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For more than 50 years, the research community has made strides to better determine the nutrient requirements for many common laboratory animal species. This work has resulted in high-quality animal feeds that can optimize growth, maintenance, and reproduction in most species. We have a much better understanding of the role that individual nutrients play in physiological responses. Today, diet is often considered as an independent variable in experimental design, and specialized diet formulations for experimental purposes are widely used. In contrast, drinking water provided to laboratory animals has rarely been a consideration in experimental design except in studies of specific water-borne microbial or chemical contaminants. As we advance in the precision of scientific measurements, we are constantly discovering previously unrecognized sources of experimental variability. This is the nature of science. However, science is suffering from a lack of experimental reproducibility or replicability that undermines public trust. The issue of reproducibility/replicability is especially sensitive when laboratory animals are involved since we have the ethical responsibility to assure that laboratory animals are used wisely. One way to reduce problems with reproducibility/replicability is to have a strong understanding of potential sources of inherent variability in the system under study and to provide “…a clear, specific, and complete description of how the reported results were reached [1].” A primary intent of this review is to provide the reader with a high-level overview of some basic elements of laboratory animal nutrition, methods used in the manufacturing of feeds, sources of drinking water, and general methods of water purification. The goal is to provide background on contemporary issues regarding how diet and drinking water might serve as a source of extrinsic variability that can impact animal health, study design, and experimental outcomes and provide suggestions on how to mitigate these effects.

A novel class of 1,3,5-pyrazoles has been discovered as potent human glucagon receptor antagonists. Notably, compound 26 is orally bioavailable in several preclinical species and shows selectivity towards cardiac ion channels, other family B receptors such hGIP and hGLP1, and a large panel of enzymes and additional receptors. When dosed orally, compound 26 is efficacious in suppressing glucagon induced plasma glucose excursion in rhesus monkey and transgenic murine pharmacodynamic models at 1 and 10 mpk, respectively.

Tissue distribution, bioavailability, and efficacy of alendronate in preventing progression of resorption of teeth were evaluated in cats. [Butyl-4-14C-]-alendronate accumulates on subgingival tooth and alveolar bone surfaces adjacent to vascularized tissue resulting in concentration of the drug around tooth roots. Three cats were treated with a 0.03 mg/kg IV bolus of [butyl-4-14C-]-alendronate followed by blood, urine, and feces collection and euthanasia 24-hours later. Drug tissue distribution was accessed by autoradiography and sample combustion. To assess bioavailability, 12 cats were administered alendronate orally (3.0 or 9.0 mg/kg in water or 9.0 mg/kg in tuna water) and urine was collected for 24-hours. In these formulations, alendronate oral bioavailability in cats was approximately 3 %. In addition, 10 cats with radiographic evidence of pre-existing tooth resorption (14 affected teeth) were treated with vehicle or 3.0 mg/kg alendronate per os once weekly for 22-weeks and, then, 9.0 mg/kg per os twice weekly for 27-weeks in a random, masked study. Radiographic area of resorption was measured and progression scored every 3 to 4-months. In placebo-treated cats, resorption progressed in five of six teeth (+ 97 % average increase in area of resorption), whereas progression of resorption was seen in only three of eight affected teeth in alendronate-treated cats with a – 22 % average change (decrease) in area (P < 0.01 difference in number of teeth showing progression; P < 0.001 difference in area of resorption). Alendronate accumulated preferentially on subgingival tooth surfaces and adjacent alveolar bone and, at a dose of 9 mg/kg twice weekly, effectively slowed or arrested the progression of resorption.

Glucose homeostasis is maintained by the combined actions of insulin and glucagon. Hyperglucagonemia and/or elevation of glucagon/insulin ratio have been reported in diabetic patients and in animal models of diabetes. Therefore, antagonizing glucagon receptor function has long been considered a useful approach to lower hyperglycemia. Dogs serve as an excellent model for studying glycemic control and various aspects of glucagon biology in vivo; however, the amino acid sequence of the dog glucagon receptor has not been reported. To better understand the pharmacology of the dog glucagon receptor and to characterize glucagon receptor antagonists, we cloned a cDNA corresponding to the glucagon receptor from dog liver RNA. The dog glucagon receptor shares a significant (>75%) homology at both nucleotide and amino acid levels with the glucagon receptor from human, monkey, mouse, and rat. The protein is highly conserved among all species in areas corresponding to the 7 trans-membrane domains. However, it shows significant divergence at the carboxy terminus such that the receptor from dog has the longest cytoplasmic tail among all species examined. When expressed in chinese hamster ovary cells, the dog glucagon receptor bound [125I]Glucagon with a K(d) of 477+/-106 pM. Glucagon stimulated the rise of intracellular cAMP levels in these cells with an EC(50) of 9.6+/-1.7 nM and such effects could be blocked by known peptidyl and non-peptidyl small molecule antagonists. In addition we show that a small molecule glucagon receptor antagonist with significant activity in cell based assays also blocked the ability of glucagon to induce elevation in blood glucose in beagle dogs. These data demonstrate that the cloned cDNA encodes a functional dog glucagon receptor. The availability of the dog cDNA will facilitate the understanding of glucagon pharmacology and aid in the characterization of novel glucagon antagonists that may serve as anti-hyperglycemic treatment for type 2 diabetes mellitus.

Dipeptidyl peptidase (DPP)-IV inhibitors are a new approach to the treatment of type 2 diabetes. DPP-IV is a member of a family of serine peptidases that includes quiescent cell proline dipeptidase (QPP), DPP8, and DPP9; DPP-IV is a key regulator of incretin hormones, but the functions of other family members are unknown. To determine the importance of selective DPP-IV inhibition for the treatment of diabetes, we tested selective inhibitors of DPP-IV, DPP8/DPP9, or QPP in 2-week rat toxicity studies and in acute dog tolerability studies. In rats, the DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality. In dogs, the DPP8/9 inhibitor produced gastrointestinal toxicity. The QPP inhibitor produced reticulocytopenia in rats only, and no toxicities were noted in either species for the selective DPP-IV inhibitor. The DPP8/9 inhibitor was also shown to attenuate T-cell activation in human in vitro models; a selective DPP-IV inhibitor was inactive in these assays. Moreover, we found DPP-IV inhibitors that were previously reported to be active in models of immune function to be more potent inhibitors of DPP8/9. These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents.

MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione-containing dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK-0767 contains a chiral center at the C-5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)-(R) to (-)-(S) enantiomers of MK-0767 were determined in plasma from humans (in vitro only) and nonclinical species used in the toxicological evaluation of rac-MK-0767, namely CD-1 mice, Sprague-Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (approximately 1.5-1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo. In mice, the ratio was higher in vitro (approximately 1) than in vivo (approximately 0.6), while in rabbits it was higher in vivo (approximately 1) than in vitro (approximately 0.5). These results suggested that differential binding of the MK-0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo, since in protein-free systems MK-0767 exists as the racemate.

The absorption, metabolism, and excretion of [14C]aprepitant, a potent and selective human substance P receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, was evaluated in rats and dogs. Aprepitant was metabolized extensively and no parent drug was detected in the urine of either species. The elimination of drug-related radioactivity, after i.v. or p.o. administration of [14C]aprepitant, was mainly via biliary excretion in rats and by way of both biliary and urinary excretion in dogs. Aprepitant was the major component in the plasma at the early time points (up to 8 h), and plasma metabolite profiles of aprepitant were qualitatively similar in rats and dogs. Several oxidative metabolites of aprepitant, derived from N-dealkylation, oxidation, and opening of the morpholine ring, were detected in the plasma. Glucuronidation represented an important pathway in the metabolism and excretion of aprepitant in rats and dogs. An acid-labile glucuronide of [14C]aprepitant accounted for approximately 18% of the oral dose in rat bile. The instability of this glucuronide, coupled with its presence in bile but absence in feces, suggested the potential for enterohepatic circulation of aprepitant via this conjugate. In dogs, the glucuronide of [14C]aprepitant, together with four glucuronides derived from phase I metabolites, were present as major metabolites in the bile, accounting collectively for approximately 14% of the radioactive dose over a 4- to 24-h period after i.v. dosing. Two very polar carboxylic acids, namely, 4-fluoro-alpha-hydroxybenzeneacetic acid and 4-fluoro-alpha-oxobenzeneacetic acid, were the predominant drug-related entities in rat and dog urine.

Substituted 4-amino cyclohexylglycine analogues were evaluated for DP-IV inhibitory properties. Bis-sulfonamide 15e was an extremely potent 2.6 nM inhibitor of the enzyme with excellent selectivity over all counterscreens. 2,4-difluorobenzenesulfonamide 15b and 1-naphthyl amide 16b, however, combined an acceptable in vitro profile with good pharmacokinetic properties in the rat, and 15b was orally efficacious at 3 mpk in an OGTT in lean mice.

The pharmacokinetics and oral bioavailability of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(trifluoromethylphenyl]thiazol-2-yl]benzenesulfonamide (1), a 3-pyridyl thiazole benzenesulfonamide beta3-adrenergic receptor agonist, were investigated in rats, dogs, and monkeys. Systemic clearance was higher in rats (approximately 30 ml/min/kg) than in dogs and monkeys (both approximately 10 ml/min/kg), and oral bioavailability was 17, 27, and 4%, respectively. Since systemic clearance was 25 to 40% of hepatic blood flow in these species, hepatic extraction was expected to be low, and it was likely that oral bioavailability was limited either by absorption or a large first-pass effect in the gut. The absorption and excretion of 3H-labeled 1 were investigated in rats, and only 28% of the administered radioactivity was orally absorbed. Subsequently, the hepatic extraction of 1 was evaluated in rats (30%) and monkeys (47%). The low oral bioavailability in rats could be explained completely by poor oral absorption and hepatic first-pass metabolism; in monkeys, oral absorption was either less than in rats or first-pass extraction in the gut was greater. In an attempt to increase oral exposure, the pharmacokinetics and oral bioavailability of two potential prodrugs of 1, an N-ethyl [(R)-N-[4-[2-[ethyl[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(trifluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide; 2] and a morpholine derivative [(R)-N-[4-[2-[2-(3-pyridinyl)morpholin-4-yl]ethyl]phenyl]-4-[4-[4-(trifluoromethyl)- phenyl]thiazol-2-yl]benzenesulfonamide; 3], were evaluated in monkeys. Conversion to 1 was low (3%) with both derivatives, and neither entity was an effective prodrug, but the oral bioavailability of 3 (56%) compared with 1 (4%) was significantly improved. The hypothesis that the increased oral bioavailability of 3 was due to a reduction in hydrogen bonding sites in the molecule led to the design of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-2-yl)ethyl]amino]ethyl]phenyl]-4-[4-(4-trifluoromethylphenyl)thiazol-2-yl]benzenesulfonamide (4), a 2-pyridyl beta3-adrenergic receptor agonist with improved oral bioavailability in rats and monkeys.

As part of our investigation into the development of orally bioavailable beta(3) adrenergic receptor agonists, we have identified a series of pyridylethanolamine analogues possessing a substituted thiazole benzenesulfonamide pharmacophore that are potent human beta(3) agonists with excellent selectivity against other human beta receptor subtypes. Several of these compounds also exhibited an improved pharmacokinetic profile in dogs. For example, thiazole sulfonamide 2e (R = 4-F(3)C-C(6)H(4)) is a potent full beta(3) agonist (EC(50) = 3.6 nM, 94% activation) with >600-fold selectivity over the human beta(1) and beta(2) receptors, which also displays good oral bioavailability in several mammalian species, as well as an extended duration of action.

Compounds containing a 1,2,3-triazole-substituted benzenesulfonamide were prepared and found to be potent and selective human beta3-adrenergic receptor agonists. The most interesting compound, trifluoromethylbenzyl analogue 12e (beta3 EC50 = 3.1 nM with >1500-fold selectivity over binding to both beta1- and beta2 receptors), stimulates lipolysis in the rhesus monkey (ED50 = 0.36 mg/kg) and is 25% orally bioavailable in the dog.

BACKGROUND Previous studies have shown that chronic treatment of castrate dogs with androgen and estrogen results in significant prostate growth. Estrogen treatment of castrate dogs in the absence of androgen has resulted in conflicting data as reported by several authors. The purpose of this experiment was to evaluate the effect of a physiological dose of estradiol on prostate growth in dogs, using ultrasound to study size changes over time. METHODS Dogs (n = 25) were randomly divided into groups (n = 5) and treated as follows: castration alone (CC), castration plus low dose estradiol (E(2) low), castration plus high estradiol (E(2) high), castration plus estradiol and androstanediol (E(2)A), or no treatment (normal controls, NC). Silastic implants containing 5alpha-androstan-3alpha-17beta-diol (3alphadiol), and/or 17beta-estradiol were used for continous delivery of steroids. Prostate volume was measured by transrectal ultrasonography, and blood was drawn for hormone and sex hormone binding globulin (SHBG) determinations. RESULTS Results show that serum estradiol and SHBG levels were fairly constant over 12 weeks in all groups. Estradiol-treated groups had mean serum estradiol values of approximately 40 and 60 pg/ml, respectively. Initially, all groups had similar prostate volumes. Over 12 weeks the castrate dogs had a decline in prostate volume, whereas the intact dogs and those treated with E(2) and 3alpha-diol maintained a constant prostate volume. Estradiol treatment caused a large, late onset (week 7), dose-dependent increase in prostate volume relative to the intact group (P < 0.01). At 12 weeks, animals were euthanized and prostates weighed. The mean prostate weights in each group were: NC 14.8 +/- 2. 9, CC 2.4 +/- 0.5, E(2)A 9.7 +/- 2.0, E(2) low 21.7 +/- 4.3, and E(2) high 63.6 +/- 12.6 g (geometric mean +/- SEM). Histologically, prostates of estrogen-treated dogs showed metaplastic squamous epithelium. CONCLUSIONS These results demonstrate that estradiol causes marked dose-dependent stimulation of prostate growth in the castrate dog.

L-770,644 (9c) is a potent and selective agonist of the human β3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.

It has been well established that the spiroindoline sulfonamide MK-0677 stimulates GH secretion from the pituitary both in vitro and in vivo. MK-0677 has also been shown to increase serum insulin-like growth factor I (IGF-I) and cortisol levels in vivo; these increases are assumed to be driven by the increased serum GH and ACTH levels, respectively. However, such increases could also be due to a direct stimulatory action of MK-0677 at the level of the liver and adrenal cortex. To address this possibility, we investigated whether MK-0677 increased IGF-I and cortisol levels in hypophysectomized dogs. Baseline GH, IGF-I, and cortisol responses to MK-0677 (1 mg/kg, orally) were initially determined. Hypophysectomy (hypox; n = 7) or sham surgery (sham; n = 5) was then carried out. Six days postsurgery, the GH and cortisol responses to MK-0677 were reevaluated in each dog. In addition, each dog was treated with porcine GH (PST; 0.1 IU/kg, s.c.) to confirm the responsiveness of the GH-IGF-I axis. The mean peak GH increases in response to MK-0677 in the presham dogs (83.7 +/- 19.2 ng/ml), post-sham dogs (108 +/- 26.2 ng/ml), and pre-hypox dogs (121.2 +/- 13.6 ng/ml) were not significantly different. Mean peak GH levels were unchanged after MK-0677 administration in the hypox dogs (2.3 +/- 0.7 ng/ml). Before surgery, serum IGF-I levels increased to 243 +/- 27 and 224 +/- 47 ng/ml in the sham and hypox groups, respectively, after MK-0677 administration. Surgery was associated with a marked (> or =50%) decrease in serum IGF-I levels. MK-0677 administration increased IGF-I levels in the sham dogs from 78 +/- 14 to 187 +/- 31 ng/ml, whereas IGF-I levels remained unchanged (17.7 +/- 2.4 ng/ml) in the-hypox dogs. PST treatment increased IGF-I levels in the sham dogs from 162 +/- 30 to 325 +/- 32 ng/ml. In the hypox dogs PST treatment restored IGF-I to physiological levels (from 17.7 +/- 2.4 to 199 +/- 41 ng/ml). Cortisol was increased after MK-0677 administration 3.7-fold in the pre-sham, 3.6-fold in the post-sham, and 3.6-fold in the pre-hypox dogs, but no increase was seen in the post-hypox dogs. ACTH GEL administration (2.2 U/kg, i.m.) to hypox dogs returned cortisol to normal physiological levels, demonstrating the functional integrity of the adrenal cortex. This study demonstrates that the GH secretagogue MK-0677 does not directly stimulate an increase in serum IGF-I or cortisol levels, but depends upon the presence of an intact pituitary.

Human beta3 adrenergic receptor agonists containing 5-membered ring ureas were shown to be potent partial agonists with excellent selectivity over beta1 and beta2 binding. L-760,087 (4a) and L-764,646 (5a) (beta3 EC50 = 18 and 14 nM, respectively) stimulate lipolysis in rhesus monkeys (ED50 = 0.2 and 0.1 mg/kg, respectively) with minimal effects on heart rate. Oral absorption in dogs is improved over other urea analogs.

The 3-pyridylethanolamine L-757,793 is a potent β 3 AR agonist (EC 50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the β 1 and β 2 ARs, respectively, L-757,793 stimulated lipolysis in rhesus monkeys (ED 50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.

The identification of L-739,943 (8b), a potent, orally bioavailable benzolactam growth hormone secretagogue, is obtained from zwitterionic L-692,429 through modification of its amino acid side chain and replacement of the acidic 2'-tetrazole with the neutral and potency enhancing 2'-(N-methylaminocarbonylamino)methyl substituent. L-739,943 is orally active for the release of growth hormone in beagle dogs at doses as low as 0.5 mg/kg. Oral bioavailability in dogs of 8b is 24% at a dose of 2 mg/kg with a mean drug Cmax of 145 +/- 46 ng/mL. L-739,943 represents a significant breakthrough in terms of both potency and oral bioavailability as compared to the prototype benzolactam L-692,429.

Estradiol (E2) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) have been implicated in prostate hyperplasia in man and dogs, but neither of these steroids bind to androgen receptors (ARs). Recently, we reported that E2 and 3alpha-diol stimulated generation of intracellular cAMP via binding to a complex of sex hormone-binding globulin (SHBG) and its receptor (R(SHBG)) on prostate cells. We speculated that this pathway, involving steroids normally found in the prostate, was involved in the indirect activation of ARs. Using the dog as a model to test this hypothesis in normal prostate, we investigated whether E2, 3alpha-diol, and SHBG stimulated the production of the androgen-responsive protein, arginine esterase (AE), the canine equivalent of human prostate-specific antigen. In cultured dog prostate tissue preincubated with SHBG, E2 and 3alpha-diol stimulated AE activity. These effects were blocked by hydroxyflutamide, an AR antagonist, and by 2-methoxyestradiol, a competitive inhibitor of E2 and 3alpha-diol binding to SHBG. In the absence of exogenous steroids and SHBG, AE also was significantly increased by treatment with forskolin or 8-Bromoadenosine-cAMP. These observations support the hypothesis that in normal prostate, E2 and 3alpha-diol can amplify or substitute for androgens, with regard to activation of the AR via the R(SHBG) by a signal transduction pathway involving cAMP. Because both E2 and 3alpha-diol are involved in the pathogenesis of benign prostatic hyperplasia in dogs and implicated in benign prostatic hyperplasia in man, antagonism of the prostatic SHBG pathway may offer a novel and attractive therapeutic target.

Twenty-two beagles were divided into two equal groups, and the right hindlimb of each animal was immobilized at 105 degrees of knee flexion by external fixation. After 10 weeks of fixation, the device was removed, allowing free mobility for the following 5 weeks. Each day throughout the 15 weeks, one group received a growth hormone secretagogue (treatment) at a dose of 5 mg/kg, and the other received a lactose placebo (control). At weeks 0, 10, and 15, strength as indicated by maximum isometric extension torque was measured in the right hindlimb, biopsies of the vastus lateralis muscle were taken, and the dogs were weighed. Weekly blood samples were analyzed for insulin-like growth factor-1, blood urea nitrogen, and creatine phosphokinase. Between weeks 0 and 10, tetanic torque declined by about 60% (p0.001) in both groups, with no significant difference between the groups (p0.7). Between weeks 10 and 15, tetanic torque in the treated group increased by 0.81 Nm; this was significantly greater than the increase of 0.25 Nm in the placebo group (p0.05). The diameters of slow (type-1) and fast (type-2) fibers measured from the vastus lateralis muscle followed the same trend. At all time points, fiber diameter correlated strongly with torque; this argues against nonmuscular causes such as nerve injury for strength loss. The mean levels of insulin-like growth factor-1 increased 100% by week 4 in the treated group and remained elevated by about 60% throughout the experiment. Levels of insulin-like growth factor-1 in the placebo group decreased 30% within week 1 and remained depressed throughout the experiment. Our interpretation of these data suggests that the growth hormone secretagogue elevated levels of serum insulin-like growth factor-1, which in turn increased the size and strength of the quadriceps muscle during remobilization. These data may ultimately have therapeutic application to humans during rehabilitation after prolonged inactivity.

We have reported that MK-0677 is a novel, orally active GH secretagogue that stimulates an immediate and longlasting increase in serum GH levels in dogs. Significant elevations in IGF-I levels were associated with the increased GH secretion. Cortisol secretion was also increased following MK-0677 administration. In the current study, we determined the effect of repeat oral administration of MK-0677 on GH, IGF-I and cortisol levels; we also investigated if the GH and cortisol responses to MK-0677 are influenced by circulating IGF-I concentrations. Following the initial oral administration of MK-0677, GH secretion (area under the time–response curve (AUC) ng/ml per h) was increased 7·9- to 9·8-fold (1·0 mg/kg), 5·6-fold (0·5 mg/kg) or 3·9-fold (0·25 mg/kg). With repeat MK-0677 administration, the GH response was decreased by 41–77%; GH concentrations remained significantly above control in the 0·5 mg/kg and 1·0 mg/kg groups. Individual beagle GH profiles indicated that the increased GH concentration was associated with an amplified GH pulsatile profile. Serum IGF-I levels were significantly increased over control levels at all dosage levels by 480 min on the first day of MK-0677 administration. With repeated administration, IGF-I levels were increased up to 126% and remained elevated through 14 days, the longest treatment period evaluated. While daily MK-0677 administration appeared to increase IGF-I levels over 24 h, as evidenced by significant increases in the pretreatment IGF-I levels on days 4–14, no such increase was noted with alternate day MK-0677 administration; thus the dosage regimen modulated circulating IGF-I levels. MK-0677 stimulated increases in cortisol secretion (AUC μg/dl per h) on the first day of treatment. A decreased cortisol response was observed following repeated daily treatment with MK-0677; in contrast, with alternate day treatment, no decrease in cortisol response to MK-0677 occurred. A marked increase in circulating IGF-I concentrations following administration of exogenous GH resulted in a significant decrease in both the GH and cortisol response to MK-0677 compared with control animals. Our findings suggested, therefore, that circulating IGF-I concentrations regulate GH and cortisol response to MK-0677. In summary, chronic oral administration of MK-0677 was associated with significant increases in GH and IGF-I levels that were maintained for the duration of the treatment. The GH profile following MK-0677 administration consisted of episodic increases above control. Compared with day 1, repeated daily treatment with MK-0677 resulted in an attenuated GH response that was associated with an increase in circulating IGF-I levels. The cortisol response was similarly reduced during chronic MK-0677 treatment, suggesting that IGF-I mediated negative feedback on both the GH and cortisol axes. The fact that similar attenuation of the GH and cortisol responses to MK-0677 on day 1 was observed if IGF-I levels were increased by treating animals with exogenous GH suggested that the attenuated response to MK-0677 that occurred during chronic treatment was mediated by increases in IGF-I rather than desensitization to MK-0677. Thus, a regulatory feedback loop apparently prevents hyperstimulation of the GH axis by MK-0677. We conclude that MK-0677 offers the potential of an orally active GH secretagogue that can maintain elevated IGF-I levels when administered chronically. Journal of Endocrinology (1997) 152, 183–192

Large animal models of heart failure are essential in preclinical device testing. In sheep, catheter-based coil embolization of the left anterior descending and diagonal artery provides a minimally invasive and reproducible model of myocardial infarction (MI). Although widely used, this model has historically been plagued with a 30% mortality rate, both in the literature and in our own experience. Our study endeavored to decrease the mortality rate by targeting the most common cause of death, intractable arrhythmias, during creation of the ovine MI model. To this end, we evaluated 2 methods of managing perioperative antiarrhythmic therapy and cardiopulmonary resuscitation during model creation. The first group of sheep was managed at the discretion of the individual operator, whereas the second group was treated according to a standardized protocol that included mandatory pretreatment with amiodarone. Sheep experiencing life-threatening arrhythmias, most commonly ventricular fibrillation, were either resuscitated according to operator-driven instructions or the standardized protocol. By comparing these 2 treatment groups, we have shown that using a standardized protocol is advantageous in reducing mortality associated with the ovine MI model. Since implementing the standardized protocol, our laboratory has lowered the expected mortality rate to 10% during catheter-based induction of ovine MI and has greatly reduced the number of animals required for study needs. In addition, the standardized protocol has proven beneficial in training new staff members. By implementing this standardized method of model management, the outcomes of model creation have been optimized.

To investigate the effect of hypophyseal transection (HST) on GH secretagogue activity of the non-peptidyl GH secretagogue L-692,585 in the conscious pig, male castrated swine were randomly assigned to either a hypophyseal stalk transection group (HST; n=3) or to a sham-operated control group (SOC; n=3). Treatments administered were L-692,585 (100 γg/kg), human GH-releasing factor(1–29)NH2 (GRF; 20 γg/kg) or L-692,585 (100 γg/kg) + GRF (20 γg/kg) on days −7 to −3 before surgery and days +3 to +8 after surgery. To evaluate the integrity of the pituitary gland, the animals were challenged with corticotropin-releasing hormone (CRH; 150 γg) or GnRH (150 ng/kg) both before and after surgery. Blood was collected from −60 to +180 min post treatment and assayed for GH, cortisol and LH. Before surgery, no significant difference (P>0·05) in peak GH response (ng/ml) was present between the two groups (SOC vs HST) in response to L-692,585 (101 ± 12 vs 71 ± 9) or L-692,585 + GRF (171 ± 21 vs 174 ± 21). Only two out of three SOC vs three out of three HST pigs responded to GRF (13 ± 2 vs 25 ± 3) resulting in a significant difference between groups. Following surgery, significant differences were present in peak GH response (ng/ml) between SOC and HST groups following L-692,585 (79 ± 6 vs 13·8 ± 1·0); however, the response to L-692,585 + GRF was similar (115 ± 8 vs 94 ± 7). All animals responded to GRF; however, a significant difference was present between groups due to the magnitude of the responses. Whereas the cortisol responses (ng/ml) to L-692,585 in the SOC and HST groups were similar before surgery, a significant difference was present after surgery (44·4 ± 6·4 vs 14·6 ± 2·1). No significant difference was noted between the HST and SOC groups in response to CRH or GnRH either before or after surgery. These results indicated that L-692,585 induced an immediate GH response in the intact animal in contrast to GRF where the GH release was variable. L-692,585 also stimulated an immediate increase in cortisol levels. Transection of the hypophyseal stalk dramatically decreased but did not ablate the GH or cortisol response to L-692,585. Co-administration of L-692,585 + GRF induced an immediate GH response of similar magnitude in the intact and HST animal. We conclude that L-692,585 has a direct but limited action at the level of the pituitary and that an intact hypophyseal stalk is required for a maximal GH and cortisol response. L-692,585 acts with GRF at the level of the pituitary to induce a maximal GH response. These findings suggest that L-692,585 stimulates GH secretion by acting in combination with GRF and interrupting the inhibitory tone of somatostatin on the somatotroph. Journal of Endocrinology (1996) 148, 371–380

Publisher Summary Chinese hamsters are small rodents with a grayish black coat and a black dorsal stripe. Adult animals weigh approximately 39–46 gm, and measure approximately 9 cm in length. This species has been shown to be susceptible to a number of experimentally induced viral, bacterial, and parasitic infections. In recent years, the Chinese hamster's contributions as a laboratory animal have been largely overshadowed by the focus on its cell lines and the role it plays in scientific research and biotechnology. The Chinese hamster used in biomedical research is traditionally classified as Cricetulus griseus. It has several biological features that have helped promote its use in biomedical research and these attributes include its small size, polyestrous cycle, short gestation period, and low chromosome number. The Chinese hamster has a low incidence of spontaneous and endogenous viral infections. This species has been shown to be susceptible to a number of experimentally induced viral, bacterial, and parasitic infections. Chinese hamster-derived cells have played a major role in cytogenetic toxicity assays and the production of glycosylated therapeutic proteins. The behavior, research uses, and general toxicology of the Chinese hamster are summarized in this chapter.

L-692,585 is a 2-hydroxypropyl derivative of L-692,429, both novel non-peptidyl growth hormone (GH) secretagogues. The effects of single and repeated intravenous administration of L-692,585 on serum or plasma GH and other hormones in beagles were evaluated. In a balanced 8-dog dose-ranging study, compared to the saline control with a mean (± s.e.m.) after-dose serum GH peak of 6·1 ± 1·3 ng/ml, L-692,585 significantly increased (P These studies demonstrated that L-692,585 is a potent GH secretagogue that induces an acute, transient increase in GH levels. The magnitude of the response is not down-regulated following repeated daily administration for 14 days. IGF-1 levels are increased for an extended period following each treatment. Journal of Endocrinology (1994) 143, 399–406

A series of thiazole benzenesulfonamide-substituted 3-pyridylethanolamines was prepared and evaluated for their human beta3 adrenergic receptor agonist activity. Incorporation of aryl and heteroaryl substitution in the 4-position of the thiazole ring resulted in a number of highly potent and selective beta3 agonists. Results of preliminary in vivo evaluation of several of these compounds is described.

Dipeptidyl peptidase (DPP)-IV inhibitors are a new approach to the treatment of type 2 diabetes. DPP-IV is a member of a family of serine peptidases that includes quiescent cell proline dipeptidase (QPP), DPP8, and DPP9; DPP-IV is a key regulator of incretin hormones, but the functions of other family members are unknown. To determine the importance of selective DPP-IV inhibition for the treatment of diabetes, we tested selective inhibitors of DPP-IV, DPP8/DPP9, or QPP in 2-week rat toxicity studies and in acute dog tolerability studies. In rats, the DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality. In dogs, the DPP8/9 inhibitor produced gastrointestinal toxicity. The QPP inhibitor produced reticulocytopenia in rats only, and no toxicities were noted in either species for the selective DPP-IV inhibitor. The DPP8/9 inhibitor was also shown to attenuate T-cell activation in human in vitro models; a selective DPP-IV inhibitor was inactive in these assays. Moreover, we found DPP-IV inhibitors that were previously reported to be active in models of immune function to be more potent inhibitors of DPP8/9. These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents.

As a part of our investigation into the development of orally bioavailable beta3 adrenergic receptor agonists, we have identified a series of substituted oxazole derivatives that are potent beta3 agonists with excellent selectivity against other beta receptors. Several of these compounds showed excellent oral bioavailability in dogs. One example, cyclopentylethyloxazole 5f is a potent beta3 agonist (EC50 = 14 nM, 84% activation) with 340-fold and 160-fold selectivity over beta1 and beta2 receptors, respectively, and has 38% oral bioavailability in dogs.

5-n-Pentyl oxadiazole substituted benzenesulfonamide 8 is a potent and selective beta3 adrenergic receptor agonist (beta3 EC50 = 23 nM, beta1 IC50 = 3000 nM, beta2 IC50 = 3000 nM). The compound has high oral bioavailability in dogs (62%) and rats (36%) and is among the most orally bioavailable beta3 adrenergic receptor agonists reported to date.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

The cyclopentylpropylimidazolidinone L-766,892 is a potent beta3 AR agonist (EC50 5.7 nM, 64% activation) with 420- and 130-fold selectivity over binding to the beta1 and beta2 ARs, respectively. In anesthetized rhesus monkeys, L-766,892 elicited dose-dependent hyperglycerolemia (ED50 0.1 mg/kg) with minimal effects on heart rate.

A transorbital approach to the pituitary gland is described in domestic swine weighing between 40 and 70 kg. A transpalpebral eye exenteration is performed and the optic canal is enlarged caudally, using a bone drill. An operating microscope is used to improve visualization of the surgical site as the pituitary stalk and anterior pituitary are exposed to the level of the optic chiasm. This approach exposes the pituitary sufficiently to perform either a hypophyseal stalk transection or a hypophysectomy or to implant cannulas for hypothalamic-hypophyseal portal blood sampling. This technique has been performed in more than 50 pigs without major complications. Postoperative recovery has been rapid and uneventful. The transorbital approach is a significant refinement of the frontal craniotomy and cerebral elevation technique previously described in the pig, and results in shortened surgery time, minimal brain manipulation, and greatly decreased morbidity.

Equilibrium binding studies on canine mononuclear and granulocytic cells allow the identification of a single high affinity receptor for the human C-C chemokine RANTES (dissociation constant, 14 +/- 8 pM), that, in contrast to the human RANTES receptor, has no affinity for human macrophage inflammatory protein 1 alpha (hMIP-1 alpha). A single intradermal injection of hRANTES in dog resulted in eosinophil- and macrophage-rich inflammatory sites within 4 h. Cell infiltration peaked at 16-24 h after hRANTES injection. There was histological evidence of intravascular activation of eosinophils at 4 h, although eosinophils in the vasculature and interstitium contained apparently intact granules. Monocytes were the predominant cells adherent to venular endothelium at 16-24 h. Human MIP-1 alpha elicited no response in canine dermis, whereas monocyte chemoattractant protein 1 caused mild perivascular cuffing with monocytes. In contrast, human interleukin 8 induced a neutrophilic dermal infiltrate that was maximal by 4 h after challenge. This provides the first direct evidence in vivo that RANTES has significant proinflammatory activity and, in addition, could be a mediator in atopic pathologies characterized by eosinophilic and monocytic inflammatory responses.

A rapid and selective semi-automated gas chromatographic method, suitable for the routine assay of tiaramide in serum samples is described. The drug and the internal standard, perphenazine, were extracted with methylene chloride from alkalinized serum samples, and the trimethylsilyl ether derivatives were quantitated by a 63 Ni electron-capture detector. Linearity was observed for the range of 0.1–6.0 μg/ml. The average coefficient of variation for all concentration points over a two-week period was 8.5 ± 1.3%. Using an auto-sampler, the assay rate was 60 to 70 unknown samples in one man-day. The serviceability of the method has been demonstrated in a trial study in which an experimental tablet was given to three dogs. By taking a larger aliquot of the organic extract, a greater sensitivity can be attained if required. This was demonstrated by a trial study with human subjects.

A sensitive and specific GLC determination of plasma bucainide was developed using a nitrogen-specific detector. The method permits the determination of bucainide as low as 5.0 ng/ml of plasma and provides sufficient sensitivity and reproducibility for clinical use.