Paul C Francis

Skeletal Changes in Rats Given Daily Subcutaneous Injections of Recombinant Human Parathyroid Hormone (1-34) for 2 Years and Relevance to Human Safety

Fischer 344 rats (60/sex/group) were given daily subcutaneou s injections of recombinant human parathyroi d hormone (PTH)(1-34) for 2 years at doses of 0, 5, 30, or 75 μg/kg. Treatment caused substantial increases in bone mass consistent with the known pharmacologic effects of once-daily administration. As determined by quantitative computed tomography (QCT) and histomorphometry, bone mass was markedly increased. Substantial new bone formation resulted in a large decrease in marrow space accompanied by altered bone architecture. Bone proliferative lesions were observed in all PTH(1-34)-treated groups. Osteosarcoma occurred in 3, 21, and 31 male rats and in 4, 12, and 23 female rats in the 5-, 30-, and 75- μg/kg treatment groups, respectively. Focal osteoblast hyperplasia, osteoma, and osteoblastoma were much less frequent. Although the specific cellular or molecular mechanisms responsible for the rat bone tumors have not been fully elucidated, the data suggest that these lesions resulted from the long duration of treatment and the exaggerated pharmacologic response of the rat skeleton to daily treatment with PTH(1-34). Important differences between the rat study and clinical use in adult humans suggest that the increased incidence of bone neoplasia in rats treated for 2 years is likely not predictive of an increased risk of bone cancer in skeletally mature adult humans being given PTH(1-34) for a limited period of time in the treatment of osteoporosis.

The selective estrogen receptor modulator, raloxifene: an overview of nonclinical pharmacology and reproductive and developmental testing.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator being developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. In the ovariectomized (OVX) rat, raloxifene prevents the loss of bone at the distal metaphysis of the femur, proximal tibia, and vertebrae; reduces cancellous bone resorption; and reduces serum cholesterol, but does not cause any significant changes in stromal eosinophilia or uterine epithelium. In estrogen-stimulated OVX rats, raloxifene prevents the morning lowering of serum luteinizing hormone levels, produces a reduction in afternoon serum prolactin levels, antagonizes pituitary weight increase, and antagonizes stimulation of mammary gland development. Raloxifene also has been shown to exhibit antiestrogenic activity in several in vivo and in vitro mammary tumor models. Raloxifene treatment results in regression of endometriosis in both a surgically prepared, rat uterine explant model and in Rhesus macaques diagnosed with spontaneous endometriosis before exposure. Also, uterine leiomyomas in estrogen-stimulated OVX guinea pigs regress after the onset of raloxifene treatment. Raloxifene antagonizes testosterone-induced increases in prostate weight of castrated rats, but does not bind to androgen receptors or affect prostatic 5-alpha-reductase or testicular steroid 17-alpha-hydroxylase activity. A series of preclinical toxicology studies was designed to characterize reproductive and developmental outcomes following various schedules of raloxifene treatment in rats or rabbits. Studies of female reproduction and developmental outcome were conducted primarily at pharmacologic doses (0.1, 1, or 10 mg/kg/d); male reproductive studies used higher doses (10, 30, or 100 mg/kg/d). In this series of studies, male reproductive end points were not affected, whereas embryo implantation, fetal rabbit morphology, and several aspects of offspring development were disrupted by the lowest dose of maternal raloxifene treatment, a profile consistent with estrogen antagonist activity.

Proliferative Lesions of Ovarian Granulosa Cells and Reversible Hormonal Changes Induced in Rats by a Selective Estrogen Receptor Modulator

This study assessed the effects of raloxifene. a selective estrogen receptor modulator (SERM), on ovarian morphology and circulating hormone levels in rats. Female Fischer-344 rats (65/group) were given dietary raloxifene for 6 months at average daily doses of 0, 15, 75, and 365 mg/kg. Morphologic evaluation of ovaries was conducted on 25 rats/group at the end of the treatment period and from 20 rats per group after 1 and 3 months withdrawal from treatment. Plasma hormone analyses were conducted on 10 rats per group at the end of the treatment period and after each withdrawal period. Treatment with raloxifene for 6 months resulted in disruption of the hypothalamic-pituitary-ovarian axis, manifested by increased plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E2), and failure of ovulation, manifested by ovarian follicular prominence (retained anovulatory follicles), lack of corpora lutea (CL), and depressed plasma progesterone (P4). Many (56% to 80%) rats in all raloxifene treated groups had focal, minimal to slight hyperplasia of granulosa cells within individual retained follicles. A few treated rats in the mid- and high-dose groups (2 of 25 and 3 of 25, respectively) had more extensive focal proliferation of granulosa cells. These foci were approximately 3 to 6 mm in overall size and were characterized by moderate papillary proliferation of large granulosa cells associated with cystic spaces, often with hemorrhage. In 4 of the 5 rats with this focal cystic granulosa cell hyperplasia, the remainder of the involved ovary and the contralateral ovary were atrophic. After 1 or 3 months of drug withdrawal, most previously treated rats examined had morphologic evidence of ovarian cyclic changes, including developing follicles, various stages of CL, and normal plasma levels of LH, E2, and P4. Continued lack of cyclic changes was limited to 4 of 20 rats from the low-dose group after 1 month of recovery and to 1 low dose rat after 3 months. Intrafollicular granulosa cell hyperplasia was not seen in rats in the reversibility phase. Areas of prior focal cystic granulosa cell hyperplasia were represented by focal sclerosis that included hemorrhage and/or hemosiderin. The foci of sclerosis were associated with cystic spaces after 1 month and were solid after 3 months. A granulosa cell tumor, approximately 12-13 mm diameter, was present in a high-dose rat in the 3-month reversibility group. This tumor effaced 1 ovary and was characterized by proliferative granulosa cells, usually in papillary formations and cords within cystic spaces. This rat had atrophy of the uninvolved ovary, excessive plasma levels of E2 and prolactin, and high P4 levels considering the absence of CL. The results of this study indicate that ovarian granulosa cells in rats are susceptible to proliferative changes when stimulated chronically with excessive trophic hormones. Most of these proliferative changes were reversible upon cessation of the hormonal stimulation. However, the proliferative lesion in one treated rat progressed to apparent autonomous (neoplastic) growth.

The Selective Estrogen Receptor Modulator, Raloxifene: A Segment II/III Delivery Study in Rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.

Integrated analysis of preclinical data to support the design of the first in man study of LY2181308, a second generation antisense oligonucleotide.

AIMS To predict the concentration and target inhibition profiles of the survivin inhibitor antisense oligonucleotide LY2181308 in humans. METHODS An indirect pharmacokinetic/pharmacodynamic (PK/PD) model was built to predict the inhibition of survivin mRNA and protein in humans following LY2181308 dosing. Plasma and tissue PK data from cynomolgus monkeys were analyzed by non-linear mixed effect modelling techniques. Human PK parameters were predicted using allometric scaling. Assumptions about the pharmacodynamic parameters were made based upon the target and tumour growth inhibition data from mouse xenograft models. This enabled the prediction of the clinical PK/PD profiles. RESULTS Following a 750 mg dose, LY2181308 tumour concentrations ranging from 18.8 to 54µgg(-1) were predicted to lead to 50 to 90% target inhibition. In humans, LY2181308 tumour concentrations fro 13.9 to 52.8µgg(-1) (n=4, LY2181308 750mg) were observed associated with a median survivin mRNA and protein inhibition of 20%±34 (SD) (n=9) and 23%±63 (SD) (n=10), respectively. The human PK parameters were adequately estimated: central V(d) , 4.09 l (90% CI, 3.6, 4.95), distribution clearances, 2.54 (2.36, 2.71), 0.0608 (0.033, 0.6) and 1.67 (1.07, 2.00)lh(-1) , peripheral V(d) s, 25 900 (19 070, 37 200), 0.936 (0.745, 2.07) and 2.51 (1.01, 2.922)l, mean elimination clearance 23.1lh(-1) (5.6, 33.4) and mean terminal half-life, 32.7 days (range 22-52 days). CONCLUSION The model reasonably predicted LY2181308 PK in humans. Overall, the integration of preclinical PK/PD data enabled to appropriately predict dose and dosing regimen of LY2181308 in humans with pharmacologically relevant survivin inhibition achieved at 750mg.

The selective estrogen receptor modulator, raloxifene: segment II studies in rats and rabbits.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company primarily as a therapeutic agent for postmenopausal osteoporosis. Two Segment II studies were conducted that examined maternal reproductive parameters and fetal outcome following gestational exposure to raloxifene. Pregnant CD rats (25/group) and New Zealand white rabbits (20/group) were dosed once daily by oral gavage with 0, 0.1, 1, or 10 mg/kg on Gestation Days (GD) 6 through 17 and 7 through 19, respectively. Maternal body weight and food consumption were monitored throughout pregnancy. Caesarean sections were performed on GD 20 and GD 28 for rats and rabbits, respectively, to evaluate fetal viability, weight, and morphology. In rats, maternal body weight, body weight gain, and food consumption were reduced in all raloxifene treatment groups. Fetal viability was depressed in the 10-mg/kg group and was often associated with signs of hemorrhaging from the vagina. Fetal growth retardation was indicated in the 1- and/or 10-mg/kg groups by increased incidences of fetal runts and the developmental deviations, wavy ribs and kidney cavitation. There was no evidence of treatment-related malformations in rat fetuses. In rabbits, depressions in body weight gain and food consumption occurred in the 10-mg/kg group, and a single abortion occurred in the 1-mg/kg group. Fetal viability and weights were not affected in any of the raloxifene treatment groups. The overall proportions of fetuses with malformations, deviations, or variations were not affected by treatment with raloxifene; however, one fetus each from the 0.1-, 1-, and 10-mg/kg groups had incomplete closure of the interventricular septum. Therefore, maternal and fetal no-effect levels were not obtained in this study of raloxifene.

The reversible effects of raloxifene on luteinizing hormone levels and ovarian morphology in mice.

Raloxifene is a selective estrogen receptor modulator that has estrogen agonist effects on bone and serum lipids and estrogen antagonist effects on breast and uterine tissues. This study assessed the effects of raloxifene hydrochloride (HCl) treatment on circulating luteinizing hormone (LH) levels and ovarian morphology in sexually mature, 15-week-old, female CD-1 mice. Mice were maintained on diets providing average daily doses of 0 or 233 mg/kg raloxifene for 2 weeks (Study 1) or 0, 7.9, or 236 mg/kg raloxifene for 4 weeks (Study 2). At the end of the treatment period, blood samples were collected every 2 hours for 24 h in Study 1 (5 mice per group) and at 10:00 a.m. and 10:00 p.m. in Study 2 (8 mice per group). Serum LH levels were measured by radioimmunoassay. Ovarian histomorphology was evaluated in the 10 mice per group (Study 1) and the 8 mice per group (Study 2). For the reversibility phase (Study 2), mice were fed untreated diets for 3 weeks; serum LH levels and ovarian histomorphology were then assessed. Raloxifene treatment at 233 mg/kg/day for 2 weeks (Study 1) significantly elevated circulating LH levels by 4- to 7-fold compared with control. Raloxifene-treated mice had elevated LH levels sustained over the 24-h sampling period and did not exhibit the preovulatory LH surge evident in some control mice at the 4:00 p.m., 6:00 p.m., and 8:00 p. m. time points. Mice treated with 236 mg/day raloxifene for 4 weeks (Study 2) had elevated LH levels (4.4-fold compared to control), whereas mice exposed to 7.9 mg/kg/day raloxifene had a slight, nonsignificant increase in LH (2-fold compared to control). In both dose groups, LH levels were indistinguishable from controls 3 weeks after raloxifene treatment was discontinued. The ovaries in six of the eight mice treated with 7.9 mg/kg/day raloxifene had dilated and/or anovulatory follicles. One mouse in this group had a single hemorrhagic follicle; however, corpora lutea distribution was normal, indicating that ovulation was occurring. Raloxifene-treated mice in Study 1 and mice treated with a comparable raloxifene dose (236 mg/day) in Study 2 had histomorphological changes in the ovary indicative of arrested follicular maturation, including anovulatory hemorrhagic follicles, some developing follicles, and very few corpora lutea. At the end of the reversibility phase, hemorrhagic follicles were no longer evident and follicular maturation and corpora lutea distribution were normal. Raloxifene treatment in mice produces a dose-dependent, sustained elevation in serum LH levels and is associated with changes in ovarian follicular morphology. These changes are reversible upon discontinuation of raloxifene treatment.

The Selective Estrogen Receptor Modulator, Raloxifene: Reproductive Assessments in Adult Male Rats

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed for postmenopausal osteoporosis. Reproductive toxicity of raloxifene was examined in adult male CD rats after the oral administration of doses of 0, 10, 30, or 100 mg/kg/d. In the first study, males (12/group) were treated for 2 weeks followed by 2 weeks without treatment. After dose administration on Day 13, 6 males/group were cohabited with untreated females (1:2) for up to 7 d. Males were killed on Day 14 or 28 (6/group each day). Sperm were collected from the right cauda epididymis and evaluated for relative concentration, motion characteristics, and breakage. The kinetics of spermatogenesis were examined by DNA flow cytometry. The left testis and epididymis were preserved for histopathologic evaluation. Females were examined for reproductive status on Gestation Day 13. In a second study, males (20/group) were treated for 7 weeks (4 weeks prior to cohabitation during a 2-week cohabitation period, and for 1 additional week). Treated males were cohabited with untreated females (1:1). On Gestation Day 20, untreated females were examined for reproductive status and fetuses were examined for viability, weight, gender, and morphology. At necropsy, male reproductive tissues were collected, weighed, and preserved for histopathologic evaluation. In both studies, male body weight gain and food consumption were depressed at all dose levels. There was no indication in either study that raloxifene caused important changes in sperm production, sperm quality, or male reproductive performance at doses as high as 100 mg/kg/d.

The Selective Estrogen Receptor Modulator, Raloxifene: Reproductive Assessments following Premating Exposure in Female Rats

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed as a therapeutic agent for postmenopausal osteoporosis. Two studies were conducted that examined the effects of premating exposure to raloxifene HCl. In the first study, adult female CD rats (20/group) were given diets containing 0, 0.01, or 0.1% raloxifene (providing an average of 0, 6, or 63 mg/kg/d, respectively) for 2 weeks, after which the treated diets were replaced with control diet. Following a 2-week period without treatment, each female that had displayed at least three conversions in vaginal cytology from cornified cells to leukocytes was cohabited for 1 to 2 d with an untreated male as she entered proestrus. Females were killed at midgestation and examined for evidence of pregnancy. In the second study, adult female CD rats (40/group) were given oral gavage doses of raloxifene (0, 0.1, 1, or 10 mg/kg/d) for 4 weeks. Immediately or following a 2-week period without treatment, 20 females/group were cohabited with untreated males (1:1) for up to 3 weeks. The females were allowed to deliver and rear their offspring until Postpartum Day 21. Progeny survival, growth, and development were evaluated. Maternal body weight, body weight gain, and food consumption were depressed in all raloxifene treatment groups. Doses > or =1 mg/kg caused disruptions in estrous cycles. In Study 1, 90% of the females treated with raloxifene resumed normal cycling, and fertility was not significantly affected. Although there were no statistically significant differences in time-to-mating, fertility, or liveborn indices in Study 2, females in the 10-mg/kg immediate-cohabitation group had slightly increased gestation lengths and smaller litter sizes. Progeny from these litters were larger on Postpartum Day 1 and had advanced incisor eruption and eye opening. In addition, slight delays were seen in physical landmark appearance in the 0.1- and 1-mg/kg immediate-cohabitation groups and in the 1- and 10-mg/kg delayed-cohabitation groups. Progeny viability, growth, and negative geotactic performance were not adversely affected. In these studies of maternal premating exposure to raloxifene, findings were consistent with established pharmacologic activity of the test chemical. Reproductive effects (disrupted estrous cycles and decreased litter size) occurred at doses > or =1 mg/kg and were generally reversible. Effects on offspring were seen at doses > or =0.1 mg/kg, were of minor importance, and were resolved during the lactation period.

The selective estrogen receptor modulator, raloxifene : Reproductive assessments following preimplantation exposure in mated female rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator being developed for postmenopausal osteoporosis. As part of an integrated reproductive toxicity assessment, two studies were conducted in which raloxifene was administered orally to CD rats during Gestation Days (GD) 0 through 5. In each study, animals received daily raloxifene doses of 0, 0.1, 1, or 10 mg/kg. In Study 1, GD 20 evaluations of maternal reproductive parameters identified dose-related increases in pre- and postimplantation loss, reductions in the numbers of corpora lutea and live conceptuses, and reduced fetal weight. The low fetal weights were consistent with an extent of morphologic development that corresponded to developmental ages up to 8 d younger than GD 20. Study 2 characterized the potential impact of this disrupted and apparently delayed implantation on gestation length, parturition, and progeny viability. Dams were allowed to deliver and rear their offspring through Postpartum Day 21. Gestation lengths were extended up to 1 week, and litter sizes were reduced in a dose-dependent manner. Nevertheless, parturition occurred normally and pup morphology, survival, and physical and behavioral development were unaffected.