binding protein 1, interferon-induced (GBP1) gene. The protein encoded by GBP1 is a negative regulator of T-cell responses. We show here that GBP1 expression is lower in liver and tonsils of pigs carrying the WUR1000125-G allele due to differential allele expression (allele A expression is 1.9-fold higher than for allele G). We also show that the GBP1 gene has two active polyadenylation signals 421 bp apart and that polyadenylation usage is dependent on the WUR1000125 genotype. The distal site is the most prevalently used in all samples, but the presence of the A allele favours the generation of shorter transcripts from the proximal site. This is confirmed by a differential allele expression study in AG genotype liver and tonsil samples. The interaction between WUR1000125 and other mutations identified in the 5'- and 3'-UTR regions of this gene needs to be studied. In conclusion, our study indicates that the WUR1000125 mutation is associated with changes in the expression of the negative T-cell regulator GBP1 gene. However, the chromosome 4 locus for PRRSV viraemia levels and weight gain contains a cluster of four other GBP genes that remain to be studied as candidate genes for this QTL.

Production parameters and pig production cost: temporal evolution 2010-2014.

BACKGROUND: The assessment of the cost of production and the relative weight of the different production parameters is very important in pig farming. The goals of the present work were 1) to describe reliable reference values for production parameters and pig production cost from 2010 to 2014, 2) to describe their temporal evolution and 3) to determine the influence of the pig company size on them. Between 61 and 107 pig production companies from Spain were included in this study from 2010 to 2014. These companies sent data on feed consumption, number of pig produced, expenses and census every month. Sip consultors SL standardized collected data and calculate cost and production parameters to obtain values comparables between the different pig production companies. The collected data each month were merged to obtain a yearly average value taking into account the pig production flow each month. A suitable statistical analysis was carried out to tackle the goals. RESULTS: The production performance has been continuously improving in the piglet production and fattening phase from 2010 to 2014. Thus, the number of piglets by sow and year will increase 0.5 pigs by year and the total feed conversion rate will decrease approximately 0.03 kg feed/kg gain by year in the future if the same tendency continues. However, feed price has been steadily increasing from 2010 to 2012 and decreasing afterwards and the total cost per kilogram produced has followed a similar pattern. This result highlights the relevance of the feed price in the final cost in spite of continuous improvement in production performance across years. Finally, pig company size affected most of the production parameters studied. Thus, the best technical parameters were obtained for companies with less than 5000 sows. However, the opposite tendency is observed for feed price where the highest value was observed for the smallest companies. CONCLUSIONS: Pig production parameters have generally improved in the last five years but this improvement did not directly imply a reduction in pig production cost due to the high feed prices during the period 2010-2013.

A common method for the determination of several calcium channel blockers using an HPLC system with ultraviolet detection.

We report a common HPLC method for the single or simultaneous determination of four calcium channel blockers (CCB), namely diltiazem (DTZ), verapamil (VER), nifedipine (NIF) and nitrendipine (NIT) and their active metabolites demetildiltiazem and deacetildiltiazem (MA and M1), norverapamil (NOR), and dehydronifedipine (DHN). DHN was first synthesised in our laboratory and different pH values of the mobil phase were subsequently prepared and tested for chromatographic separation. The detection system and the environmental light conditions were optimised. The best separations of all analytes were obtained using a C(18) column and a mobile phase of methanol, 0.04 M ammonium acetate, acetonitrile and triethylamine (2:2:1:0.04 v/v). Quantitation was performed using imipramine (IMI) as the internal standard. For DTZ and its metabolites (M1 and MA), the wavelength chosen was 237 nm; for VER and its metabolite NOR, it was 210 nm; and, finally for NIF and its metabolite DHN and NIT it was 216 nm. When a simultaneous analysis was carried out the wavelength was of 230 nm. The optimum pH were 7.90 and 7.10 when the separation of NIT and DTZ or VER and NIF were carried out, respectively, and 7.90 when a simultaneous separation was carried out. The detection limit of the assay was less than 8 ng ml(-1) for all compounds, with coefficients of variation less than 7% (for inter- and intra-day) over the concentration range of 1-1000 ng ml(-1). The retention times were less than 11 min. When NIF or NIT were studied, it was necessary to use a sodium vapour lamp in order to avoid the photodegradation which takes place under daylight conditions.

Placental transfer of enrofloxacin and ciprofloxacin in rabbits.

Placental transfer of enrofloxacin and ciprofloxacin was evaluated, using a rabbit in situ perfusion model. A two-step infusion program was carried out to obtain steady-state maternal plasma concentrations of these drugs. For each compound, the placenta in 5 rabbits was perfused for 200 minutes with Earle's enriched bicarbonate buffer at flow rate of 1.5 ml/min. To assess reliability of the model, most of the determinants of placental transfer (maternal and fetal pH, gas balance, heart status, rectal temperature, and protein binding) were controlled. In addition, the infusion program included administration of antipyrine, a commonly used indicator of placental exchange. Drug concentrations were measured in maternal plasma and perfusate by use of a high-performance liquid chromatographic assay. Plasma protein-binding estimation indicated no differences between the drugs. Placental clearance of the drugs was significantly (P < 0.01) different (0.88 +/- 0.13 ml/min for enrofloxacin and 0.06 +/- 0.02 ml/min for ciprofloxacin). These values accounted for 81 and 5%, respectively, of the placental clearance found for antipyrine. These results indicate that caution must be taken when enrofloxacin is to be used during pregnancy, and suggest the need to extend this type of experiment to species that can be exposed to these drugs used for therapeutic or prophylactic purposes.

Penetration of enrofloxacin and ciprofloxacin into breast milk, and pharmacokinetics of the drugs in lactating rabbits and neonatal offspring.

OBJECTIVE: To determine the pharmacokinetics and milk penetration of enrofloxacin (ENR) and ciprofloxacin (CIP) in lactating rabbits and their disposition in suckling rabbits. DESIGN: Prospective cross-over study. ANIMALS: 6 lactating New Zeland White rabbits and their offspring (16 days after parturition). PROCEDURE: Serial plasma and milk samples were assayed by use of a high-performance liquid chromatography technique. In vitro protein binding in plasma and skim milk was measured by ultrafiltration. Skim-to-whole milk ratio also was determined. The time course of ENR and CIP was fitted by nonlinear least squares regression analysis, and the pharmacokinetic variables were compared. RESULTS: The time courses of ENR and CIP in plasma were similar in lactating adult rabbits (mean body clearances, 23.9 and 27.2 ml/min/kg of body weight, for ENR and CIP, respectively). Observed milk-to-plasma ratios (M/P) were determined, using the area under the milk and plasma concentration versus time profiles (ENR, 2.59; CIP, 3.61). Predicted M/P (ENR, 6.35; CIP, 3.04) were calculated from in vitro measurements. Body clearance calculated for ENR and CIP in suckling rabbit pups involved a decrease of 80 and 74%, respectively, over that found in lactating animals. CONCLUSIONS: Observed CIP M/P were correlated to predicted values, which strengthens the argument that CIP passes into the milk by nonionic diffusion. The lack of correlation between observed and predicted ENR M/P pointed out that ENR undergoes faster elimination from milk than that predicted by the diffusional model. Diminished elimination capacity observed in suckling rabbits would result in greater exposure than that predicted from concentrations alone.

Limited capacity of neonatal rabbits to eliminate enrofloxacin and ciprofloxacin.

The pharmacokinetics of enrofloxacin (ENR) and ciprofloxacin (CIP) in newborn and young rabbits were studied. Rabbits of different ages (1-, 8-, 16-, and 30-day-old) were administered, by the intraperitoneal route (i.p.), a dose of 7.5 mg of either drug/kg. In 1-, 8-, and 16-day-old rabbits, blood samples were drawn by cardiac puncture, under light ether anaesthesia, at predetermined times after drug administration. In 30-day-old rabbits, serial blood samples were drawn through an arterial catheter. Plasma was immediately obtained and analysed using an HPLC method. ENR and CIP plasma protein binding was also determined. The plasma pharmacokinetic profiles of ENR and CIP obtained for 30-day-old rabbits agreed with those reported in the literature for healthy adult rabbits. Nevertheless, significant differences were observed for the body clearance, the slope of the terminal phase, the volume of distribution, and the area under the curve when compared with those for younger animals (1-, 8-, and 16-day-old rabbits), indicating a limited capacity of neonatal rabbits to eliminate ENR and CIP. No differences were found when we compared the calculated values for ENR or CIP plasma protein binding as a function of the postnatal age, indicating that development does not seem to alter the free fraction of these drugs in the rabbit. Taking into account that extensive placental and milk transfer has been reported for these drugs after administration to pregnant or nursing rabbits, a cautious, attitude regarding their use in these animals must be adopted.

Pharmacokinetics of verapamil in New Zealand white rabbits during ontogeny.

The pharmacokinetics of verapamil during ontogeny in rabbits and in vitro verapamil demethylase activity have been investigated in the liver and whole blood. In vivo experiments revealed that the slope of the postdistributive phase as well as the area under the curve and clearance showed significant differences when newborn and adult rabbits were compared. Other pharmacokinetic parameters, such as volume of distribution and plasma protein binding did not show any statistical differences. Liver microsomal preparation samples from 1-, 8- and 16-day-old rabbits displayed approximately 20% of the activity observed in adults. A significant verapamil demethylase activity in the whole blood of rabbits was also noted. The in vivo results show that newborn rabbits have a capacity to eliminate verapamil that is similar or even higher than that of adults. These findings could not be explained with regard to the in vitro liver metabolism of verapamil, although they could with respect to blood metabolism.

Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection.

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane-isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1-1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.

Comparison of the pharmacokinetics of verapamil in the pregnant and non-pregnant rabbit: study of maternal and foetal tissue levels.

1. The comparison of the pharmacokinetics of verapamil (VER) has been studied between the non-pregnant and pregnant rabbit following VER intravenous (i.v.) bolus administration. Also studied has been VER tissue distribution in the non-pregnant and pregnant rabbit and its foetuses following an i.v. infusion of VER. 2. When the pharmacokinetic variables were compared between the pregnant and non-pregnant rabbit, it was observed that t(1/2)lambda2 V1 and V(D) were significantly higher in the non-pregnant than in the pregnant rabbit. Moreover, lambda(z) was significantly lower in the non-pregnant than in the pregnant rabbit. However, AUC and CL showed no significant differences between the pregnant and non-pregnant rabbit. 3. When tissue concentrations were examined, it was found that in most of the tissues studied high concentrations of VER were found both in the pregnant and non-pregnant rabbit. Furthermore, VER concentrations in the uterus, heart, spleen and kidney were significantly higher in the non-pregnant than in the pregnant rabbit. 4. The results suggest that VER diffuses poorly through the placenta, given that VER blood concentrations were lower in blood foetuses than in maternal blood. Moreover, the concentrations of VER in the selected foetal tissues were either similar (brain and liver) or lower than those observed in maternal tissues.

Diltiazem blood pharmacokinetics in the pregnant and non-pregnant rabbit: maternal and foetal tissue levels.

1. The aim was to investigate the pharmacokinetics of diltiazem (DTZ) and its metabolites, deacetyldiltiazem (M1) and N-demethyldiltiazem (MA), in the pregnant rabbit following DTZ intravenous administration. In addition, DTZ tissue distribution in both the non-pregnant and pregnant rabbit and foetuses was also studied. 2. The slope of the alpha- and beta-phases increased slightly in six of the eight pregnant rabbits as compared with the non-pregnant animal, but the other pharmacokinetic parameters that largely determine drug disposition (AUC, V(n), CL) showed no significant differences. 3. MA blood disposition was unaltered by pregnancy. However, all the pharmacokinetic parameters calculated for the deacetylated metabolite of DTZ were significantly modified in the pregnant as compared with the non-pregnant rabbit. 4. DTZ tended to concentrate in most of the tissues examined. Significant differences were observed in the DTZ concentration in the uterus and kidney from the pregnant as compared with the non-pregnant rabbit. 5. The findings suggest that DTZ diffuses easily through the placenta, reaching DTZ blood concentrations equivalent to that observed in maternal blood. However, the concentration of DTZ and its metabolites in the selected foetal tissues was either higher (in brain and muscle) or lower than that observed in maternal tissues, suggesting a different tissue affinity and/or a different metabolic activity in the foetuses as compared with the mothers.

Enhanced diltiazem deacetylase activity in pre-term and full-term rabbits compared with adult rabbits.

The in vitro metabolic activity of the esterase responsible for the hydrolysis of diltiazem (DTZ) to its deacetylated metabolite (M1) was determined in an age-dependent fashion using the rabbit as an animal model. The presence of the enzyme in several tissues (liver, lung, small intestine, and brain) and in whole blood from pre-term and full-term fetuses, full-term newborns, yound and adult rabbits was examined. To this end, DTZ was spiked to 10,000-g tissue homogenates and whole blood to yield a final concentration of 1 microgram/ml. Serial samples were withdrawn from the incubation medium up to 240 min and assayed for DTZ and M1 concentration. In all tissues examined there was a net production of M1. Chemical breakdown and stability studies confirmed the metabolic origin of the M1 formed throughout the incubation. In pre-term fetuses (25 days of gestation) the brain was found to be the most active tissue in eliminating DTZ (brain > liver > lung > small intestine). This trend changed in young and adult rabbits (lung = brain > liver > small intestine). Although an important age-dependent DTZ deacetylase activity was observed in blood, it was not included in the comparison between organs because of the unequal composition of the incubation medium. In conclusion, results showed that fetuses and newborn rabbits have a similar, and in some instances higher, DTZ deacetylase activity to that in adults (p < 0.05). In vitro findings were further confirmed by in vivo experiments.

Development of diltiazem deacetylase and demethylase activities during ontogeny in rabbit.

1. Diltiazem (DTZ) undergoes extensive metabolism in hepatic and extrahepatic tissues. Deacetyldiltiazem (M1) and N-desmethyldiltiazem (MA) are two of the main basic metabolites of DTZ that retain pharmacological activity. The development of DTZ deacetylase and demethylase activities through ontogeny has not been addressed. In order to address this issue, in vitro studies have been carried out using the blood and several tissues of rabbit as enzyme sources. In addition, in vivo studies using a pharmacokinetic approach were carried out to support the in vitro findings. 2. DTZ was incubated with homogenates of selected tissues and in whole blood and DTZ, and its metabolites were assayed by HPLC. In addition, a pharmacokinetic study after intraperitoneal administration of DTZ in the 1-, 8-, 16-, 30-day-old and adult rabbit were also carried out. 3. DTZ deactylase activity was detected whatever the age and tissue examined (including blood). Except in gut homogenates, this activity was shown to be higher at earlier postnatal ages. DTZ demethylase activity was only detected in the liver and gut homogenates and in whole blood. This activity increases from the 1- to 30-day-old rabbit (except for blood), after which it decreases slightly to reach the adult level. 4. In vivo experiments showed a close pharmacokinetic profile throughout ontogeny (except for the 30-day-old rabbit) after DTZ intraperitoneal administration. 5. Extrahepatic metabolism may play a more significant role in the overall metabolism and pharmacokinetics of DTZ at earlier stages of development. 6. Finally, in vivo studies Suggest that age does not seem to modify DTZ disposition and, for this reason, dosage may not have to be taken into account as a function of age.

Penetration of diltiazem into breast milk and its pharmacokinetics in the lactating rabbit.

1. The aim was to investigate the milk transfer and pharmacokinetics of diltiazem (DTZ) in the lactating rabbit following DTZ intravenous (i.v.) administration. In addition, DTZ metabolism in mammary tissue and milk was also studied. 2. The pharmacokinetic parameters that largely determine drug disposition (AUC, VD, CL) showed no significant differences between the non-lactating and lactating rabbit. 3. When DTZ was administered to the lactating rabbit, the observed DTZ milk-to-blood AUC ratio (M/B) closely correlated with the calculated ratio, as predicted by a diffusional model, suggesting that DTZ passes into milk via non-ionic diffusion and that other factors which may affect the milk transfer seem to have limited relevance. 4. After a single intravenous dose of DTZ to the lactating rabbit, deacetyldiltiazem (M1) and demethyldiltiazem (MA) were observed in blood, but only M1 could be detected in milk. 5. In conclusion, DTZ seems to diffuse freely into milk after its i.v. administration to the lactating rabbit and should not be given to nursing mammals because of the potential risk for their young. This risk may be even higher because of the presence of M1 (a pharmacologically active metabolite) in milk after administration of the parent drug.

Altered diltiazem metabolism in the neonatal rabbit following intra-uterine chronic exposure to diltiazem.

1. Diltiazem undergoes extensive metabolism in hepatic and extrahepatic tissues. Deacetyldiltiazem (M1) and N-demethyldiltiazem (MA) are two of the main basic metabolites of diltiazem that retain pharmacological activity. This drug impairs its own metabolism after chronic administration in the adult patient. The study examines the possibility that intra-uterine exposure following chronic maternal therapy with DTZ from mid-gestation to term also impairs DTZ metabolism of its offspring. 2. DTZ was incubated in homogenates from liver, lung, brain and gut and in the whole blood of animals whose mothers were exposed to chronic treatment with diltiazem or unexposed (placebo). DTZ and its metabolites were assayed by HPLC. 3. DTZ deacetylase activity observed in liver, lung and brain homogenates from 1-, 8- and 16-day-old rabbits was significant lower in exposed animals. In gut homogenates, this age-dependent effect was not so clear. This inhibition could not be detected in any organ of 30-day-old rabbits. On the other hand, the activity observed in whole blood was not altered by intra-uterine chronic exposure to DTZ. 4. DTZ demethylase activity showed no differences in tissue homogenates and in whole blood from exposed compared with the unexposed rabbit. 5. In conclusion, the findings suggest that intra-uterine chronic exposure to DTZ has a large and prolonged effect on newborn metabolism deacetylase activity compared with the unexposed rabbit.

Pharmacokinetics of verapamil in lactating rabbits. Prediction of verapamil distribution into rabbit milk.

In this work, we have studied the pharmacokinetics and milk penetration of verapamil following intravenous administration in lactating rabbits. Milk-to-serum drug concentration ratios (M/B(obs)) have been determined using area under the milk and serum concentration-time profiles, and the resulting values have then been compared with those obtained by theoretical classical diffusion milk transfer models that were described by Fleishaker et al. [J. Pharm. Sci. 76 (1987) 189.], Atkinson and Begg [Br. J. Clin. Pharmacol. 25 (1990) 495.], and Stebler and Guentert [Pharm. Res. 9 (1992) 1299.]. The pharmacokinetic profile of verapamil in lactating rabbits following endovenous administration is described in the form of a two-compartment model. Moreover, we detected an important milk transfer after endovenous administration of verapamil in lactating rabbits. M/B(obs) was near 15. The classical diffusional models mentioned were not able to predict this extensive transfer of verapamil into rabbit milk. However, when the classical Fleishaker equation was modified and a stepwise regression was carried out, we found that the M/B(obs) value could be predicted using the plasma and milk protein binding.

Deacetylation of diltiazem by several rabbit tissues.

PURPOSE: Diltiazem (DTZ) undergoes extensive metabolism yielding several metabolites, some of which retain a certain degree of pharmacological activity. N-demethylating activity has been detected mainly in the liver. Nevertheless, the organs involved in the formation of the deacetylated metabolite of DTZ (M1) have not been fully elucidated. In order to address this issue, we have carried out in vitro studies using the blood, lung, brain, small intestine, and liver as enzyme sources. METHODS: DTZ (1,000 ng/ml) was incubated in 10,000 x g supernatant homogenates of selected tissues or in whole blood for 240 minutes at 37 degrees C. Multiple samples were withdrawn, and DTZ and its metabolite M1 were assayed by HPLC. RESULTS: The apparent degradation rate constant of DTZ was in the rank order blood > lung > brain > liver > small intestine. This trend can also be observed for the AUC and for the percentage of DTZ metabolized. In all the tissue homogenates examined there was a net production of the deacetylated metabolite. The M1 metabolite was also detected in the blood (500 ng/ml after 240 minutes of incubation). CONCLUSIONS: The widespread distribution of the DTZ deacetylase activity described in this study suggests that extrahepatic metabolism of DTZ to M1 may play a relevant role in the overall pharmacokinetics of DTZ.

Pharmacokinetics of fosfomycin in chickens after a single intravenous dose and tissue levels following chronic oral administration.

This paper describes the pharmacokinetics of fosfomycin following a single i.v. bolus dose (10 mg/kg of body weight) in broiler chickens. Serial blood samples were collected up to 5 h post-administration. Fosfomycin serum concentrations were determined by a microbiological method, using Proteus mirabilis as the test microorganism. The serum concentration versus time curves after i.v. administration followed a biexponential decline. The main pharmacokinetic variables were t1/2 lambda l = 23 min, t1/2 lambda n = 112 min, VDarea = 575 +/- 190 mL/kg and CLb = 3.12 +/- 0.44 mL.min-1.kg-1. Tissue levels of fosfomycin in kidney, liver, lung, muscle, heart, fat, gizzard and serum were also determined after oral chronic administration of the drug in drinking water (150 micrograms/mL). During the oral chronic administration period, high FOS concentrations in serum were maintained (mean 6.1 +/- 1.1 micrograms/mL), but a significant decline over time could be observed (P < 0.05). Fosfomycin was detected in all tissues except muscle, with mean concentrations ranging from 0.63 microgram/g in fat to 13.48 micrograms/g in kidney. Twenty-four hours after the treatment was finished, fosfomycin levels were below the assay detection limit in all tissues tested.