Natan Patlas

Transplacental effects of bisphosphonates on fetal skeletal ossification and mineralization in rats

Bisphosphonates are clinically used mainly to reduce bone resorption. We studied the transplacental effects of two bisphosphonates on the fetal skeleton in rats. Pregnant rats were treated during days 11-20 of pregnancy with daily subcutaneous injections of 0.1 mg/kg of alendronate or a newly synthesized bisphosphonate, VS-b6. This period of pregnancy was chosen because the active development of bones from mesenchyme through cartilaginous models occurs during that time. Histological examination of midlongitudinal sections of the 21-day-old fetuses showed an increase in the amount of diaphyseal bone trabeculae with slight shortening of the diaphysis in the experimental fetuses, in comparison to controls. Computerized histomorphometric studies similarly showed an increase in the amount of diaphyseal bone trabeculae with a concomitant decrease in bone marrow volume, but no change in cartilage volume. In addition, chemical analysis of the fetal bones showed an increase in calcium content in the treated fetuses. 14C-alendronate was shown to pass through the rat placenta and accumulate in the fetuses, most probably in their bones. This is presumed because bisphosphonates are known to accumulate in bone, being stored there for long periods of time. It is important, in light of our results, to give careful consideration to the treatment of women with bisphosphonates at childbearing age, whenever this is needed.

Synthesis and preclinical pharmacology of 2-(2-aminopyrimidinio) ethylidene-1,1-bisphosphonic acid betaine (ISA-13-1)-a novel bisphosphonate.

AbstractPurpose. To validate our hypothesis that a bisphosphonate (BP) having a nitrogen-containing heterocyclic ring on the side chain, and with no hydroxyl on the geminal carbon would possess increased activity, and better oral bioavailability due to enhanced solubility of its calcium complexes/salts and weaker Ca chelating properties. Methods. A novel BP, 2-(2-aminopyrimidinio)ethylidene-l,l-bisphosphonic acid betaine (ISA-13-1) was synthesized. The physicochemical properties and permeability were studied in vitro. The effects on macrophages, bone resorption (young growing rat model), and tumor-induced osteolysis (Walker carcinosarcoma) were studied in comparison to clinically used BPs. Results. The solubility of the Ca salt of ISA-13-1 was higher, and the log βCa: BP stability constant and the affinity to hydroxyapatite were lower than those of alendronate and pamidronate. ISA-13-1 exhibited effects similar to those of alendronate on bone volume, on bone osteolysis, and on macrophages, following delivery by liposomes. ISA-13-1 was shown to have 1.5−1.7 times better oral absorption than the other BPs with no deleterious effects on the tight junctions of intestinal tissue. Conclusions. The similar potency to clinically used BPs, the increased oral absorption as well as the lack of effect on tissue tight junction of ISA-13-1 warrant its further consideration as a potential drug for bone diseases.

Oophorectomy-Induced Osteopenia in Rats in Relation to Age and Time Postoophorectomy

Oophorectomized (OVX) rats served for many years as a popular model for ‘postmenopausal’ osteoporosis in spite of the fact that the rat continues to grow during these experiments. We performed OVX in rats at 1, 3, 6 and 10 months of age and compared the histomorphometric (bone size, bone trabecular and cartilage volume in different areas) and chemical (ash, Ca, P and Mg content) parameters at 2, 6, 8 and 20 weeks post-OVX (2–20 weeks) to those of sham-operated rats. Significant differences were observed only in the animals that were OVX at a young or young-mature age, i.e. mainly at 1 and 3 months and some of the rats at 6 months of age. There were no changes in bone ash and mineral contents in the OVX animals in comparison to sham-operated rats, except 2 weeks post-OVX in the 1-month-old rats where these variables were reduced. The most significant finding was a reduction in the metaphyseal bone volume. This was observed in the rats OVX at 1, 3 and 6 months of age, but not in those OVX at 10 months. In the young OVX rats there was also an increase in epiphyseal cartilage volume. The epiphyseal and diaphyseal bone volumes were not different between the groups at any time postsurgery, explaining the lack of differences in bone ash and mineral contents. There were no significant changes in the results of the histomorphometric studies between OVX and sham-operated rats when surgery was performed at 10 months of age. Since OVX exerts significant changes only in young rapidly growing rats, this approach is an inappropriate model for postmenopausal osteoporosis, which occurs long after bone growth has ended.

Specific β Estradiol Binding in Cartilage and Serum from Young Mice and Rats is Age Dependent

Various studies have shown a direct effect of beta estradiol on cartilage and bone. Such effects point to the possibility that specific receptors to estradiol exist in the growth plate cartilage as well as in bone. 3H-estradiol specific binding (EB) was therefore investigated in the supernatant of cartilage homogenates from the epiphyses and ribs of young growing mice and rats. High levels of EB were observed in the cytosol fraction of cartilage homogenates in the late fetal stage and in young rats and mice. The EB levels decreased gradually from late fetal stage up to 14 days of age in both groups of animals independent of their sex. Nuclear binding of 3H estradiol was also demonstrated by autoradiography in the chondrocytes of proliferating and hypertrophic zones. Estradiol binding was inhibited by high doses of unlabelled beta-estradiol, but not by alpha estradiol. Binding was also inhibited by tamoxifen and DES but not by testosterone. High levels of estradiol binding (EBS) were also observed in serum from young animals, but not in animals 2 months of age or older. Study of estradiol binding in cartilage and in serum of rats of the same age showed a significant difference in estradiol binding between these two systems. The difference in estradiol binding between serum and cartilage was seen in the response to inhibitors, Scatchard analysis, and temperature dependence. The results of our study imply that there are specific receptors for 17 beta estradiol in growth plate cartilage; they originate from chondrocytes, and their amount decreases with age. The effects of estradiol on endochondral bone growth seems therefore to be receptor mediated.

A high oleic sunflower oil fatty acid esters of plant sterols mixed with dietary diacylglycerol reduces plasma insulin and body fat accumulation in Psammomys obesus

BackgroundMetabolic syndrome is associated with subsequent development of cardiovascular diseases and type 2 diabetes. It is characterized by reduced response to insulin, central obesity, and dyslipidemia. Intake of plant sterols (PS) has been shown to confer a healthier lipid profile and ameliorate cardiovascular disease risk factors in experimental animals and humans. In this study we used an animal model of type 2 diabetes to assess the effects of a preparation of PS esterified to high oleic sunflower oil fatty acids mixed with dietary diacylglycerol (PS-HOSO) on diabetic related metabolic parameters. Psammomys obesus (P. obesus) were fed high energy (HE) diet supplemented by either PS-HOSO or control oil. Following 4.5 weeks of intervention, animals were divided into fasting and non-fasting modes prior to outcome measurements. Glucose and insulin levels as well as blood lipid profile, body weight, and fat accumulation were evaluated in fasting and non-fasting modes.ResultsP. obesus fed with a HE diet displayed a characteristic heterogeneity in their blood glucose and insulin levels with a subset group displaying type 2 diabetes symptoms. PS-HOSO treatment significantly reduced total cholesterol (24%, P < 0.001) and non-HDL cholesterol (34%, P < 0.01) compared to the control diet. Among fasting animals, body weight at end point and epididymal fat-to-liver weight ratio were significantly (P < 0.05 each) reduced (7% and 16%, respectively) compared to controls. Interestingly, fasting blood glucose levels were similar between groups, whereas plasma insulin level at end point was 44% lower in the PS-HOSO group compared to control group (P < 0.0001)ConclusionPS-HOSO supplementation to diabetes-prone gerbils counteracts the increase in body weight and epididymal fat accumulation, and also results in a drop in circulating insulin levels. These effects are pointing out that PS-HOSO may serve as a functional ingredient for metabolic syndrome or diabetic sufferers, which not only influences body weight, but also prevents or reverses insulin resistance and hyperlipidemia.

The response to sex steroid hormones and vitamin D of cultured osteoblasts derived from ovariectomized mice with and without 17β-estradiol pretreatment

This study investigated whether 17β-estradiol (E2) may have different effects on osteoblasts derived from estrogen-deficient ovariectomized (OVX) mice compared to sham-operated normal animals. We studied the specific effects of 17β-estradiol on the differentiation and function of cultured osteoblasts derived from these groups of animals, with or without estrogen replacement treatment. One-month-old mice were ovariectomized or sham-operated, and treated (every second day) for 4 weeks with 0.5 mg/kg 17β-estradiol or with vehicle alone. At the end of the experiment, bones were removed for primary osteoblast cultures or for morphological and chemical evaluation. In cells from untreated OVX animals, alkaline phosphatase (ALP) specific activity was reduced, while collagen production and mineralization were unchanged when compared to cells from controls. In vivo estrogen pretreatment of the OVX mice elevated ALP activity and mineralization of the cells, while collagen production was reduced. The addition of 17β-estradiol to the culture medium increased ALP activity, collagen production, and mineralization by all cultured osteoblasts, except in those derived from sham-operated, estrogen-pretreated mice, where these features remained unchanged. Osteocalcin production was unchanged. Addition of testosterone or 1,25(OH)2D3 to the culture medium induced changes that differed among the groups depending on the source of the cultured cells. It seems that ovariectomy in mice prior to culture affected the phenotype of the cultured osteoblasts and their response to estradiol, testosterone, and 1,25(OH)2D3, depending on whether animals were pretreated with estradiol or not. These results imply that the animal’s estrogen status prior to culture can influence the response to estrogens; this finding may have important implications for hormone replacement therapy (HRT) in postmenopausal women.

Pregnancy outcome in the Psammomys obesus gerbil on low- and high-energy diets

Introduction: Diabetes mellitus (DM) during pregnancy is associated with an increased risk for poor reproduction and a high rate of congenital malformations. The gerbil Psammomys obesus is a unique model for nutritionally induced Type 2 DM (T2DM) that enabled us to study the outcome of uncontrolled T2DM during pregnancy. Methods: Female Psammomys on low-energy (LE) or high energy (HE) diet were studied. The blood glucose levels and weights of pregnant animals were determined. The offspring from the different groups were followed-up to weaning. Results: Most of the HE-diet animals were diabetic (77%). There were no differences in the pregnancy rates in animals on both diets (32.7% in HE vs. 38.3% in LE). Pregnancy of the HE-diet group was longer than the LE-diet group (26.7 vs. 26.1 days), and litter average was reduced (2.7 vs. 3.0). At birth, the offspring of the HE-diet dams weighed less (5.2 vs. 7.2 g) and had smaller crown rump length (4.0 vs. 4.6 cm) These offspring also presented a 1–3 days delay in neuro-developmental parameters (first turn over, hair appearance, eye-opening and response to noise). However, from the fourth week of life they became diabetic, and from the third week they weighed more than the LE offspring. Conclusion: HE-diet caused diabetes, maternal complications and altered reproduction in Psammomys animals. The offspring of diabetic Psammomys presented birth weight and length changes as well as developmental delay.

Effects of Continuous or Cyclic Administration of Pamidronate on the Skeleton in Intact and Oophorectomized Young Rats

Bisphosphonates are potent inhibitors of bone resorption, and are therefore used for the treatment of various bone diseases including osteoporosis. We examined whether cyclic therapy with bisphosphonates in oophorectomized osteoporotic rats had any advantage over continuous treatment. We therefore treated intact and oophorectomized young female rats for 8 weeks with 1 and 5 mg/kg/day of pamidronate. The 8-week treatment was given continuously for 6 days/week or intermittently, i.e. 6 days of pamidronate (APD) and 3 weeks off, for 2 cycles. We found an increase in tibial wet and ash weight and in the mineral content in oophorectomized rats treated continuously or intermittently with APD in comparison to nontreated oophorectomized animals. Histomorphometric analysis showed an increase in the volume of metaphyseal cartilage and bone. No changes were found in the volume of epiphyseal or diaphyseal bone. Pamidronate had very little effect on the bone of intact rats. Pamidronate seems to be more effective in inhibiting bone resorption in bone that undergoes rapid turnover (i.e. in oophorectomized animals) when compared to bone with low turnover (intact rats). Although the results of cyclic treatment were similar to those of continuous treatment, we have to remember that cyclic therapy may be more advantageous since animals receiving cyclic therapy received only 25% of the dose of rats continuously treated.