Philippe Laroque

The Effects of Diet, Ad Libitum Overfeeding, and Moderate Dietary Restriction on the Rodent Bioassay: The Uncontrolled Variable in Safety Assessment

Ad libitum (AL) overfeeding is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. There is a highly significant correlation between AL food consumption, the resultant obesity and body weight, and low 2-yr survival in rodents. AL feeding of diets with lowered protein, metabolizable energy (ME), and increased fiber does not improve survival. Only dietary restriction (DR) of all diets tested significantly improves survival and delays the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. Moderate DR results in an incidence of spontaneous tumors similar to AL-fed rats, but the tumors are found incidentally and do not cause early mortality. There is a decreased age-adjusted incidence of pituitary and mammary gland tumors in moderate DR-fed rats, but tumor growth time is similar between AL and DR rats with only a delay in tumor onset time seen in DR-fed groups. Moderate DR does not significantly alter drug-metabolizing enzyme activities nor the toxicologic response to 5 pharmaceuticals tested at maximum tolerated doses (MTDs). However, moderate DR-fed rats did require much higher doses of 4 additional pharmaceutical compounds before classical MTDs were produced. Toxicokinetic studies of 2 of these compounds demonstrated equal or higher steady-state systemic exposures to parent drug and metabolites in moderate DR-fed rats. Markers of oxidative stress (lipid peroxidation, protein oxidation) are decreased and cytoprotective anti-oxidant markers are preserved in moderate DR-fed rats. But moderate DR does not delay reproductive senescence in female rats. Only marked DR delays reproductive senescence compared to AL and moderate DR-fed female rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for the rodent bioassay when used to assess pharmaceuticals for human safety and compounds for risk assessment.

The Novel γ Secretase Inhibitor N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) Reduces Amyloid Plaque Deposition without Evidence of Notch-Related Pathology in the Tg2576 Mouse

There is a substantial body of evidence indicating that β-amyloid peptides (Aβ) are critical factors in the onset and development of Alzheimers disease (AD). One strategy for combating AD is to reduce or eliminate the production of Aβ through inhibition of the γ-secretase enzyme, which cleaves Aβ from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant γ-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Aβ(40) and Aβ(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Aβ levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible—a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of γ-secretase should provide the basis for the next generation of γ-secretase inhibitors.

Chronic Nephropathy in Ad Libitum Overfed Sprague-Dawley Rats and Its Early Attenuation by Increasing Degrees of Dietary (Caloric) Restriction to Control Growth

The early development and progression of chronic nephropathy and its amelioration by moderate and marked dietary restriction (DR) was determined in Sprague-Dawley (SD) rats at 20, 33, 60, and 113 weeks of age. Both sexes of SD rats were overfed ad libitum (AL) or DR-fed at 72-79%, 68-72%, or 47-48% of the adult AL intake. The AL-fed rats rapidly developed increased body and kidney size, increased glomerular area (GA) and urinary protein loss, followed by declining creatinine clearance. Early increased kidney growth and glomerular hypertrophy by 20 weeks preceded increases in glomerular sclerotic index (GSI), 7-day BrdU tubular labeling index (TLI), and the lesions associated with chronic nephropathy. The glomerular number (GN) or the number of nephrons did not differ between the groups over the course of the study. Moderate DR (68-79% of AL) prevented the increased kidney size and GA at 20 weeks and delayed increases in GSI and TLI until 60 weeks of age. Marked DR (47—48% of AL) prevented increases in kidney size, GA and TLI at 20 weeks, and GSI at 60 weeks of age. In AL-fed rats, the early increase in GA predicted the early onset of proteinuria and the later decrease in creatinine clearance, and increased GSI, TLI, and mortality from severe nephropathy. The temporal and dose-related effects of increasing degrees of DR demonstrated that while nephron numbers were unchanged with age, the early development of glomerular hypertrophy was the critical morphological biomarker predicting the progression and severity of chronic nephropathy. Caloric restriction by DR prevented or delayed the development of glomerulosclerosis, tubulointerstitial damage, functional changes, morbidity, and mortality associated with chronic nephropathy in AL-overfed SD rats by controlling initial body and kidney growth, glomerular size, and nephron hypertrophy. These results indicate that control of body and renal growth by DR may be essential to prevent the development and progression of glomerulosclerosis in spontaneous nephropathy of laboratory rats.

Mechanisms of action of the antidepressants fluoxetine and the substance P antagonist L-000760735 are associated with altered neurofilaments and synaptic remodeling

Antidepressants are widely prescribed in the treatment of depression, although the mechanism of how they exert their therapeutic effects is poorly understood. To shed further light on their mode of action, we have attempted to identify a common proteomic signature in guinea pig brains after chronic treatment with two different antidepressants. Both fluoxetine and the substance P receptor (NK(1)R) antagonist (SPA) L-000760735 altered cortical expression of multiple heat shock protein 60 forms along with neurofilaments and related proteins that are critical determinants of synaptic structure and function. Analysis of NK(1)R-/- mice showed similar alterations of neurofilaments confirming the specificity of the effects observed with chronic NK(1)R antagonist treatment. To determine if these changes were associated with structural modification of synapses, we carried out electron microscopic analysis of cerebral cortices from fluoxetine-treated guinea pigs. This showed an increase in the percentage of synapses with split postsynaptic densities (PSDs), a phenomenon that is characteristic of activity-dependent synaptic rearrangement. These findings suggest that cortical alterations of the neurofilament pathway and increased synaptic remodeling are associated with the mechanism of these two antidepressant drug treatments and may contribute to their psychotherapeutic actions.

The Effects of Ad Libitum Overfeeding and Moderate and Marked Dietary Restriction on Age-Related Spontaneous Pituitary Gland Pathology in Sprague-Dawley Rats

This study compared the effects of ad libitum (AL) overfeeding and moderate or marked dietary restriction (DR) on the pathogenesis of aged-related pituitary gland changes in Sprague—Dawley (SD) rats. SD rats were fed Purina Certified Rodent Diet AL (group 1), DR at 72—79% of AL (group 2), DR at 68—72% of AL (group 3) or DR at 47—48% of AL (group 4) for 106 weeks. Interim necropsies were performed at 13, 26, and 53 weeks, after a 7-day 5-bromo-2-deoxyuridine (BrdU)-filled minipump implantation. Body weights, organ weights and insulin-like growth factor 1 (IGF-1) serum levels were measured at interim and final necropsies. Serum levels of prolactin (PRL), progesterone, estradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured at 53 and/or 106 weeks. In addition to the routine histopathologic examination, determination of 7 stereologic parameters after pituitary immunohistochemistry of PRL, growth hormone (GH) and BrdU was done in both sexes at 13, 26, and 53 weeks. Body and pituitary weights were proportional to the food intake. In AL-fed rats, hyperplastic and neoplastic changes developed early and progressed with age, affecting almost all animals by 106 weeks. These changes were associated with high PRL serum levels. Pituitary adenomas were the most common cause of death in both sexes. In DR rats, a delayed onset and a decreased incidence of pituitary tumors were observed in association with decreased serum IGF-1, PRL, estradiol, and LH levels. The results of the stereological analysis demonstrated that, compared to AL-fed rats, pituitary glands from DR rats contained lower PRL and GH secreting cell volumes, and a lower epithelial cell BrdU labeling index, which correlated with a lower incidence of pituitary tumors at study termination. Moderate and marked degrees of DR delayed the onset of pituitary tumors in a temporal-and dose-related manner. In contrast to marked DR, which dramatically reduced the incidence of hyperplastic and neoplastic pituitary gland changes, moderate DR delayed the onset but did not prevent the development of pituitary tumors.

Effect of chronic growth hormone administration on skeletal muscle in dogs.

Administration of growth hormone (GH) results in increased body weight gain in dogs. Increased body weight gain is believed to be a result of the trophic effect of GH on the musculoskeletal system. However, edema is one of the side effects described in man following exogenous GH administration. Thus, the objective of this study was to determine if the expected increased weight gain in GH-treated dogs is a result of increased muscle mass. Porcine growth hormone (pGH), administered subcutaneously to beagle dogs at doses of 0.025, 0.1, and 1 IU/kg/day for 14 wk, resulted in elevated serum GH and insulin-like growth factor-1 (IGF-1) levels (see accompanying paper, Prahalada et al). This was associated with a significant increase in body weight gain and weights of the cranial tibialis muscle in both male and female dogs. The increased muscle mass likely contributed to the significant increase in body weight gain seen in both sexes. Quantitative analysis of skeletal muscle sections stained for ATPase activity showed increases in type I (slow twitch) and type II (fast twitch) myofiber sizes in mid- and high-dose males and in high-dose females. The ratio of type I and type II muscle fibers remained unchanged. Hypertrophic myofibers were enlarged but had a normal histologic and ultrastructural organization when observed by light and transmission electron microscopy. The results of this study have demonstrated that increased muscle mass in pGH-treated dogs is related to hypertrophy of muscle fibers and not due to edema. Exogenous GH administration has an anabolic effect on skeletal muscle in dogs.

Morphological Changes in the Pituitary Gland of Dogs Chronically Exposed to Exogenous Growth Hormone

Growth hormone (GH) synthesis and release from the pituitary is regulated by hypothalamic releasing hormone, insulin-like growth factor-1 (IGF-1), and somatostatin. However, the potential effects of pharmacological doses of exogenous GH on the pituitary are not well studied. To determine the potential chronic effects of exogenous GH on pituitary morphology in dogs, porcine GH (pGH) was administered subcutaneously to 3 groups of dogs (4 animals/sex/group) at doses of 0.025, 0.1, and 1.0 IU/kg/day for 14 wk. A group (4/sex) of dogs served as the vehicle control. The pituitaries from all dogs were weighed and fixed in appropriate fixatives for light and electron microscopic examination; in addition, cells of the pars distalis were quantitated by a point counting method following immunostaining to identify cells containing GH, prolactin (PRL), and adrenocorticotrophic (ACTH) hormones. Administration of pGH resulted in a statistically significant (p ≤ 0.05) increased pituitary weight through the high dose. By light microscopy (LM), hypertrophy of pars distalis cells was evident in mid- and high-dose female dogs. The pituitaries of dogs given the lowest dose (0.025 IU/kg/day) of pGH were not remarkable based on weight and LM findings. In addition, transmission electron microscopic (TEM) examination of the pituitary gland of high-dose dogs demonstrated, in both sexes, pituitary cells with variably dilated rough endoplasmic reticulum and decreased numbers of secretory granules; some of these cells reacted positively to GH immunostaining. Quantitative analysis of the pituitary gland of high-dose males and females showed an increase in the absolute volume of all cell populations studied: GH-, PRL-, and ACTH-positive cells. Based on the LM and TEM findings, the increased volume of the cell populations studied is likely related to cellular hypertrophy. The expected elevation in serum GH levels following repeated administration of pGH and an associated elevation in serum IGF-1 levels resulted in morphologic changes in the pituitary gland of dogs given high doses (≥0.1 IU/kg/day) of pGH; these observations differed from the reported findings in pituitaries of transgenic mice secreting large quantities of bovine GH.

Morphological Changes in the Kidney of Dogs Chronically Exposed to Exogenous Growth Hormone

Porcine growth hormone was administered subcutaneously to beagle dogs at doses of 0.025,0.1, and 1 IU/kg/d for 14 weeks, markedly elevating serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. This was accompanied by a significant increase in body weight gain and kidney weights in both male and female dogs. The increase in kidney weight (6 to 54%) was slightly greater than the increase in body weight (6 to 40%). By light microscopy, glomerular deposits, mesangial thickening, and very slight cellular infiltration in glomeruli were seen in mid- and high-dose groups. Based on morphometric evaluation, there was an increase in the renal glomerular area, which was statistically significant (p ≤ 0.05) in the mid- and high-dose males and in the high-dose females. This was associated with a statistically significant (p ≤ 0.05) increase in the number of total glomerular cells in the mid- and high-dose males. By transmission electron microscopy, thickening of the glomerular basal lamina and diffuse increase of the mesangial matrix were observed in both male and female dogs in the mid- and high-dose groups. Immunohistochemical reactions were negative for IgG, IgM, and C3. The morphological changes in the kidney of dogs resemble the diffuse glomerulosclerosis described in human diabetic nephropathy.

The effects of ad libitum feeding and marked dietary restriction on spontaneous skeletal muscle pathology in Sprague-Dawley rats.

The effects of ad libitum (AL) feeding and marked dietary restriction (DR) on spontaneous age-related skeletal muscle changes in male Sprague–Dawley (SD) rats were evaluated at 1 and 2 years. SD rats were fed Certified UAR A04C Rodent Chow ad libitum (AL), or DR at 50% of AL for (106 weeks). Body weights and organ weights were measured at the 1-year interim and 2-year final necropsies. In addition to the routine histopathologic examination, determination of 5 stereologic parameters was done in the vastus lateralis muscle after histochemistry of ATPase activity at 1 and 2 years. Body and skeletal muscle weights were proportional to the food intake. In AL-fed rats, muscle weights decreased between 1 and 2 years, in correlation with decreased type 2 myofiber numbers. In this group, fibrovascular index markedly increased with aging and muscle degeneration occurred at 2 years. In DR rats, there were no significant changes in muscle weights between 1 and 2 years. No histopathological changes were observed and the fibrovascular index was unchanged. These results demonstrated a protective effect of DR on the age-related skeletal muscle pathology in SD rats.