Time-release Pellets Research Articles

Chronic ethanol–clonidine hemodynamic interaction in telemetered spontaneously hypertensive rats

Our previous studies have established that ethanol, administered acutely or chronically, attenuates the hypotensive action of the centrally acting drug clonidine. In this study, we employed the radiotelemetry technique to evaluate the long-term hemodynamic interaction between the two drugs administered simultaneously to spontaneously hypertensive rats (SHRs). Changes in blood pressure (BP), heart rate (HR), and their variability was determined in pair-fed rats receiving ethanol (5%, w/v), clonidine pellets (10 mg/pellet, s.c.), or their combination for 28 days. Ethanol feeding caused significant decreases and increases in BP and HR, respectively. The time-domain variability indices of BP and HR were also reduced by ethanol. Clonidine produced significant reductions in BP and HR that were evident for only 1–2 days and disappeared thereafter. In rats receiving the combined ethanol and clonidine treatment, hemodynamic changes were identical to those produced by ethanol alone. These findings suggest (i) long-term exposure of SHRs to moderate amounts of ethanol reduces HR variability, possibly due to diminished cardiac vagal modulation, and (ii) the lack of a maintained hypotensive response to clonidine, administered via timed-release pellets, made the evaluation of its chronic interaction with ethanol unfeasible.

Thymus-derived glucocorticoids are insufficient for normal thymus homeostasis in the adult mouse

BackgroundIt is unclear if thymus-derived glucocorticoids reach sufficient local concentrations to support normal thymus homeostasis, or if adrenal-derived glucocorticoids from the circulation are required. Modern approaches to this issue (transgenic mice that under or over express glucocorticoid receptor in the thymus) have yielded irreconcilably contradictory results, suggesting fundamental problems with one or more the transgenic mouse strains used. In the present study, a more direct approach was used, in which mice were adrenalectomized with or without restoration of circulating corticosterone using timed release pellets. Reversal of the increased number of thymocytes caused by adrenalectomy following restoration of physiological corticosterone concentrations would indicate that corticosterone is the major adrenal product involved in thymic homeostasis.ResultsA clear relationship was observed between systemic corticosterone concentration, thymus cell number, and percentage of apoptotic thymocytes. Physiological concentrations of corticosterone in adrenalectomized mice restored thymus cell number to normal values and revealed differential sensitivity of thymocyte subpopulations to physiological and stress-inducible corticosterone concentrations.ConclusionThis indicates that thymus-derived glucocorticoids are not sufficient to maintain normal levels of death by neglect in the thymus, but that apoptosis and possibly other mechanisms induced by physiological, non stress-induced levels of adrenal-derived corticosterone are responsible for keeping the total number of thymocytes within the normal range.

Estradiol treatment redirects the isotype of the autoantibody response and prevents the development of autoimmune arthritis.

A number of clinical and experimental observations have been made relating elevated estrogen levels with the amelioration of autoimmune diseases, yet questions remain about the levels required for efficacy as well as the mechanism of disease inhibition. Using the collagen-induced arthritis (CIA) model, we have studied the effects of physiological, sustained levels of 17beta-estradiol in preventing the development of autoimmune arthritis and analyzed the changes in the autoimmune response. Using time-release pellets of 17beta-estradiol, arthritis development was significantly inhibited in three different strains of CIA-susceptible mice compared with the effect of placebo treatment, and serum estradiol levels similar to those of mice in estrus were found to be equally effective as higher estradiol concentrations. Analysis of the autoimmune response in the estradiol-treated mice indicated that T cell production of IFN-gamma was markedly decreased, and significant decreases were also observed in levels of IL-10 and GM-CSF produced by lymph nodes cells from estradiol-treated mice. Although the total IgG anti-CII response was only minimally affected by estrogen treatment, a significant reduction in the levels of IgG2a anti-CII Abs and an increase in the levels of IgG1 anti-CII Abs were observed in estradiol-treated mice. These data indicate that estradiol treatment altered the Th profile of the autoimmune T cell response, which, in turn, altered the production of IgG Abs to an isotype that is poor at fixing complement, an important component in the immunopathogenesis of CIA.

17beta-estradiol upregulates the expression of peroxisome proliferator-activated receptor alpha and lipid oxidative genes in skeletal muscle.

This study examined the actions of 17beta-estradiol (E(2)) and progesterone on the regulation of the peroxisome proliferator-activated receptors (PPARalpha and PPARgamma) family of nuclear transcription factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically, carnitine palmitoyltransferase I (CPT I), beta-3-hydroxyacyl CoA dehydrogenase (beta-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague-Dawley rats were ovariectomized and treated with placebo (Ovx), E(2), progesterone, or both hormones in combination (E+P). Additionally, sham-operated rats were treated with placebo (Sham) to serve as controls. Hormone (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E(2) treatment increased PPARalpha mRNA expression and protein content (P<0.05), compared with Ovx treatment. E(2) also resulted in upregulated mRNA of CPT I and PDK4 (P<0.05). PPARgamma mRNA expression was also increased (P<0.05) by E(2) treatment, although protein content remained unaltered. These data demonstrate the novel regulation of E(2) on PPARalpha and genes encoding key proteins that are pivotal in regulating skeletal muscle lipid oxidative flux.

Tamoxifen, a Selective Estrogen Receptor Modulator, Reduces Ischemic Damage Caused by Middle Cerebral Artery Occlusion in the Ovariectomized Female Rat

Previous work has demonstrated that physiological concentrations of 17β-estradiol can protect the female rat brain against middle cerebral artery occlusion (MCAO)-induced ischemic damage. The present study examined whether therapeutic doses of the clinically relevant selective estrogen receptor modulator (SERM), tamoxifen, can similarly protect the female rat brain against ischemic stroke damage. Adult female rats were bilaterally ovariectomized and implanted subcutaneously with either a placebo or tamoxifen time-release pellet (0.1, 0.8 or 2.4 mg/kg/day). One week later, the animals underwent permanent MCAO to assess the protective ability of the different tamoxifen doses on brain infarct size. As expected, MCAO produced a large infarct (∼53%) of the affected cerebral hemisphere in placebo (control) animals. The 0.1 mg/kg/day dose of tamoxifen did not exhibit any significant protective effects, however; the 0.8 and 2.4 mg/kg/day doses of tamoxifen, which are in the therapeutic range, dramatically reduced infarct of the affected cerebral hemisphere (∼70% reduction) as compared to the controls. The reduction of infarct size was primarily due to protection of two major structures, the cerebral cortex and striatum. Laser Doppler analysis further revealed that tamoxifen had no significant effect on cerebral blood flow either before or after MCAO, suggesting that tamoxifen protection is independent of cerebral blood flow changes. Further studies showed that tamoxifen pellets implanted at the time of MCAO did not reduce infarct size, suggesting that pretreatment with tamoxifen is necessary to observe a protective effect. These studies suggest that clinically important SERMs may have an additional unrecognized beneficial effect of protection of the female brain.

Effects of tamoxifen on ischemia-induced angiogenesis in the mouse lung.

Obstruction of pulmonary blood flow in the mouse lung causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 5-6 days [Mitzner et al. Am J Pathol 2000; 157(1): 93-101]. Tamoxifen, a triphenylethylene antiestrogen, has been shown to be effective in limiting tumor growth, possibly because of inhibition of angiogenesis. We investigated the effects of tamoxifen on blood vessel development after left pulmonary artery ligation (LPAL). Timed-release pellets of either tamoxifen (free base/15 mg over 21 days) or placebo carrier were implanted subcutaneously in male C57BL/6J mice 6-8 weeks of age. Two days after pellet implantation, the left pulmonary artery was permanently obstructed by suture ligation. New systemic vessel growth was assessed after left ventricular injection of fluorescence labeled microspheres. Tamoxifen slowed the formation of functional blood vessels seven days after LPAL. By 14 days, however, no difference was observed between tamoxifen and placebo treated mice with systemic perfusion to the left lung reaching a maximum of 3.8% and 4.7% of cardiac output respectively. No change in VEGF mRNA expression was observed until 14 days after LPAL when a small increase (2-fold) was observed in both placebo and tamoxifen treated lungs. However, VEGF protein was elevated in both tamoxifen and placebo lungs 24 h after LPAL (approximately 4-fold). These changes in VEGF protein may be due to the presence of trapped inflammatory cells observed in lung sections at this early time point. Although tamoxifen appeared to slow the progression of blood vessel formation, it did not affect VEGF mRNA, therefore likely acting through an estrogen receptor-independent mechanism.

17β-estradiol attenuates intimal hyperplasia and macrophage accumulation with a reduction in monocyte chemoattractant protein 1 expression in a vein graft model

Objective: Autogenous vein grafts are commonly used for arterial reconstructive procedures. Their success is limited by the development of intimal hyperplasia, a fibroproliferative disease that predisposes the grafts to occlusive stenosis. Our goal was to assess whether 17β-estradiol (E 2) inhibits vein graft intimal hyperplasia coincident with a reduction in monocyte chemoattractant protein 1 (MCP-1) expression and macrophage accumulation. Method: Male Lewis rats were implanted with time-release pellets that contained 0.5 mg E 2 (E5 group) or placebo (PL group). Epigastric vein to common femoral artery interposition grafts were harvested at 2, 4, 8, and 12 weeks after surgery. We assessed macrophage/monocytes numbers, proliferating cell nuclear antigen, MCP-1, and transforming growth factor-β1 with use of immunohistochemistry. MCP-1 message expression was quantified by real-time polymerase chain reaction. Results: The time-release pellets raised the serum E 2 level to greater than 250 pg/mL on the day of surgery. Serum E 2 level declined to 43 ± 13 pg/mL by 4 weeks and to baseline by 6 weeks. We found that the neointimal area ratio was reduced significantly in the E5 group at 2 and 4 weeks (45%, P < .05, and 68%, P < 0.05, respectively) relative to that in the PL group. The number of proliferating cells was reduced in the E5 group. There was a significant attenuation of MCP-1 expression and of the number of macrophages accumulating in the graft with E 2 treatment. Furthermore, MCP-1 messenger ribonucleic acid expression was also significantly attenuated in the E5 group at 4 weeks when compared to the PL group. There was no significant difference between the two groups in the expression of transforming growth factor-β1. Conclusions: E 2 treatment reduces vein-graft intimal hyperplasia coincident with a reduction in MCP-1 expression, macrophage accumulation, and cell proliferation. (J Vasc Surg 2002;36:613-21.)

Chronic Continuous Nicotine Exposure During Periadolescence Does Not Increase Ethanol Intake During Adulthood in Rats

Background Nicotine has been considered as the gateway drug, because many teenagers experience cigarette smoking before seeking out other drugs. By using an animal model system, we assessed the effects of chronic continuous nicotine exposure during periadolescence on ethanol intake during young adulthood.Methods Periadolescent Sprague‐Dawley rats (35 days old) were used at the beginning of this study. These animals received subcutaneous implantation of nicotine pellets (15 or 25 mg in 21‐day time‐release pellets) or placebo pellets (0 mg of nicotine) on postnatal day 35. Beginning on postnatal day 53, the animals received various concentrations of ethanol solution during their active period (5:00 PM to 9:00 AM) starting with 2% (v/v, 4 days), then 5% (5 days), 8% (6 days), and 10% (6 days). Between 9:00 AM and 5:00 PM of the same day, the ethanol solution was replaced by regular tap water. The amounts of ethanol solution and regular water were measured daily.Results The analyses showed that ethanol intake (grams per kilogram of body weight) in the nicotine 15 and 25 mg groups did not differ from that in the nicotine 0 mg group, and no sex difference was found in ethanol intake. However, ethanol intake was increased as a function of the treatment days.Conclusions The results showed that chronic continuous nicotine exposure during adolescence did not increase ethanol intake in rats during young adulthood.

Effect of the ovarian hormones on GLUT4 expression and contraction-stimulated glucose uptake.

This study examined the roles of the female sex steroids, 17beta-estradiol (E(2)) and progesterone (Prog), on glucose uptake and GLUT4 protein expression. Female Sprague-Dawley rats were either sham operated (C) or ovariectomized and treated with placebo (O), E(2) (E), Prog (P), or both hormones at physiological doses (P + E) or the same dose of Prog with a high dose of E(2) (P + HiE) via timed-release pellets inserted at the time of surgery, 15 days before metabolic testing. On the morning of day 15, animals received a 300-microCi injection (ip) of 2-deoxy-[(14)C]glucose and then either exercised on a motorized treadmill for 30 min at 0.35 m/s or remained sedentary in their cages for the same period. Basal glucose uptake was not different between the treatment groups in either the red or white quadriceps. However, glucose uptake was decreased (P < 0.05) in O, P, and P + E rats during exercise in the red quadriceps compared with C rats, whereas E and P + HiE treatment restored glucose uptake. Glycogen content in skeletal muscle followed similar trends, with no differences seen in resting animals. Postexercise red quadriceps glycogen levels were higher (P < 0.05) in the E and P + HiE rats compared with O and P. Treatment of ovariectomized rats with progesterone (P rats) decreased (P < 0.05) GLUT4 content in the red quadriceps by 21% compared with C rats. These data demonstrate that estrogen-deficient animals have a decreased ability for contraction-stimulated glucose uptake and increased glycogen use during aerobic exercise. However, changes in contraction-stimulated glucose uptake could not be explained by altered transporter protein content, since the absence of E(2) had no effect on GLUT4 protein.

Administration of exogenous endothelin-1 following vascular balloon injury: early and late effects on intimal hyperplasia.

Administration of exogenous endothelin-1 (ET-1) has been shown to stimulate neointimal hyperplasia following arterial balloon angioplasty (BA). However, the specific effects of ET-1 on the cellular and extracellular matrix response of the vessel wall after balloon injury and the persistence of these ET-1 effects have not been studied. The objectives of this study were to determine the acute (1 week) and long term (10 weeks) effects of administering exogenous ET-1 after arterial BA on neointimal hyperplasia, collagen synthesis and content, cellular proliferation, and ET(A) and ET(B) receptor expression. Thirty-one rabbits were randomized to receive subcutaneous ET-1 (500 pmol/kg/day for 1 week) or placebo time-release pellets and sacrificed at either 1 or 10 weeks after BA. At 1 week, there was a significant two-fold increase in intimal cross-sectional area (CSA) in ET-1 treated animals compared with placebo. ET-1 treated animals showed significant increases in collagen synthesis (ten-fold) and collagen content (three-fold) compared to placebo treated animals. ET-1 treated animals also had a significant increase (two-fold) in proliferation rates. In addition, ET(A) and ET(B) receptor expression were significantly upregulated in ET-1 treated animals. By 10 weeks these stimulatory effects on intimal CSA and collagen content were no longer evident with a 'catch up' phenomenon observed in the placebo treated animals. Similarly, ET(A) and ET(B) mRNA levels had declined significantly in both groups. Therefore, exogenous ET-1 acutely stimulates extracellular and cellular processes including increased expression of ET(A) and ET(B) receptors contributing to intimal hyperplasia. However, these effects are transient and not maintained long term after withdrawal of exogenous ET-1 stimulation.

Acute and chronic estrogen supplementation decreases uterine sympathetic innervation in ovariectomized adult virgin rats.

Uterine innervation undergoes substantial reorganization associated with changes in reproductive status. Nerves innervating the uterus are decreased in pregnancy and puberty, and even the normal rodent estrous cycle is characterized by fluctuations in numbers of myometrial nerve fibers. During the follicular (proestrus/estrous) phase of the estrous cycle, intact nerves are rapidly depleted and then return over the next 2-3 days in the luteal (metestrus/diestrus) phase. We hypothesize that uterine nerve depletion is initiated by increased circulating estrogen in the follicular phase. However, studies have not shown whether estrogen can reduce uterine innervation and, if so, whether the time course is compatible with the rapid changes observed in the estrous cycle. These questions were addressed in the present study. Mature ovariectomized virgin rats received 17-beta-estradiol as a single injection (10 microg/kg s.c.) or chronically from timed-release pellets (0.1 microg/pellet for 3 weeks sustained release). Total (protein gene-product 9.5-immunoreactive) and sympathetic (dopamine beta-hydroxylase-immunoreactive) uterine innervation was assessed quantitatively. Both total and sympathetic innervation was abundant in uterine longitudinal smooth muscle of ovariectomized rats. However, following acute or chronic estrogen administration, total and sympathetic fiber numbers were markedly decreased. This was not due to altered uterine size, as reductions persisted after correcting for size differences. Our results indicate that sympathetic nerves are lost from uterine smooth muscle after estradiol treatment in a manner similar to that seen in the intact animal during estrus and pregnancy. This suggests that the rise in estradiol prior to estrus is sufficient to deplete uterine sympathetic innervation.

Effect of ovarian hormones on mitochondrial enzyme activity in the fat oxidation pathway of skeletal muscle.

To examine the roles of 17beta-estradiol (E(2)) and progesterone (Prog) in lipid metabolism, skeletal muscle enzyme activities were studied in female Sprague-Dawley rats. Groups included sham-operated rats (C) and ovariectomized rats treated with placebo (O), E(2) (E), Prog (P), both hormones at physiological doses (P + E), or both hormones with a high dose of E(2) (P + HiE). Hormone (or vehicle only) delivery was via time-release pellets inserted at the time of surgery, 15 days before metabolic testing. Results demonstrated that carnitine palmitoyltransferase maximal activity was 19, 21, and 19% lower (P < 0.01) in O, P, and P + E rats, respectively, compared with C rats. Conversely, activity in E and P + HiE rats was 14 and 19% higher (P < 0.01) than in C. beta-Hydroxyacyl-CoA dehydrogenase (beta-HAD) maximal activity was 20% lower (P < 0.01) in O than in C rats; similarly, P and P + E rats were 18 and 19% lower, respectively (P < 0.01); however, treatment with E(2) returned beta-HAD activity to C levels. These results suggest that E(2) plays a role in lipid metabolism by increasing the maximal activity of key enzymes in the fat oxidative pathway of skeletal muscle.

Estrogen augments the vasodilatory effects of vascular endothelial growth factor in the uterine circulation of the rat

Objective: Pregnancy augments uterine artery vasodilatation in response to vascular endothelial growth factor, although the underlying mechanism is not known. The aim of this study was to test the hypothesis that estrogen and progesterone, the primary sex steroids of pregnancy, are responsible for this effect through increased endothelial secretion of nitric oxide. Study Design: Adult female Sprague-Dawley rats underwent oophorectomy at 9 weeks of age with concomitant placement of 21-day timed-release pellets containing either 17β-estradiol (n = 6) or progesterone (n = 6), or a combination of these (n = 6). Control rats also underwent oophorectomy but did not receive hormone replacement (n = 6). Two to 3 weeks after oophorectomy the rats were killed and the main uterine artery was dissected free, cannulated in an arteriograph, and pressurized to 50 mm Hg. After constriction with phenylephrine, concentration-response curves to vascular endothelial growth factor (0.1-20 nmol/L) were performed to compare arterial sensitivity to and maximal effects of vascular endothelial growth factor among the 4 treatment groups. Vessels were then treated with N ω-nitro-L -arginine (0.24 mmol/L), an inhibitor of nitric oxide synthase, and the maximally effective concentration of vascular endothelial growth factor was reapplied to evaluate the relative contribution of nitric oxide to the overall effect. Results: Comparisons of the effective concentration of vascular endothelial growth factor that elicited 50% of the maximal dilatation revealed the vessels of the estrogen group to be approximately 10 times more sensitive than the control group (0.4 ± 0.11 nmol/L vs 4.2 ± 1.13 nmol/L, respectively; P <.05). Responses of vessels from the progesterone and combined groups were intermediate (progesterone, 2.3 ± 0.66 nmol/L; combined, 1.1 ± 0.28 nmol/L). Maximal vasodilatory responses were greatest in the groups with treatment including estrogen (estrogen, 61% ± 3.1%; combined, 54% ± 3.4%; progesterone, 42% ± 5.8%, control, 40% ± 3.5%; P <.05). Addition of N ω-nitro-L -arginine inhibited maximal vascular endothelial growth factor–induced dilatation by approximately 40% irrespective of treatment group. Conclusion: The presence of estrogen rather than progesterone leads to an enhancement of vascular endothelial growth factor–induced arterial dilatation during pregnancy. This effect results from a proportional increase in endothelial nitric oxide secretion, along with that of another, as yet unidentified vasodilatory substance. (Am J Obstet Gynecol 2000;183:449-53.)

Insulin and glucocorticoid-dependent suppression of the IGF-I system in diabetic wounds

Background: Growth-promoting polypeptides, including insulin-like growth factor-I (IGF-I), orchestrate different biochemical events that culminate in the restoration of functional integrity of wounded skin. The nonhealing cutaneous wound is a well-documented phenomenon in experimental and clinical diabetes. Accordingly, we undertook this study to ascertain whether diabetes impairs the healing process by suppressing the wound microenvironmental IGF-I system (eg, IGF-I; IGF-I receptor [IGF-I R]; and IGF-I binding protein [IGF-BP3]). Methods: The induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol sponge and stainless steel mesh chamber models were used to study wound healing. Nondiabetic and diabetic animals received, respectively, subcutaneous 30-day time-release pellets of glucocorticoid (200 mg) and mifepristone (RU-486, 25 mg). Corresponding control animals received placebo pellets. Polyvinyl alcohol sponge and wound fluid expression of the IGF-I system were evaluated by using ligand blotting, radioimmunoassay, and reverse transcriptase polymerase chain reaction-based techniques. Results: Polyvinyl alcohol sponge contents of messenger RNA (mRNA) transcripts encoding for IGF-I, IGF-I R, and IGF-BP3 were reduced in diabetic and glucocorticoid-treated control animals. A similar pattern of changes in protein levels of IGF-I and IGF-BP3 occurred in wound fluid collected from these animals. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the glucocorticoid receptor blocker RU-486 (hypercortisolemia) ameliorated the diabetes-related decrease in the IGF-I system during wound healing. Conclusions: The current data, together with data garnered from the literature, support the concept that the state of hypercortisolemia in diabetes mellitus impairs the healing process, at least in part, by suppressing the wound microenvironmental IGF-I system. Confirmation regarding this premise awaits further investigation. (Surgery 2000;127:687-95.)

Combined effects of AT 1 and ET A receptor antagonists, candesartan, and A-127722 in DOCA–salt hypertensive rats

Several recent studies have provided evidence that many of the hemodynamic and mitogenic actions of angiotensin II (Ang II) are mediated by endothelin-1 (ET-1). We hypothesized that Ang II and ET-1 act synergistically to promote a decline in renal function and the development of renal fibrosis in the deoxycorticosterone acetate (DOCA)-salt model of malignant hypertension and renal dysfunction. Experiments were conducted to determine the effects of ET A receptor antagonism (A-127722) and AT 1 receptor antagonism (candesartan cilexetil) on the development of renal fibrosis and the decline of renal function. Surgery was conducted on male, Sprague–Dawley rats to remove the right kidney and implant subcutaneously a time-release pellet containing DOCA. DOCA-treated rats were also given 0.9% NaCl to drink. After recovery from surgery, rats received one of four treatments via the drinking solution: (1) candesartan cilexetil (10 mg/kg/day), (2) A-127722 (10 mg/kg/day), (3) candesartan cilexetil plus A-127722, or (4) untreated controls. Over the course of a 3-week treatment period, systolic arterial pressure in all groups were elevated. However, this increase was significantly attenuated in the group given combined A-127722 and candesartan, but not with candesartan alone. Creatinine clearance, used as a measure of GFR, was significantly higher in rats treated with either or both drugs. At the end of the study, renal medullary tissue was harvested for determination of TGF-β and fibronectin content (ELISA). TGF-β levels were not reduced by either ET A, AT 1, or combined ET A and AT 1 receptor blockade. Likewise, fibronectin content was similar among groups. These studies indicate that combined ET A and AT 1 receptor blockade may produce some improvement on hemodynamics, but have no effect on progression of renal damage in this non-renin-dependent model of hypertension.

Effect of farnesyltransferase inhibitor FTI-276 on established lung adenomas from A/J mice induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The Ras protein undergoes a series of post-translational modifications at the C-terminal CAAX motif, which culminates with the anchoring of p21 Ras to the plasma membrane where it relays growth regulatory signals from receptor tyrosine kinases to various pathways of cell signal transduction. FTI-276 is a CAAX peptidomimetic of the carboxyl terminal of Ras proteins. Pharmacokinetic analysis of FTI-276 in A/J mice with a time-release pellet system showed a dose of 50 mg/kg body wt achieved an average serum level of 1.68 microg/ml for up to 30 days following implantation. In the present study, 4 week old A/J mice were initiated with a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (100 mg/kg), and monitored for 18 weeks. Mice were grouped for daily delivery (time-release pellet) of 50 mg/kg of FTI-276 for 30 days (n = 12) and the control group (n = 12). Analysis of tumors from time-release pellet treated animals showed a 60% reduction in tumor multiplicity and a 42% reduction in tumor incidence. Moreover, FTI-276 treatment resulted in a significant reduction in tumor volume (approximately 58%). Mutation analysis of the lung tumors from both treatment groups revealed that most of the tumors harbored mutations in the codon 12 of K-ras and there is no significant difference in the incidence and types of mutations between tumors from the treated and control animals. This is the first demonstration of chemotherapeutic efficacy of a synthetic CAAX peptidomimetic farnesyltransferase inhibitor in a primary lung tumor model.

CHEMOPREVENTIVE EFFICACY OF PROMISING FARNESYLTRANSFERASE INHIBITORS

The studies presented were designed to test the efficacy of farnesyltransferase inhibitors (FTIs) as potential chemopreventive compounds in the mouse lung tumor model, and in tumor cell lines. The compounds included manumycin, gliotoxin, dihydroepiandrosterone (DHEA), perillyl alcohol (POH), and FTI-276. Each of these compounds had the potential, based on in vitro and limited in vivo evidence, to inhibit mouse lung tumorigenesis. In vitro studies were conducted with both K-ras-transformed NIH-3T3 cells and mouse lung tumor epithelial cell lines. Weutilized 2 primary mouse lung tumor models that reliably produce lung tumors with an oncogenic K-ras mutation when induced by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Manumycin, gliotoxin, DHEA, and POH were administered 3 times per week peritoneally (IP), starting 1 week prior to carcinogen treatment, and throughout the test period (4.5 months). FTI-276 was delivered daily for 4 months by a time-release pellet method. Both the manumycin and gliotoxin treatment groups demonstrated 100% incidence and an increase in tumor multiplicity over control, of 66% and 58% increase respectively (P<.05). Although DHEA showed no significant chemopreventive effect, POH treatment demonstrated a 22% reduction in tumor incidence (P<.05) and a 58% reduction in tumor multiplicity (P<.05). Finally, FTI-276 reduced both the tumor multiplicity by 41.7% (P <.005), and the total tumor volume/burden per mouse by 79.4% (P<.0001). The apoptotic index in FTI-276-treated tumors showed an increase of 77% over control tumors (P<.05). In vitro, all compounds demonstrated growth inhibition at a dose-response manner; however, manumycin, gliotoxin, and DHEA demonstrated an initial increase in growth rate at lower doses. In summary, we have shown that POH and FTI-276 are chemopreventive in a primary mouse lung tumor model. In contrast, DHEA was not significantly chemopreventive at the dosage utilized, and treatment of an immunocompetent host with manumycin or gliotoxin demonstrated a significant increase in tumorigenicity over carcinogen control.

Tissue distribution and pharmacological potential of SM-16896, a novel oestrogen-bisphosphonate hybrid compound.

Postmenopausal osteoporosis is caused mainly by a deficiency of oestrogen with rapid bone loss. To target oestrogen to the bone effectively, we have synthesized and evaluated the effects of a novel hybrid compound of oestrogen and bisphosphonate, SM-16896. The tissue distribution pattern and pharmacological potential are reported. Although the affinity for calf uterine oestrogen receptor was very low (IC50: 73.3 microM; 1/25000 of that of 17beta-oestradiol (2.84 nM)), SM-16896 showed oestrogenic activity. SM-16896 (1 microM) induced a 4.5-fold transcriptional activity in rat osteosarcoma UMR-106 cells compared with vehicle-treated control, when we used the expression vector for human oestrogen receptor and a CAT reporter plasmid containing an oestrogen-responsive element. The distribution of SM-16896 after a subcutaneous administration to 7-week-old female rats was examined by radioluminography using 3H-labelled SM-16896. At 30 min after the administration, significant radioactivity was detected in the bone. At 24 h after administration, a high level of radioactivity was detected in the bone, but in the uterus it was only at a background level. Daily subcutaneous administration of 0.5 mgkg(-1) SM-16896 for 12 weeks (five times per week) to 13-week-old ovariectomized rats suppressed the ovariectomized-induced reduction in bone mineral density. A bone mineral density ratio of 120% was maintained compared with sham-operated rats, whereas a relatively low suppression of uterine weight was observed (about 50% loss compared with sham-operated rats). In the same experiment, the implantation of a 17beta-oestradiol time-release pellet (0.25 mg/pellet/90 days) almost completely suppressed the reduction of both the bone mineral density and uterine tissue weight. It is likely that the effect of SM-16896 on bone was due to its oestrogenic activity, since 1.0 mgkg(-1) SM-18108, the bisphosphonate moiety of this compound, had no effect on bone in 7-week-old ovariectomized rats. The results suggest that SM-16896, a bisphosphonate-conjugated oestrogen, showed a preference profile in the uterus and bone due to its characteristic distribution pattern compared with the natural oestrogen analogue 17beta-oestradiol. Thus, bisphosphonate-conjugated oestrogens have the potential to improve patient compliance in oestrogen therapy by minimizing adverse effects and reducing the frequency of medication.

Opposing actions of dehydroepiandrosterone and corticosterone in rats.

The purpose of this study was to determine the impact of dehydroepiandrosterone (DHEA) and corticosterone (CORT) treatment, using implants as a route of administration, on specific hormones, metabolites, and enzymes involved in energy metabolism. Sixty male Sprague-Dawley rats, 325 g initial weight, were implanted subcutaneously for 3 weeks with time-release pellets containing either DHEA or CORT at doses of 0, 10, 25, 50, or 100 mg in this 2 x 5 factorial experiment. In general, body weights and food intakes decreased as the level of steroid hormones increased. In contrast to DHEA treatment, rats receiving the 50- and 100-mg doses of CORT had lighter thymus glands and spleens and heavier epididymal and retroperitoneal fat pads than their controls. Rats treated with 100 mg of DHEA had lowered serum levels of triglycerides and lipid hydroperoxides whereas rats treated with 100 mg of CORT had higher levels of these blood lipids compared to their respective controls. In contrast to DHEA treatment, there was a dose-dependent increase in liver lipid content and the specific activities of the hepatic lipogenic enzymes glucose-6-phosphate dehydrogenase, malic enzyme, and fatty acid synthase in response to CORT treatment. Rats treated with 100 mg of DHEA had higher serum levels of IGF-1 than control rats. Conversely, rats treated with 100 mg of CORT had lower serum levels of IGF-1 and higher serum levels of testosterone, progesterone, and insulin than their controls. These data demonstrate the lipogenic actions of corticosterone in rats. Conversely, DHEA treatment reduced serum and hepatic lipids. Furthermore, these data suggest that using implants instead of bolus injections of steroids may be a more physiological approach for studying the influence of these steroids on lipid metabolism.

Opposing Actions of Dehydroepiandrosterone and Corticosterone in Rats

The purpose of this study was to determine the impact of dehydroepiandrosterone (DHEA) and corticosterone (CORT) treatment, using implants as a route of administration, on specific hormones, metabolites, and enzymes involved in energy metabolism. Sixty male Sprague-Dawley rats, 325 g initial weight, were implanted subcutaneously for 3 weeks with time-release pellets containing either DHEA or CORT at doses of 0, 10, 25, 50, or 100 mg in this 2 × 5 factorial experiment. In general, body weights and food intakes decreased as the level of steroid hormones increased. In contrast to DHEA treatment, rats receiving the 50- and 100-mg doses of CORT had lighter thymus glands and spleens and heavier epididymal and retroperitoneal fat pads than their controls. Rats treated with 100 mg of DHEA had lowered serum levels of triglycerides and lipid hydroperoxides whereas rats treated with 100 mg of CORT had higher levels of these blood lipids compared to their respective controls. In contrast to DHEA treatment, there was a dose-dependent increase in liver lipid content and the specific activities of the hepatic lipogenic enzymes glucose-6-phosphate de-hydrogenase, malic enzyme, and fatty acid synthase in response to CORT treatment. Rats treated with 100 mg of DHEA had higher serum levels of IGF-1 than control rats. Conversely, rats treated with 100 mg of CORT had lower serum levels of IGF-1 and higher serum levels of testosterone, progesterone, and insulin than their controls. These data demonstrate the lipogenic actions of corticosterone in rats. Conversely, DHEA treatment reduced serum and hepatic lipids. Furthermore, these data suggest that using implants instead of bolus injections of steroids may be a more physiological approach for studying the influence of these steroids on lipid metabolism.