1 Introduction
Viral hepatitis B, caused by hepatitis B virus (HBV), remains a global health concern due to its high morbidity and mortality. With clear action mechanisms and efficacies, nucleoside analogs have become the main first-line drugs for combating viral hepatitis B [
1,
2]. Some newly licensed nucleoside analogs have showed strong inhibitory effects on HBV; however, high chance of recurrence after drug withdrawal and high resistance rate also restrict their application. Therefore, it is a high priority to develop a new nucleoside analog with high efficiency, low toxicity, high bioavailability, and low resistance to combat HBV [
3].
Antiretroviral drugs function by reducing the residual rate of HIV vertical transmission [
4], and to improve maternal health [
5,
6]. Some studies suggest that the combination of antiretroviral therapy in pregnant women with human immunodeficiency virus type 1 (HIV-1) infections, increases the risk of premature birth and other adverse outcomes of pregnancy. However, data on complications of pregnancy, associated with monotherapy or combination therapy with antiretroviral agents, are limited. In 1998, a retrospective Swiss study consisting of 30 women with HIV-1 who had received a combination of different antiretroviral therapy during pregnancy (with protease inhibitors in 13 women and without protease inhibitors in 17) showed that such treatment was associated with a 33% risk of premature delivery [
7]. Contemporaneously, the Pediatric AIDS Clinical Trials Group (PACTG) observed that in phase 1 trials of nelfinavir, ritonavir, or indinavir, given in combination with zidovudine and lamivudine during pregnancy, 4 of 11 deliveries (36%) occurred before 37 weeks of gestation. While Ruth [
8] reported that as compared with no antiretroviral therapy or monotherapy, combination therapy for HIV-1 infection in pregnant women is not associated with increased rates of premature delivery or with low birth weight, low Apgar scores, or stillbirth in their infants. Thus, the association between combination therapy with protease inhibitors and an increased risk of very low birth weight requires confirmation.
Metacavir, a prodrug of 2′,3′-dideoxyguanosine (ddG), is a novel synthetic nucleoside analogue for the treatment of hepatitis B virus (HBV) [
9]. Many preliminary studies have showed that Metacavir displays good anti-HBV activities, both
in vitro and
in vivo [
10−
13]. It can remarkably inhibit the Hbs Ag and Hbe Ag secreted by 2215 cells and cell total HBV DNA. In ducks infected by duck hepatitis B virus (DHBV), Metacavir at the dosages of 40 and 80 mg/kg showed anti-DHBV activities, and significantly decreased the serum DHBV-DNA and intra-hepatic DHBV-DNA levels [
10]. Monkey PK/PD results showed that Metacavir is highly concentrated in liver (or liver-enriched), and thus can effectively combat HBV in the target organ with fewer side effects [
11]. The LD
50s for the orally, intravenously, and intraperitoneally administered Metacavir were 1500, 800, and 700 mg/kg, respectively, in rats. In a monkey acute toxicity experiment, a dosage of 800 mg/kg was associated with gastrointestinal toxicities and liver dysfunction; however, no death was noted. All these preliminary studies have demonstrated that Metacavir has a potential to become a new anti-HBV agent. Furthermore, we found the main target organs of the toxic effects of Metacavir were the gastrointestinal tract, liver, blood, and kidneys, and the no-observed-adverse-effect-level (NOAEL) of Metacavir for rhesus monkey was considered to be 50 mg/(kg·d) [
12]. Another of our studies found that mitochondrial injuries in a 40 mg/kg Metacavir-treated group were mild in each type of tissue, without any obvious change in mitochondrial function. At week 4 in the recovery phase, results showed that all these injuries were reversible after drug withdrawal [
13]. Nowadays, drugs like nucleoside analogues are used in pregnant women according to hazard rating classifications (A, B, C, D, X) based on risk caused by drugs in fetal rat (according to the U.S. Food and Drug Administration, FDA). It is necessary to evaluate the level of their reproductive toxicity, so we did a reproductive toxicity experiment to evaluate some related indicators. In the present study, the potential reproductive toxicity of Metacavir, including maternal toxicity, embryo-fetal developmental toxicity and teratogenicity, was assessed during gestation.
2 Materials and methods
2.1 Drugs
Metacavir (100% in powder form) were provided by Nanjing Chang’ao Company (Lot No. 20080412). This product is highly hydroscopic and should be stored at 4°C under dry conditions.
2.2 Animal grouping and treatment
Sprague-Dawley (SD) rats (160 females and 80 males, 11 weeks old) were obtained from the Institute of Laboratory Animal, Sichuan Academy of Medical Sciences (SCXK: Chuan 2004-16), and animals were raised in a 12-h light/ 12-h dark cycle with free access to food and water. During the quarantine taming stage, each cage contained 5 rats. Seven days later, the quarantine period ended. One male and two females were housed together in each cage for mating. Vaginal plugs and sperms were monitored by vaginal smears every morning. Based on this, the start of pregnancy was defined as the day when sperms were discovered. Trinitrophenol was used to do body surface symbol during mating period and administration period. Finally, we obtained 81 female rats that mated successfully. These rats got divided into 4 groups randomly: a low dose group (20), a middle dose group (20), a high dose group (21), and a solvent control group (20). Based on previous results [12] as well as the clinically recommended dose, we prepared 5, 10, and 20 mg/ml Metacavir solutions respectively, with double distilled water (according to the manufacturers recommendations), which were intragastrically administered as with10 mg/kg dose, that was 50, 100, 200 mg/kg.body weight respectively, corresponding to 10, 20, and 40 times the recommended clinical dosage for rat. In the solvent control group, the drug had been substituted with double distilled water. During the 6−15 days of pregnancy, Metacavir was administered intragastrically once every day. Females were sacrificed in the afternoon when reaching 20 days of pregnancy, and subsequently gross examination and dissection was carried out. The experimental procedures for the care and use of laboratory animals were in accordance with the guidelines of the Ministry of Science and Technology of China [
14].
2.3 F0 female observations
During the administration period (6−15 pregnant days), rats were observed once every day prior to administering the drug, as well as after the administration). The body weight at the 0 and 3 days of pregnancy, during the administration period (6, 10, 13, 16 days of pregnancy, and at 20 days of pregnancy (dissection day) were recorded.
2.4 Gross necropsy and histopathology
All F0 animals were sacrificed by decapitation under anesthesia at 20 days of pregnancy and complete gross necropsies were conducted. Ovaries, uterus (with cervix), pituitary, liver, and spleen weights were measured. All of the above-mentioned organs, as well as the vagina, were preserved in a neutral, phosphate buffered 4% formaldehyde solution. Organs or tissue showing macroscopic abnormalities were also preserved.
Fixed tissue and organs from F0 animals were processed, embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. All collected samples of the high-dose and control groups, as well as the organs and tissue showing macroscopic abnormalities, were subjected to microscopic examination.
Observations in reproductivity were recorded, which entails: gravid uterus weight, placenta gross weight, sex of live births, numbers of live births vs. stillbirths, as well as the numbers of corpus luteum, including early and late absorption fetal.
2.5 F1 fetal observation
The head, neck, pars trunk and limbs of fetal rats were examined in order to detect abnormalities. Furthermore, the weight of each rat was measured.
F1 fetal organs observation: Fetal rats were obtained in even fetal position of each litter and fixed with Bouin’s fluid. Observation was carried out to all fetal rats [
15], and sex was checked again while taking internal organs observation.
F1 fetal skeletal observation: Fetal rats were obtained in odd fetal position of each litter, then fixed with 70% alcohol. Alizarin Red S was used to dye transparent skeletal specimen according to Staples’s method [
16]. Skeletal deformation, variation and ossification were observed via dissecting microscope, and sex was checked again while removing internal organs.
2.6 Evaluation criteria
F0 female toxicity evaluation included clinical manifestation, weight and reproductive parameters. Embryo-fetal survival, fetal weight, placental weight and appearance, size, and sex proportion were evaluated to determine developmental toxicity.
2.7 Statistical analysis
Pregnant female mean weight, weight gain, fetal weight, placenta mean weight, number of corpus luteum and live births were analyzed by t-test. The numbers of stillbirths, absorption fetal rats (early, late and total numbers) were analyzed by nonparametric statistics chi-square criterion. The abortion rate, premature birth rate, maternal mortality rate and incidences of fetal malformation appearance were analyzed by t-test and χ2-test.
3 Results
3.1 F0 female rats observation
The pregnant rats of each group that had been treated with Metacavir showed no serious abnormalities. Rats of the control group were in good health, had good appetite, and behaved normal. Compared with the control group, all pregnant rats of the low dose group were normal while 5/21 in high and 2/20 in middle dose groups respectively, exhibited slower weight gain, lethargy and lack of activities. In addition, the fur on these rats appeared rough and is partly retrorse.
The mean weight and change of weight of F0 female rats can be seen in Table 1. In the present study, we observed Metacavir-associated weight changes; each group treated with Metacavir had a lower calibration weight gain than the control group during 6−20 days. Compared with the control group, the calibration weight gain were not significantly different in low dose group. However, in the middle and high dosage groups, the differences were indeed significant (P<0.05, P<0.01, respectively).
3.2 Gross necropsy and histopathology
No significant differences were observed in the relative weights of any of the reproductive organs in female rats. No Metacavir-related macroscopic or microscopic findings were observed in the F0 female rats (Table 2).
3.3 Influence to reproductivity of pregnant rats
The reproductivity of pregnant rats was listed in Table 3. There were no miscarriages or premature births, whether in the control group or the treated groups. Each treated group had no Metacavir-associated changes in uterine implantation. The mean corpus luteum, nidation, live births, absorption fetal rat, placental weight, number of F0 female rats with live births, and the percentage of F0 female rats with absorption fetal rat, were not significantly different from the control group (P>0.05). There was one F0 female rat with 6 stillbirths in the high dose group, causing the number of stillbirths in the group to become significantly different from the control group (P<0.05). The group incidence was 1/20, and there was no significant difference compared with control group.
3.4 Influence to F0 fetal rat growth and development
Mean fetal weight of the low and middle dosage groups were not significantly different from the control group. The high dose group was lower than the control group, but the difference was not significant. There were no Metacavir-associated changes in sexuality, compared to the control group, and the ratio of female to male were similar (Table 4). No abnormal appearances caused by Metacavir were observed in the present study. Fetal rats in both the control group and treated groups were normal in appearance.
No visceral abnormalities involving Metacavir were observed in the present study (Tables 5 and 6). The control group had 5 fetal rats with pyelectasis and 3 fetal rats with cerebral ventricle broadening; low dose group had 7 fetal rats with pyelectasis and 5 fetal rats with cerebral ventricle broadening; middle dose group had 5 fetal rats with pyelectasis and 3 fetal rats with cerebral ventricle broadening; high dose group had 7 fetal rats with pyelectasis and 2 fetal rats with cerebral ventricle broadening. The cleft palate, single nasal meatus, nanophthalmos, exencephaly, cardiac anomalies, bladder broadening, and so on, were all normal, except for cerebral ventricle and pelvis.
No Metacavir-associated skeletal deformities were found in any treated group (Tables 6 and 7). While some fetal rats in each treated group displayed ossification variations, the incidence of litter showed no statistically significant difference, which was unrelated to administration.
4 Discussion
Compared with the control group, the calibration weight gain of F0 rats were lower in all treated groups. In the middle and high dose groups, the differences were significant, and they showed slower weight gain, reduced activity, and depression, suggesting that these were Metacavir-associated toxic effects. The results, showing that the main target organs of the toxic effects of Metacavir were the gastrointestinal tract and the liver, were in accordance with our previous repeated dose toxicity study on Metacavir in rhesus monkeys [
12].
There was no Metacavir-associated toxic effect on organ indexes, nor were there any pathological changes, showing that the drug has no obvious side effects on pregnant rats’ genital systems. These findings are consistent with our long-term toxicity study on Metacavir in rhesus monkeys [
12]. As for digestive system pathology changes observed in the previous study in a high dosage group (200 mg/kg) were not seen in the present study. This may however be related to the difference in administration time and/or animal species. Reproductivity of each treated group did not show any statistical difference related to administration, compared with the control group, thus indicating that Metacavir has no toxic effects on reproductivity of pregnant rats. Although the number of stillbirths in the high dosage group was significantly higher than the control group (
P<0.05), which was caused by one pregnant rat whose fetal rats were all stillbirths, the litter incidence rate was low (1/20). There was no significant difference in the incidence rate compared with the control group, thus we speculate that the observed results are not associated with the administration. Our previous work demonstrated that lamivudine had no effect on reproductivity (regardless of sex), but when pregnant rabbits were treated with dose equivalent to human, the risk of early embryonic death would increase. In addition, there is no evidence that telbivudine impairs reproductivity when female rats are exposed to telbivudine at a concentration equal to 14 times that of a human therapeutic dose. All these results together with the present findings indicate that the effect on reproductivity caused by using nucleoside analogues during the gestational period is controversial, requiring more research, especially direct clinical data from pregnant women.
The weight of live births of each treated group were not significantly different from the control group (P>0.05), and no fetal rats showed signs of visceral abnormalities caused by the administration, thereby indicating that Metacavir has no teratogenic effect on fetal’s growth and development. Furthermore, no Metacavir-associated skeletal deformities were found in any treated group. As these anomalies occurred sporadically in each group and had no relation, we concluded they had no relation to the administration [17]. The neonatal physical check-up revealed no congenital anomalies, and fetal growth was within normal reference ranges, suggesting that lamivudine may be safely used in the treatment of chronic hepatitis B with acute exacerbation during the second trimester of pregnancy. Vein administration of adefovir to pregnant rats at a dose that can produce obvious maternal toxicity (equivalently to 38 times of the human recommended dose), will increase the incidence of embryo toxicity and fetal abnormity (general, eye-vesicle introcession, exomphalocele, and tail kink). In preclinical studies, the telbivudine-associated teratogenic effects and any harmful effects to embryo and fetal growth have not been seen. The rate of birth defects in more than 400 women exposed to zidovudine or lamivudine during the first trimester of pregnancy, as reported to the Antiretroviral Pregnancy Registry, was no higher than the rate of birth defects of women who were exposed to similar drugs after the first trimester, as reported in the Metropolitan Atlanta Congenital Defects Program of the Centers for Disease Control and Prevention [18]. These data suggested that our evaluated drug and those already on the market are all safe to human. According to hazard rating classifications (A, B, C, D, X) based on the risk caused by drugs in fetal rat (FDA), the nucleoside analogue telbivudine belongs to B, while the others belong to C. The newly investigated drug, Metacavir, should belong to C since there is a lack of evidence showing side effects in humans. It should be noted that there has not been enough control studies regarding gravidity. Since animal toxicity studies do not always prognosticate human effects, we could only utilize the new drugs during the gestational period, as long as the benefits exceeded the risks.
5 Conclusions
In the present study we concluded that Metacavir may induce maternal toxicity at 200 mg/kg body weight(i.e., 40 times the recommended clinical dosage for rats) to Sprague-Dawley rats, and that the major effect was slower weight gain. The embryo toxicity showed slight weight loss between each individual, but the statistics were not significantly different from the control group. Compared with the control group, the 200 mg/kg body weight group had an increased rate of stillbirths by 2.87% (P ≤0.05), although the litter incidence showed no statistical significance. The appearance, organs and skeleton of each treated group were all normal; indicating that a dose lower than 200 mg/kg.body weight of Metacavir is neither maternally toxic, teratogenic, nor affects embryo-fetal development in pregnant Sprague-Dawley rats after oral administration during the first 6−15 days of pregnancy.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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