Highly active antiretroviral therapy during gestation: effects on a rat model of pregnancy

Summary

Objective:
To assess the adverse effects of the chronic use of zidovudine/lopinavir/ritonavir in a rat pregnancy model.

Type of article:
Original paper.

Design:
A prospective experimental study.

Setting:
Department of Obstetrics, São Paulo Federal University (UNIFESP).

Methods:
40 pregnant EPM-1 albino rats were randomly allocated into four groups of 10 animals each: control (Ctrl) group (untreated) and three experimental groups (Exp1, Exp2 and Exp3), which received zidovudine/lopinavir/ritonavir in the corresponding doses of 10/13.3/3.3; 30/39.9/9.9 and 90/119.7/29.7 mg/Kg/day from the first up to the 20th day of pregnancy, respectively. The rats were treated by gavage daily. Body weights were recorded on days 0, 7, 14 and 20. At term, the rats were sacrificed and the implantation sites, number of live and dead fetuses and placentas, resorptions and fetal and placental weights were recorded. The fetuses were evaluated for external abnormalities under a stereomicroscope. The chi-square test was used to compare death rates between groups.

Results:
Weight gain during pregnancy no showed significant differences between groups. Average weight gains between the 7th and 20th day were 45.70 ± 5.27 g for Ctrl; 48.49 ± 3.64 g for Exp1; 45.39 ± 6.22 g for Exp2 and 44.19 ± 6.78 g for Exp3. However, the percentage weight gain in the 7th was lower in groups Exp2 and Exp3 and in the 14th in the Group Exp2. All other parameters assessed did not differ significantly between groups. Exp2 and Exp3 in relation of the others.

Conclusions:
The chronic exposure of pregnant rats to high doses of zidovudine/lopinavir/ritonavir in association resulted in a significant reduction in maternal body weight gain but was not associated with significant adverse fetal parameters.

Keywords:
biological assay, antiretroviral, pregnancy, rat, teratology

INTRODUCTION

The human immunodeficiency virus (HIV) over time has shown a tendency to stabilize the number of infected persons. However, in women, the rate of the epidemic is growing. In the 80s, beginning of the pandemic period, the ratio of men / women infected was 20:1. Currently, for every 1.6 infected men, there is 1 infected woman [4].

Disturbing fact has been the predominant age in females, between 13 to 19 years. This fact is associated with unprotected sex, early onset of sexual activity, cultural, socioeconomic and biological conditions of women themselves, which makes it vulnerable to HIV. The prevalence of infection in the reproductive period favors vertical transmission [5]. Most studies suggest that vertical transmission occurs in 70% of cases the intrapartum, and 30% of cases occurring in utero. Breastfeeding has 15 % additional risk of transmission [9, 10, 23].

In 90s began the study of antiretroviral in HIV positive pregnant women as Protocol 076, verifying the reduction of transmission of 25.5% among newborns of women who used placebo to 8.3% for children of mothers who used AZT [11]. Since then, several studies have demonstrated the effectiveness of protective measures for vertical transmission. In Brazil, in 2011, 745 cases of acquired immunodeficiency syndrome (AIDS) in children under 5 years were reported with an incidence rate of 5.4 / 100,000. The use of antiretroviral drugs for pregnant women and newborn, the indication for cesarean delivery and breast feeding with artificial milk, decreased by 25% AIDS cases among children under 5 years in the last ten years in our country [4].

Among the several treatment protocols, highly active antiretroviral therapy (HAART), usually includes one nucleoside analog (DNA chain terminator), one protease inhibitor and either a second nucleoside analog (“nuke”) or a non-nucleoside reverse transcription inhibitor [14]. Though the association of antiretroviral drugs has been shown to be largely safe for the concept [31], it is supposed that association of multiple drugs may introduce changes in their pharmacokinetics [19] and carry on unpredictable results on maternal and/or fetal compartments. The association resulted in a significant reduction of viral load in plasma in such an extent that sometimes it leads to undetectable levels of the virus [13].

Despite the reduction of vertical transmission, some antiretroviral drugs may be associated with teratogenicity, preterm delivery, intrauterine growth restriction, and may be developing resistance to antiretroviral use [22, 23]. Carvalho et al. [7] founded deleterious effects on fertility, high maternal mortality with the administration of ritonavir to pregnant rats, suggesting an important compartmentation of the drug. Cunha et al. [12] showed that the administration of a combination of lopinavir plus ritonavir to pregnant rats can causes morphological as well as functional changes in maternal and fetal liver and kidneys and, in higher than therapeutic doses, might be toxic to those animals. Though one cannot extrapolate drug effects from animals to human beings, the study of the effect of drugs on animal pregnancy is the best model to understand the effect of such drugs on humans. Carvalho et al. [8] showed that high dosing of zidovudine/lopinavir/ritonavir association during the entire rat pregnancy period can cause morphological changes in maternal liver, kidneys and pancreas. On the other hand, the corresponding fetal organs were not affected.

This experimental way of studying the effect of drugs on pregnancy has been adopted by the Experimental Obstetrics Discipline, Department of Obstetrics, São Paulo Federal University (UNIFESP), Brazil. Drugs have been studied alone or in association with others. The aim of such studies is to verify any pharmacokinetic change in the antiretroviral drugs that can provoke adverse effects. It is well known that there are many trials of drug effects on rat pregnancy. We decided to test lower doses of zidovudine/lopinavir/ritonavir association, corresponding to 1, 3 and 9 times the human therapeutic doses. These same doses have previously been investigated in an experimental model study that appraised the effects on maternal and fetal prole.

MATERIALS AND METHODS

Female Wistar rats (Rattus norvegicus albinus) of the EPM-1 variant, weighing approximately 200g each, were provided by the Center for Development of Experimental Models of UNIFESP. The study was approved by the local Animal Care Committee (Report 0402/09) and followed the guidelines proposed by the Canadian Council on Animal Care [24].

The animals were kept in plastic cages under controlled room temperature (22°C) and artificial light by fluorescent lamps with a constant day/night cycle (lights on 07:00–19:00), with free access to pelleted Purina rat food (Agribrands Purina do Brasil Ltda, Vilhena, RO, Brazil) and tap water. After a 7-day period of adaptation, the animals were mated in the proportion of one healthy male to three females during two hours. The immediate 24-hour period after mating was taken as day 0 (zero) of pregnancy if spermatozoids were detected in vaginal smears [16]. Forty pregnant rats were randomly divided into four groups. The control group (Ctrl) received distilled water and the experimental groups (Exp1, Exp2 and Exp3) received zidovudine/lopinavir/ritonavir in the corresponding doses of 10/13.3/3.3; 30/39.9/9.9 and 90/119,7/29.7 mg/Kg/day by gavage from the first up to the 20th day of pregnancy. Body weights were recorded for all animals at days 0, 7, 14 and 20 of pregnancy and expressed as percentage of body weight gain [26, 27].

At term, the animals were weighed; anesthetized using a mixture of xylazine (20 mg/kg) and ketamine (100 mg/kg) administered by the intraperitoneal route and submitted to laparotomy and hysterotomy. The following parameters were recorded: ovular implantations, ovular reabsorptions, number of live and dead fetuses and fetal and placental weights. The fetuses were inspected in detail under a stereoscopic microscope for gross external malformations: limb shortening, spina bifida, cleft lip, cleft palate, polydactyly and rachisquisis [27].

Data were expressed as mean ± standard deviation (SD). ANOVA and the Kruskal-Wallis multiple comparisons test were used for statistical analyses. The Chi-square test was used to compare death rates between groups. Significance was set at 5%.

RESULTS

Mean weight gain in the control group ranged from 12.77 g (± 2.93) to 58.47 g (± 8.73). In the experimental groups, mean weight gain from day 1 to 20 of gestation was 11.73 g (± 2.54) to 60.22 g (±5 .52), 8.78 g (± 0.67) to 54.17 g (± 12.71) and 9.01 g (± 0.88) to 53.20 g (± 14.85) in groups Exp1, Exp2 and Exp3, respectively. As shown on Table 1, these differences were significant in the Exp2 and Exp3 groups on the 7th day and Exp2 on the 14th day in relation to the others. None of the other parameters analyzed (number of fetuses, number of placentas, fetal weight and placental weight) differed significantly between the groups (Table 2).

DISCUSSION

Although funding for high-power anti-HIV therapy has increased in recent years, public and private health resources are still limited, especially in developing countries. The costs of AIDS treatment are especially high. Thus, the agents of first choice most used in developing countries are the most common and low-cost drugs, such as AZT and ritonavir, usually produced by pharmaceutical companies located in China, where the production costs is much cheaper. In view of this, we decided to study the action of the combination of zidovudine/lopinavir/ritonavir on rat pregnancy in order to ascertain the possible side effects of this association.

Studies using zidovudine in monotherapy have failed to find changes in maternal weight gain, maternal mortality or fetal malformation, even at high doses (60 and 100 mg/kg, by day) [26, 32]. However, a study with isolated use of ritonavir, using nine times the therapeutic dose, found high maternal mortality (40%) with the probable cause of death being multiple organ failure [7]. The use of lopinavir/ritonavir in the same doses used in this study showed no maternal mortality or fetal malformation [32]. This may be due to the use of ritonavir associated with another antiretroviral, because the association with lopinavir acts only as an enhancer, through the inhibition of cytochrome P450 3A [19].

Since maternal weight gain depends on the initial and final weight of the rats, we considered the most reliable weight percentage difference in order to analyze such data, because there is a weight variation in the beginning of the experiment between the different groups investigated [32]. As shown in Table 1, the pregnant rats treated with zidovudine/lopinavir/ritonavir in the beginning of the pregnancy (to the 7th day) showed less body weight gain in the Exp2 and Exp3 groups and in the 14th less gain in the Exp2 group compared to the others. These changes were statistically significant, though at the end of the experiment there was compensation and the final gain was similar in the various groups of the experiment.

A fixed dose combination of the protease inhibitors lopinavir and ritonavir is currently available for clinical use, with a recommended daily dose of 800mg of lopinavir plus 200 mg of ritonavir, given once or in two divided doses. However, in our experiment we gave them only once a day [3, 12].

This weight reduction is presumably due to dose-dependent gastrointestinal side effects as a function of ritonavir exposure through the 7th–14th day period of gestation, and can be related to the fact that rats lack some of the multiple factors involved in the pathogenetic mechanisms proposed for humans, which include the interference of several regulatory proteins such as sterol regulatory enhancer binding protein-1, the proteasome, mitochondrial DNA polymerase gamma and GLUT-4 [6]. Also, the short time of exposition to the drugs (3 weeks) could have played some role in this result [21]. Other authors have suggested the association of the drugs used in this study cab cause neuritis for a short time. Therefore, the animals can slowly move in their cages during the first and second part if pregnancy. This fact may explain our data on the weight of pregnant rats [1].

In rats, ritonavir markedly enhanced the pharmacokinetics of lopinavir, probably due to inhibition of presystemic and systemic metabolism, leading to increased exposure to this potent HIV protease inhibitor [30]. Few pharmacokinetic data during pregnancy are available for the dual protease inhibitor combinations that include low doses of ritonavir to boost concentrations of the coadministered protease inhibitor [2]. This finding applies to two relevant issues. First, no clear-cut dose-dependent effect can be inferred from this result. However, it can be related to the individual sensitivity of the animals to the drugs. Such individual variation could be linked to the functional levels of P glycoprotein (P-gp) [18], a membrane efflux pump pertaining to the superfamily of ATP binding cassette proteins [17]. As known, this protein is encoded by the ‘multidrug resistence’ genes Mdr 1a and Mdr 1b [15] and is expressed in tissues involved with drug absorption, metabolism and excretion [28]. P-gps limit penetration of potentially harmful or therapeutic hydrophobic compounds, thus providing protection of an organism against potentially toxic compounds in the environment. Accordingly, a placental P-gp may play an important role in the protection of the developing fetus [20]. Other barriers in which P-gps are importantly involved are the blood-brain, the blood-nerve; the blood-testis and the intestinal barrier (see Smit et al. [29]). Predictably, the absence of P-gp can lead to adverse effects when an organism is exposed to drugs such as antineoplastic agents, cardiac glycosides, beta-blockers, calcium channel blockers and HIV-protease inhibitors [25].

CONCLUSION

Taking into consideration the modern antiretroviral triple therapy for HIV infection, it is frequently difficult to predict the sort of interactions among the drugs used and which drug might be responsible for a particular effect. This challenge can be overcome by future biological assays using animal models to shed more light on the effect of associated antiretroviral drugs on the mother and fetus. However, our observations that zidovudine/lopinavir/ritonavir at doses of just one order of magnitude higher than those used in humans can cause effects on rat pregnancy outcome should not be underestimated. Finally, the chronic exposure of pregnant rats to high doses of associated zidovudine/lopinavir/ritonavir resulted in a significant reduction in maternal body weight gain, an effect that was not associated with significant adverse affects on the fetal parameters.

Prof. Edward Araujo Júnior, PhD (Corresponding author)

Department of Obstetrics, Federal University of São Paulo (UNIFESP)

Rua Carlos Weber, 956 apt. 113 Visage

Vila Leopoldina

São Paulo – SP, Brazil

CEP 05303-000

e-mail: araujojred@terra.com.br