Hypocholesterolemic activity of raloxifene (LY139481): pharmacological characterization as a selective estrogen receptor modulator.

After once-daily oral dosing in ovariectomized rats, raloxifene (LY139481) hydrochloride produced dose- and time-dependent reductions in serum cholesterol and high-density lipoprotein-cholesterol. Paired-feeding studies demonstrated that effects of raloxifene on serum lipids were not secondary to effects on food consumption. Maximal reductions in serum cholesterol occurred within 4 days of raloxifene administration or sooner, depending on the administered dose. The ED50 for 50% reduction in serum cholesterol by raloxifene was 0.13 +/- 0.04 mg/kg/day (mean +/- S.E.M., n = 17); maximal cholesterol reduction by raloxifene (68%) was significantly less than that produced by estrogen (17 alpha-ethinylestradiol; 89%) after 4 to 7 days of daily dosing. Dose-response curves for cholesterol lowering by raloxifene were generated in the presence of varying doses of 17 alpha-ethinylestradiol; two-way analysis of variance revealed significant interactions between estrogen and raloxifene with respect to cholesterol lowering (P < .001). Furthermore, a high dose of raloxifene (10 mg/kg/day) prevented further reduction of serum cholesterol by estrogen (1-100 micrograms/kg/ day) beyond that produced by raloxifene alone. For a series of closely related structural analogs of raloxifene, log(ED50) values for cholesterol lowering were highly correlated with log(relative binding affinity) for the estrogen receptor (r = 0.93; P < .0001). Thus, cholesterol lowering by raloxifene in ovariectomized rats is mediated primarily via partial agonist effects at estrogen receptors. Taken together with previous observations in uterine tissue of estrogen antagonism by raloxifene in the absence of significant agonism, the present findings support the classification of raloxifene as a selective estrogen receptor modulator.

Serotonin-induced paw edema in the rat: pharmacological profile.

1. Serotonin (5-HT) induced a linear increase in paw weight in rats within 1 hr of an intraplantar injection (50 microliters vol) over a concentration range of 0.005-0.2 mg/ml. At the 0.2 mg/ml concentration, a 16-fold increase in paw weight was observed as compared to saline-injected controls. 2. Serotonin antagonists, such as LY53857, were the most effective antagonists of 5-HT induced paw swelling, producing near complete antagonism and an approximate ED50 of 0.1 mg/kg. A mixed 5-HT/histamine antagonist, cyproheptadine, also produced a nearly complete inhibition of the 5-HT response with an approximate ED50 of 1.3 mg/kg. 3. Dopamine agonists (pergolide, quinpirole), yohimbine, dexamethasone and nifedipine also produced a significant degree of antagonism of the 5-HT response. 4. Clonidine, prazocin, chlorpheniramine, cimetidine, various dopamine antagonists, imipramine, cyclosporine A, piroxicam and superoxide dismutase were all ineffective at altering the paw swelling response to 5-HT.

Effect of weight manipulation on bone loss due to ovariectomy and the protective effects of estrogen in the rat.

While characterizing the effects of estrogen on an ovariectomized (OVX) rat model of bone loss, we examined several weight-matching regimens [e.g., ad libitum (feed bins continually full), weight matched (rate of weight gain for OVX and Sham-OVX groups was equalized), and weight restricted (weight gain rates for all groups were equalized to that of estrogen-treated OVX rats)] for possible effects. Bone loss following ovariectomy is primarily the result of an increase in bone resorption and is extremely sensitive to the effects of estrogens. Thus, in all of our analyses, treatment with 17 beta-estradiol served as a positive control for the prevention of bone loss. Each weight-matching study had three groups: control (Sham-OVX), OVX, and OVX + 17 beta-estradiol (0.1 mg/kg/day), and lasted for either 2, 4, or 6 weeks. Throughout the study, each Sprague Dawley rat was weighed every other day, and following sacrifice, a femur was removed for bone mineral density (BMD) analysis at the distal metaphysis by single photon absorptiometry. Following 2 weeks of dietary modifications, no significant differences were detected in BMD among the ad lib or weight matched groups. However, an estradiol-preventable reduction in BMD in restricted OVX rats was detected at 2 weeks postovariectomy. Additionally, OVX rats in all three dietary regimens displayed an estrogen-preventable reduction in proximal femur BMD at 4 and 6 weeks postovariectomy. These results indicate that a 4-week rat ovariectomized model of bone loss, under conditions of ad libitum feeding, shows great potential for pharmacologic manipulation.

Pharmacologic manipulation of a four day murine delayed type hypersensitivity model.

A murine delayed-type hypersensitivity (DTH) model was developed as a tool for drug discovery. Time course studies indicated that hind paw swelling was maximal at four days post-sensitization with picryl chloride. A pharmacological survey involving daily administration of drugs revealed that as a class, the glucocorticoids (e.g. dexamethasone and corticosterone) were the most potent inhibitors of the DTH response. The immunosuppressants, methotrexate, cyclosporine A, cyclophosphamide, and azathioprine, were all able to suppress the DTH response, with methotrexate being the most potent suppressor of paw swelling. Likewise, non-steroidal anti-inflammatory agents (e.g. indomethacin, piroxicam, diclofenac, and naproxen) all suppressed the DTH response, with indomethacin and piroxicam being the most potent suppressors. A series of central nervous system affecting drugs, including serotonin agonists [e.g. trifluromethylphenylpiperazine (tfMPP), 1-(3-chlorophenyl)piperazine (mCPP), quipazine, and 8-hydroxy-DPAT hydrobromide (8-OH DPAT)], and serotonin antagonists (e.g. cyproheptadiene, ketanserin, and mianserin) were examined in the 4 day DTH model. Except for 8-OH DPAT, all of the serotonin agonists were able to suppress the DTH response, with mCPP being the most potent suppressor. In contrast, none of the tested serotonin antagonists had any effect on the DTH response. The histamine antagonists (e.g. cimetidine and chlorphineramine) were largely ineffective in suppressing the DTH response. These data provide a pharmacological profile for a series of immunomodulator, non-steroidal anti-inflammatory, and central nervous system active compounds in a classic immunologic model.

Pharmacologic manipulation of graft versus host induced splenomegaly.

A murine graft versus host (GVH) model was developed as a tool for drug discovery. A pharmacological survey revealed that as a class the anti-rheumatics (e.g., auranofin, azathioprine, and methotrexate) were the most potent inhibitors of GVH induced splenomegaly. The immunosuppressants, cyclophosphamide and cyclosporine A, and the glucocorticoids (e.g., dexamethasone, hydrocortisone, and corticosterone) were all able to suppress the GVH response. Anti-inflammatory agents (e.g., indomethacin and piroxicam), and a series of central nervous system affecting drugs, including serotonin agonists (e.g., trifluromethylphenylpiperazine (tfMPP), 1-(3-chlorophenyl)piperazine (mCPP), and quipazine), and tricyclic antidepressants (e.g., amitriptyline, desipramine, imipramine, and nortriptyline) typically were ineffective at doses up to 10 mg/kg. However, at high dose levels (30 mg/kg) piroxicam enhanced while amitriptyline and cyproheptadine (a mixed serotonin and histamine antagonist) suppressed GVH induced splenomegaly. These data provide a pharmacological profile for a series of immunomodulator, anti-inflammatory, and central nervous system active compounds in a classic immunologic model.

Conditioned immunosuppression of a murine delayed type hypersensitivity response: dissociation from corticosterone elevation.

The mechanisms involved in behavioral modulation of immunity by Pavlovian conditioning have not been delineated, although an elevation in adrenocortical steroids has been invoked as an explanation. Therefore, we investigated whether or not a 4-day, murine delayed type hypersensitivity (DTH) response could be modified by a taste aversion conditioning paradigm. Mice were conditioned by the pairing of their saccharin (0.1%) drinking water (SAC) with a cyclophosphamide (CY) injection (50 mg/kg) on Day 0. Conditioned mice that were exposed on Day 3 to SAC + CY, SAC + normal saline, or water + CY exhibited significant suppression of DTH induced paw swelling when compared to nonconditioned controls. Conditioned immunosuppression was demonstrated, since the DTH response was suppressed by SAC without a concomitant reexposure to the immunosuppressant, CY. However, when dexamethasone (3 mg/kg) was used as the conditioning agent, the SAC + vehicle group showed no reduction in paw swelling. A serum corticosterone time course study was performed to examine possible involvement of glucocorticoids in conditioned immunosuppression. On Day 3, mice were sacrificed 30, 60, 90, 120 min and 24 h after reexposure to SAC or water. No significant differences in serum corticosterone levels were detected between nonconditioned controls and any conditioned group at any time point. These results demonstrate conditioned immunosuppression of a cell-mediated immune response that is not linked to a rise in glucocorticoid levels.

Dissociation of immunosuppression by chlorpromazine and trifluoperazine from pharmacologic activities as dopamine antagonists.

Neuroleptic compounds may affect the immune system through a variety of mechanisms. Most possess a complex pharmacology, which makes specific, causal relationships difficult to discern. In this study, a series of experiments was performed to examine the effects of dopamine antagonists on a battery of immunologic parameters. Mitogen-induced lymphocyte proliferation in vitro was inhibited by haloperidol, chlorpromazine, and trifluoperazine at 10, 1 and 1 microM concentrations, respectively. Sulpiride and metoclopramide had no direct effect in vitro. In vivo lymphocyte proliferation was significantly reduced by chlorpromazine at the highest tested doses (12.5 and 15 mg/kg) and by trifluoperazine at the highest tested dose (30 mg/kg). All other dopamine antagonists had no significant effect on in vivo lymphocyte proliferation. A murine graft vs host (GVH) response was unaffected by haloperidol, sulpiride, and metoclopramide. Chlorpromazine and trifluoperazine exhibited significant inhibition of the GVH response at the highest doses only (15 and 30 mg/kg, respectively). In a picryl chloride induced delayed type hypersensitivity (DTH) assay, haloperidol, metoclopramide, and sulpiride had no effect. However, both chlorpromazine and trifluoperazine significantly reduced DTH-induced paw swelling at the higher doses (7.5 mg/kg, and 10 and 30 micrograms/kg, respectively). These studies indicate that the more specific dopamine antagonists (e.g. sulpiride, metoclopramide, and haloperidol) do not share the immunologic profiles of chlorpromazine and trifluoperazine, suggesting that these effects of chlorpromazine and trifluoperazine are not related to their dopamine antagonist properties.

Suppressive effects of morphine pellet implants on in vivo parameters of immune function.

Chronic morphine treatment elicits a variety of immunosuppressive effects in mice. Most of the work describing this immuno-suppressive activity of the opioid is based on in vitro assessments of the performance of certain components of the immune system in morphine-treated animals. Relatively little has been done by way of tracking the effects of chronic morphine treatment on immunologic parameters in the intact animal. Therefore, this study used several classic in vivo determinations of immune function in mice treated chronically with morphine. Morphine pellet (75 mg) implantation led to a significant inhibition (91%) of paw swelling in a picryl chloride-induced delayed type hypersensitivity response. Uptake of iododeoxyuridine in an in vivo lymphocyte proliferation assay and splenomegaly in a graft vs. host reaction were also significantly suppressed by morphine pellet implantation (34 and 52%, respectively). Coimplantation of a naltrexone pellet (10 mg) completely reversed the suppressive responses to morphine in each assay. Naltrexone alone had no significant effect in any of the assays. The suppressive effects of morphine were less pronounced in adrenalectomized mice in the graft vs. host assay (51% vs. 9% reduction in morphine-pelleted shams relative to morphine-pelleted adrenalectomized mice). These findings indicate the pathophysiologic significance of the previously reported suppression of in vitro correlates of immune function in morphine-pelleted mice. The results further demonstrate that the immunosuppressive effects observed after morphine pellet implantation are naltrexone reversible and suggest that activation of the adrenal is one potential mechanism for this effect.