Vascular alteration in relation to fosfomycine: In silico and in vivo investigations using a chick embryo model.

Fosfomycin residues are found in the egg following administration in the layer hen. In this regard, some aspects of embryo-toxicity of fosfomycin have been documented previously. The exact mechanism by which fosfomycin causes embryo-toxicity is not clearly understood. We hypothesis that fosfomycin may alter vasculature as well as normal expression of genes, which are associated with vascular development. Therefore, the present study aimed to address these issues through in silico and in vivo investigations. At first, embryo-toxicity and anti-angiogenic effects of fosfomycin were tested using computerized programs. After that, fertile chicken eggs were treated with fosfomycin and chorioallantoic membrane vasculature was assessed through morphometric, molecular and histopathological assays. The results showed that fosfomycin not only interacted with VEGF-A protein and promoter, but also altered embryonic vasculature and decreased expression level of VEGF-A. Reticulin staining of treated group was also confirmed decreased vasculature. The minor groove of DNA was the preferential binding site for fosfomycin with its selective binding to GC-rich sequences. We suggested that the affinity of fosfomycin for VEGF-A protein and promoter as well as alteration of the angiogenic signaling pathway may cause vascular damage during embryonic growth. Hence, veterinarians should be aware of such effects and limit the use of this drug during the developmental stages of the embryo, particularly in breeder farms. Considering the anti-angiogenic activity and sequence selectivity of fosfomycin, a major advantage that seems to be very promising is the fact that it is possible to achieve a sequence-selective binding drug for cancer.

Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report the application of two amino acid based prodrug approaches on a fosmidomycin surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei malaria mouse model. These results indicate good in vivo antiplasmodial potential.

Effects of different antibiotics on the uterine contraction and the expression of aquaporin 5 in term pregnant rat.

Aquaporin (AQP) water channels are small hydrophobic integral membrane proteins. AQP5 expression, which is regulated by oxytocin, showed a dramatic down-regulation at the term and preterm uterus. Since antibiotics are among the drugs to treat intrauterine infections, our aim was to study the effects of antibiotics on AQP5 and uterine contractility on 22-day pregnant rats. The change in uterine AQP5 expression was investigated by PCR and Western blot techniques. Uterine contractility was tested in an organ bath system. 7 days of pre-treatment with amoxicillin or single dose of fosfomycin decreased the AQP5 protein level, while 7 days of treatment with doxycycline had no effect. Fosfomycin or amoxicillin pre-treatments enhanced, while doxycycline pre-treatment did not alter the oxytocin-induced contractions. Amoxicillin and fosfomycin may sensitize the uterus to oxytocin via the reduction of AQP5 expression. This synergism might have importance during the pharmacotherapy of infection-related preterm birth.

FR-900098, an antimalarial development candidate that inhibits the non-mevalonate isoprenoid biosynthesis pathway, shows no evidence of acute toxicity and genotoxicity.

FR-900098 is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase, the second enzyme in the non-mevalonate isoprenoid biosynthesis pathway. In previous studies, FR-900098 was shown to possess potent antimalarial activity in vitro and in a murine malaria model. In order to provide a basis for further preclinical and clinical development, we studied the acute toxicity and genotoxicity of FR-900098. We observed no acute toxicity in rats, i.e. there were no clinical signs of toxicity and no substance-related deaths after the administration of a single dose of 3000 mg/kg body weight orally or 400 mg/kg body weight intravenously. No mutagenic potential was detected in the Salmonella typhimurium reverse mutation assay (Ames test) or an in vitro mammalian cell gene mutation test using mouse lymphoma L5178Y/TK(+/-) cells (clone 3.7.2C), both with and without metabolic activation. In addition, FR-900098 demonstrated no clastogenic or aneugenic capability or significant adverse effects on blood formation in an in vivo micronucleus test with bone marrow erythrocytes from NMRI mice. We conclude that FR-900098 lacks acute toxicity and genotoxicity, supporting its further development as an antimalarial drug.

[Experimental study of the ototoxicity of fosfomycin].

Two tenths ml of 5% fosfomycin sodium solution (10 mg) was injected into the tympanic cavity of guinea pigs once a day for 7 days. Auditory brain stem response (ABR) was performed to examine the effect of the drug on the experimental animals. Additionally, a scanning electron microscopic study was also performed to observe the effect of the drug on outer hair cells of the organ of Corti and on vestibular sensory epithelia. No degenerative changes were observed in outer hair cell cilia of the organ of Corti and the vestibular sensory cilia at 1 day, 7 days, 14 days and 1 month after injection. No changes of the threshold of ABR were also noticed.

[Effect of fosfomycin-calcium on reproductive performance of rats. III. Peri- and post-natal examination (author's transl)].

The peri- and post-natal examination of fosfomycin-Ca (FOM-Ca) was undertaken in Wistar strain rats. Rats were treated orally at dose levels of 140, 1,400 and 2,800 mg/kg/day from the 14th day of gestation to 21st day after delivery. One-third of pregnant rats in each group were sacrificed on 20th day of gestation and then their fetuses were examined for external, visceral and skeletal observation. The remaining mothers were allowed to deliver naturally, and then their offsprings were examined for postnatal development. No effect of FOM-Ca treatment to rat mothers was found except soft stool was seen in 1,400 and 2,800 mg/kg groups. No effect of FOM-Ca on rat fetuses and newborns was found except fetal body weight and survival rate decreased and skeletal variation increased in maximum dose. Consequently, it can be concluded that FOM-Ca has no effect on rat mothers, fetuses, and newborns treated during peri- and post-natal period.

[Effect of fosfomycin-calcium on reproductive performance of rats. II. Fertility test (author's transl)].

Fosfomycin calcium (FOM-Ca) was orally administered to Wistar rats for 60 consecutive days in males and 14 consecutive days in females in varying doses of 140 mg/kg, 700 mg/kg and 1,400 mg/kg, and then the animals were subjected to mating. It was further administered to pregnant female rats for another week to investigate its effects upon the embryos and fetus. The following results were obtained. 1) In parent animals, soft stools were noted in both sexes of the groups with higher doses, but otherwise there was no remarkable abnormality noted. 2) No increase was seen in the rates of development of dead embryos or externally abnormal fetus. 3) Skeletal abnormality was seen only slightly in the animals of the group with 700 mg/kg, but otherwise all the other groups were similar to the control group, showing that there would be no adverse effects by the tested drug upon the fetal skeletal structure. 4) On the ground of the above-mentioned test results, it was claimed that FOM-Ca exerts adverse effects upon the fetus of rats even in the dose of 700 mg/kg and that the safe dose to fetus of rats would be 140 mg/kg. It was also judged that it has no teratogenicity through administration before or at the initial stage of gestation.

[Effect of fosfomycin-calcium on reproductive performance of rat and rabbit: teratogenicity test (author's transl)].

The teratogenicity study of fosfomycin-Ca (FOM-Ca) was undertaken in Wistar strain rats and JW strain rabbits. Rats were treated orally at dose levels of 140,700 and 1,400 mg/kg/day from 7th to 17th day of gestation, and rabbits were treated orally at dose levels of 80, 140 and 420 mg/kg/day from 6th to 18th day of gestation. In the case of rats, two-thirds of pregnant mothers in each group were sacrificed on 20th day of gestation and then their fetuses were examined for external, visceral and skeletal observation. The remaining mothers were allowed to deliver naturally, and then their offsprings were examined for postnatal development. In the case of rabbits, all pregnant mothers were sacrificed on 29th day of gestation and their fetuses were examined. No effect of FOM-Ca treatment to rat and rabbit mothers was found except soft stool was seen with maximum dose in rats. Dead or resorbed rate of fetuses increased, external anomalies (short tail, abdominal hernia) were found and skeletal anomalies slight increased with maximum dose in rats. However, there was no significant difference from the control or background data. While, no effect of FOM-Ca treatment was observed in rabbits and rat offsprings. Consequently, it can be concluded that FOM-Ca has no teratogenicity effects on rats and rabbits.

[Effect of fosmocyin-Na on reproductive performance of rats and rabbits. I. Teratogenicity test (author's transl)].

The teratogenicity study of fosfomycin-Na (FOM-Na) was undertaken in Wistar rats and New Zealand white rabbits. Rats were treated intraperitoneally at dose levels of 125, 250, 750 and 1,500 mg/kg/day from day 7 to day 17 of gestation, and rabbits were treated intravenously at dose levels of 80, 100, 200, 400 and 800 mg/kg/day from day 6 to day 18 of gestation. In the case of rats, two-thirds of pregnant mothers in each group was sacrificed on day 20 of gestation and then their fetuses were examined for external, visceral and skeletal observation. The remaining mothers were allowed to deliver naturally, and then their offsprings were examined for postnatal development. In the case of rabbits, all pregnant mothers were sacrificed on day 29 of gestation and their fetuses were examined. Body weight of rat mothers during gestation were decreased and 4 mothers were dead until day 20 of gestation in the maximum dose. In this dose, foetal toxicity was recognized too. However, external, visceral and skeletal anomalies related with FOM-Na treatment were not observed in all groups. No effect on development of offsprings was observed. No effect of treatment of FOM-Na to rabbits was found except foetal body weight was slightly decreased in the maximum dose.

[Effect of fosfomycin-Na on reproductive performance of rats. II. Fertility test (author's transl)].

The fertility study of fosfomycin-Na (FOM-Na) was undertaken in Wistar rats. FOM-Na was administered intraperitoneally at dose levels of 125, 250, 750 and 1,500 mg/kg/day. The male rats were continuously treated with FOM-Na from 63 days before mating and the females were treated from 14 days before mating. The males and females were treated through the mating period and then the pregnant rats were treated until 7 days of gestation. All pregnant rats were sacrificed on day 20 of gestation and then their fetuses were examined for external, visceral and skeletal observation. Mean body weight of males decreased and 6 males and 3 females died in the maximum dose. However, no significant differences were found between treated groups and the control with regard to fertility rate and pregnant rate. External, visceral and skeletal anomalies related with FOM-Na treatment were not observed. In this experiment, no effects of FOM-Na for reproductive performance in rats were observed.

[Effect of fosfomycin-Na on reproductive performance of rats. III. Peri- and post-natal examination (author's transl)].

The peri- and post-natal study of fosfomycin-Na (FOM-Na) was undertaken in Wistar rats. FOM-Na was administered intraperitoneally at dose levels of 250, 750 and 1,500 mg/kg/day from day 14 of gestation to day 21 after delivery. It could be concluded that delivery rate in the 750 and 1,500 mg/kg groups decreased, but no influence of FOM-Na was found on postnatal development of offspring (F1) and neonate (F2) without maximum dose.

[Toxicological studies on fosfomycin-Na salt. II. Subacute toxicity in rats and rabbits (author's transl)].

FOM-Na solution in distilled water for injection, J.P., with its pH adjusted to 7.0 +/- 0.2 with diluted hydrochloric acid, was administered to rats and rabbits for subacute toxicity test. The results revealed the following: 1. It was intraperitoneally administered to Wistar rats each weighing 100 +/- 10 g at their age of 5 weeks, and intravenously into auricular veins of male albino rabbits each weighing about 3 kg, for 35 successive days except Sundays through one administration per day. There was no death in rats of both sexes with the doses less than 1,000 mg/kg, while 3/10 males and 5/10 females died with the dose of 2,000 mg/kg. In terms of general conditions, both stretched physical position and vocalization were noted, which were presumed to be attributable to the stimuli of the administration. In the postmortem examination mutual adhesion of organs in the peritoneum was noted, while in the lightmicroscopic examinations histological proliferation and adhesion were found out in the hepatic capsules or serous membrane of the intestine etc., but no abnormalities were detected in the other organs. In the mean body weights, there were no significant differences between the control group and the groups of males with doses less than 500 mg/kg and females with doses less than 1,000 mg/kg whereas in the groups of males with doses more than 1,000 mg/kg and the females with a dose of 2,000 mg/kg, a trend of reduced weight gain was noted in comparison with the control group. The feed intake also was reduced as the dose was elevated. In terms of the male group hematology, the In. P increased with doses higher than 250 mg/kg, while BUN was reduced in the groups with a dose of 250 mg/kg and doses higher than 1,000 mg/kg, and Na was reduced in the groups with doses from 125 up to 500 mg/kg. In the female groups, the loss of Hgb and rise in the Cl were noted in the doses higher than 500 mg/kg while the loss of WBC was noted in almost all the treated groups. However, none of these changes was suggestive of specific abnormalities when compared with the photomicroscopic findings and our hematological background data. 2. There were no significant changes in the general conditions of any group of rabbits. Their mean body weights and their mean feed intakes proceeded almost similarly with those of the control group. In the hematological and histopathological tests also, no specific abnormal finding was experienced, which were deemed to be attributable to the administration of FOM-Na.

[Toxicological studies on fosfomycin-Na salt. I. Acute toxicity in mice and rats (author's transl)].

The acute toxicity of sodium fosfomycin (FOM-Na), a new antibiotic in ICR mice and Wistar rats has been investigated. The LD50 values in mice were: 1,230 (male), 1,225 (female) mg/kg by i.v.; 2,175 (m), 2,467 (f) mg/kg by i.p.; 2,625 (m), 2,662 (f) mg/kg by i.m.; 5,100 (m), 6,150 (f) mg/kg by s.c. and 8,020 (m), 7,300 (f) mg/kg by p.o. The LD50 values in rats were: 1,650 (m), 1,560 (f) mg/kg by i.v.; 2,060 (m), 2,000 (f) mg/kg by i.p.; 2,630 (m), 2,460 (f) mg/kg by i.m.; 5,100 (m), 4,320 (f) mg/kg by s.c. and 4,700 (m), 4,550 (f) mg/kg by p.o. Animals dosed only i.v. died within 2 minutes. Signs of toxicity in mice or rats given FOM-Na were similar and included motor activity depression, reduced respiration and occasionally tremors. Surviving mice or rats given FOM-Na developed no pathological changes of the drug specificity.

Fosfomycin, antimicrobial activity in vitro and in vivo.

Antibacterial activities of fosfomycin were investigated both in vitro and in vivo for the purpose of comparative evaluation on its fundamental properties with other antimicrobial agents. The MIC was determined with nutrient agar (Difco) inoculated with one loopful of 1,000-fold dilution (about 10(6) cells/ml) of bacterial suspension cultured overnight in nutrient broth. This substance showed antibacterial activity to most gram-positive and gram-negative bacteria, being strongest to Enterobacteriaceae with a peak of the MIC at 1.56 mug/ml in Salmonella. It was also active against P. aeruginosa with a peak of the MIC at 6.25 mug/ml in its sensitivity distribution. Intravenous and subcutaneous fosfomycin Na salt and oral fosfomycin Ca salt were given to 5-week-old ddN strain male mice challenged with clinical isolates of P. aeruginosa, E. coli and P. mirabilis. Therapeutic effect was observed in all these test organisms. In P. aeruginosa, it was more effective than carbenicillin.