Development of a Mechanism-Based Dosimetry Model for 2,4,4-Trimethyl-2-pentanol-lnduced {alpha}2u-Globulin Nephropathy in Male Fischer 344 Rats

A mechanism-based dosimetry model was developed to describe2,4,4-trimethyl-2-pentanol (TMP-2-OH) dosimetry and renal 2u-globulin(2u) nephropathy in the male Fischer 344 rat. Experimental datawere collected to estimate the chemical-specific parameters(metabolic constants, tissue solubility, and oral absorptionrate) necessary to describe TMP-2-OH dosimetry in male rats.The concentrations of 2u and TMP-2-OH were measured in malerats up to 64 hr after a single oral dose of TMP-2-OH (6, 60,or 600 mg/kg). The model predicted the time course behaviorof TMP-2-OH and 2u in the kidney, but overestimated their renalconcentrations by two or threefold. Simulations of renal 2uconcentration were sensitive to changes in TMP-2-OH-2u-bindingaffinity and degradation rate of the TMP-2-OH-protein complex.In contrast, simulation of the concentration of TMP-2-OH inthe kidney was most sensitive to the amount of protein present.Oral absorption of TMP-2-OH was dose dependent. The model predictedthat 2u and TMP-2-OH concentration in the kidney is sensitiveto changes in the rate of TMP-2-OH absorbed after oral administration.This model permitted a more rigorous evaluation than has previouslybeen possible of the combination of protein characteristicsand chemical dosimetry required for the accumulation of 2u inthe kidney of male rats. The behavior of the model is consistentwith the qualitative aspects of the 2u hypothesis. However,further characterization of 2u distribution and renal hydrolysiswill be required in order to fully characterize the hypothesisat the quantitative level.     

Increased Hyaline Droplet Formation in Male Rats Exposed to Decalin Is Dependent on the Presence of {alpha}2u-Globulin

Increased Hyaline Droplet Formation in Male Rats Exposed toDecalin Is Dependent on the Presence of _2u-Globulin. RIDDER,G. M., VON BARGEN, E. C., ALDEN, C. L., AND PARKER, R. D. (1990).Fundam. Appl. Toxicol. 15, 732–743. A peculiar decalin-inducedmale rat nephropathy associated with the altered renal handlingof filtered protein appears limited to the accumulation of theprotein, _2u-globulin. Several strains of male rats that produce_2u-globulin (Fischer-344, Sprague-Dawley, Buffalo, and NorwayBrown) demonstrate spontaneous renal cortical hyaline dropletswhich are exacerbated after exposure to decalin. In all cases,a close correlation exists between hyaline droplet formationobserved histologically and _2u-globulin accumulation measuredbiochemically. In stark contrast, the NCI-Black-Reiter strain,which does not produce measurable quantities of _2u-globulin,neither forms hyaline droplets nor accumulates any filteredprotein in its kidney cortex either spontaneously or after exposureto decalin. Also, female rats injected ip with male rat _2u-globulinexhibit increased hyaline droplet formation and _2u-globulinaccumulation when treated with decalin. These data provide evidencethat the presence of _2u-globulin is key in understanding whythis nephropathy appears unique to the male rat.     

Degeneration of the Rat and Canine Adrenal Cortex Caused by {alpha}-(1,4-Dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone (DMNM)

Degeneration of the Rat and Canine Adrenal Cortex Caused by-(1,4-Dioxido-3-methylquin-oxalin-2-yl)-.Nmethylnitrone (DMNM).YARRINGTON, J. T., LOUDY, D. E., SPRINKLE, D. J., GIBSON, J.P., WRIGHT, C. L, and JOHNSTON, J. O. (1985). Fundam. Appl.Toxicol. 5, 370–381. The antibacterial druga -(1,4-dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone(DMNM) given at a dose of 22.5 mg/kg bid to four dogs for 14days caused diminished adrenal cortical reserves as determinedby decreased plasma cortisol (three dogs) and lower aldosteronelevels (four dogs) following the intravenous infusion of ACTH.A dose of 100 mg/kg/day of DMNM administered to rats for 31or 35 days resulted in significant decreases in blood glucose.Histologically, the adrenal glands of both species treated withDMNM for a maximum period of 21 days (dogs) and 35 days (rats)had widespread granular and vacuolar degeneration of the cortex.This degeneration in treated rats began in the zona reticularisand inner regions of the zona fasciculata and eventually involvedthe entire cortex including the zona glomerulosa. As a resultof treatment, significant ultrastructural alterations withincells of the rat and canine adrenal cortex consisted of degenerationof the mitochondria and an increase in the numbers and lipolysisof lipid droplets. The ultrastructure of the zona reticularisand fasciculata was most severely affected.     

Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.     

Specific binding of threo 1-{1-[2-(1,4-benzo-dioxane-2-yl)-2-hydroxyethyl] 4-piperidyl}-2-benzimidazolinone (R 29814), to rat brain membranes

The binding of ³[H]R 29814, the pharmacologically less active threoisomer of the potent hypotensive agent erythro-R 28935, was assayed in rat brain membranes and compared with published data on ³[H]R 28935 binding. The ³[H]R 29814 bound to homogenates of rat brain with one saturable component with high affinity (K_D = 1.1 nM). The specific binding was rapid and reversible. It was not affected by sodium or magnesium ions, or by guanosine triphosphate. The maximum number of binding sites amounted to approximately 135 fmol/mg protein. The drug specificity of the ³[H]R 29814 binding site was not associated with that of any known drug recognition site, but corresponded to that of erythro ³[H]R 28935. A linear relationship was derived between the affinities of drugs for inhibiting ³[H]R29814 and ³[H]R 28935 binding. The results suggest that identical sites were labeled by ³[H]R 29814 and ³[H]R 28935 in rat brain membranes with comparable affinity. Since the hypotensive activities of R 29814 and R 28935 greatly differ, it is concluded that the high affinity binding sites of 3[H]R 29814 as well as those of ³[H]R 28935 are not compatible with the sites responsible for the hypotensive effect.     

Diffuse Vacuolar Epithelial Degeneration in Rats Receiving Subchronic Vapor Exposure to Bis[2-(Dimethylamino)Ethyl] Ether (Dmaee)

Abstract

Four groups of Sprague-Dawley rats (15 rats/sex/group) were exposed by whole body to analytically measured concentrations of 0 (control), 0.23, 1.25, or 5.8 ppm bis/2-(dimethylamino)ethyl ether (DMAEE) vapor for 6 h/day, 5 days/wk, for 14 wk. Ten rats/sex/group were euthanized after the exposure regimen, and 5 rats/sex/group were euthanized after a 6-wk recovery period. An additional 3 rats/sex, exposed to 0 and 5.8 ppm, were sequentially sacrificed following 1, 3, or 5 exposures for electron microscopic evaluation. The only systemic toxicity was reduced body weight gain for the 5.8 ppm group throughout the exposure regmen. Clinical signs of ocular and nasal tract irritation (swollen eyelids and periocular and perinasal encrustation) were observed, primarily in the 5.8 ppm group, during the exposure and recovery periods. An ophthalmic examination, following 13 wk of exposure, revealed keratitis in males from the 5.8 ppm group and females from the 1.25 and 5.8 ppm groups. At necropsy, diffuse corneal color change and/or swollen eyelids were observed for the 1.25 and/or 5.8 ppm groups. Exposure-related microscopic lesions were seen only in tissues having direct contact with DMAEE, including nonhaired skin, eyes, and the upper respiratory tract. These consisted of necrosis, inflammation, and vacuolar degeneration of the nasal cavity mucosa and submucosa, and vacuolar degeneration of the larynx, trachea, bronchial epithelium, corneas, and glabrous or sparsely haired skin (pinna, muzzle, and eyelids). Nasal cavity lesions were present in rats from all exposure groups including the recovery period, while lesions in other tissues were observed only in the 5.8 ppm group at the 14-wk sacrifice. Electron microscopy of the nasal mucosa of rats from the 5.8 ppm group showed the development of intracytoplasmic membrane-bound vacuoles in the mucosal epithelium after a single exposure, and in the mucosa and submucosa after 3 and 5 exposures. Vacuoles developed from the coalescence of small secretory vesicles budding from the Colgi apparatus.     

Structure-activity relationships in 1,4-benzodioxan-related compounds. 8.(1) {2-[2-(4-chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-dimethoxyphenoxy)ethyl]amine (clopenphendioxan) as a tool to highlight the involvement of alpha1D- and alpha1B-adrenoreceptor subtypes in the regulation of human PC-3 prostate cancer cell apoptosis and proliferation

A series of new alpha1-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). All the compounds synthesized were potent antagonists and generally displayed, similarly to 4, the highest affinity values at alpha1D- with respect to alpha1A- and alpha1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB) assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity (estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration, with 7 ("clopenphendioxan") exhibiting the highest efficacy. Moreover, this study highlighted for the first time alpha1D- and alpha1B-AR expression in PC3 cells and also demonstrated the involvement of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction of alpha1D- and alpha1B-AR expression in PC3 cells was associated with the apoptosis induced by 7. This depletion was completely reversed by norepinephrine.     

Synthesis and structure-activity relationship of fluoro analogues of 8-{2-[4-(4-methoxyphenyl)piperazin-1yl]ethyl}-8-azaspiro[4.5]decane-7,9-dione as selective alpha(1d)-adrenergic receptor antagonists

We have discovered high-affinity antagonists (exemplified by 11 and 12) that are the most selective for alpha(1d)-adrenergic receptors (alpha(1d)-AR) reported to date. In cloned receptor assay systems, 12 displays at least 95-fold selectivity for the alpha(1d)-AR over all other G-protein-coupled receptors tested, and the subtype selectivity of 11 was confirmed in pharmacologically defined isolated tissue preparations.     

Identification of a novel selective peroxisome proliferator-activated receptor alpha agonist, 2-methyl-2-(4-{3-[1-(4-methylbenzyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]propyl}phenoxy)propanoic acid (LY518674), that produces marked changes in serum lipids and apolipoprotein A-1 expression

Low high-density lipoprotein-cholesterol (HDL-c) is an important risk factor of coronary artery disease (CAD). Optimum therapy for raising HDL-c is still not available. Identification of novel HDL-raising agents would produce a major impact on CAD. In this study, we have identified a potent (IC50 approximately 24 nM) and selective peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, 2-methyl-2-(4-{3-[1-(4-methylbenzyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]propyl}phenoxy)propanoic acid (LY518674). In human apolipoprotein A-1 (apoA-1) transgenic mice, LY518674 produced a dose-dependent increase in serum HDL-c, resulting in 208 +/- 15% elevation at optimum dose. A new synthesis of apoA-1 contributed to the increase in HDL-c. LY518674 increased apoA-1 mRNA levels in liver. Moreover, liver slices from animals treated with LY518674 secreted 3- to 6-fold more apoA-1 than control liver slices. In cultured hepatocytes, LY518674 produced 50% higher apoA-1 secretion, which was associated with increase in radiolabeled methionine incorporation in apoA-1. Thus, LY518674 is a potent and selective PPARalpha agonist that produced a much greater increase in serum HDL-c than the known fibrate drugs. The increase in HDL-c was associated with de novo synthesis of apoA-1.     

Development of LC/MS/MS assay for the determination of 5-ethyl-2-{5-[4-(2-hydroxyethyl)piperazine-1-sulfonyl]-2-propoxyphenyl}-7-propyl-3,5-dihydropyrrolo[3,2-d]pyrimidin-4-one (SK3530) in human plasma: application to a clinical pharmacokinetic study

5-Ethyl-2-{5-[4-(2-hydroxyethyl)piperazine-1-sulfonyl]-2-propoxyphenyl}-7-propyl-3,5-dihydropyrrolo[3,2-d]pyrimidin-4-one (SK3530) is a new phosphodiesterase type-5 inhibitor currently undergoing a Phase III investigation for the treatment of male erectile dysfunction. This study first describes a rapid and sensitive LC/MS/MS assay method for the quantification of SK3530 and its major metabolite, SK3541, in human plasma. The assay was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy, and precision. The multiple reaction monitoring was based on the transition of m/z=532.5-->99.1 for SK3530, 488.6-->295.5 for SK3541, and 520.3-->99.1 for SK3304 (internal standard). The assay utilized a single liquid-liquid extraction and isocratic elution, and the LLOQ was 1 ng/ml using 0.2 ml human plasma. The assay was linear over a range from 1 to 1000 ng/ml for both SK3530 and SK3541, with correlation coefficients >0.9999. The mean intra- and inter-day assay accuracy ranged from 94.7 to 101.6% and 96.8 to 101.1% for SK3530 and 92.6-105.7% and 97.4-107.8% for SK3541, respectively. The mean intra- and inter-day precision was between 7.2-12.1% and 5.7-7.4% for SK3530 and 4.6-13.2% and 5.0-14.1% for SK3541, respectively. The developed assay was applied to a clinical pharmacokinetic study after oral administration of SK3530 in healthy male volunteers (dose 100 mg).