Chronic Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta) with Cisterna Magna Ports: Update on Refinements.

More than 20 y ago, we developed an animal model for chronic and continuous collection of cerebrospinal fluid (CSF) from conscious rhesus macaques. Since our previous publication in 2003, we have successfully implanted 168 rhesus macaques using this approach. Our experience enables us to provide up-to-date information regarding the model, including refine- ments to our implant design, reductions in maintenance, and new procedures for dealing with contamination. The results of our experiences have reduced the number of surgeries required and helped to increase the longevity of the implant, with some functioning for more than 18 y. Building on our success in rhesus macaques, we attempted to develop similar animal models in the African green monkeys and dogs but have been unable to develop reliable chronic models for CSF collection in these species.

High-resolution peripheral quantitative computed tomography and finite element analysis of bone strength at the distal radius in ovariectomized adult rhesus monkey demonstrate efficacy of odanacatib and differentiation from alendronate.

Translational evaluation of disease progression and treatment response is critical to the development of therapies for osteoporosis. In this study, longitudinal in-vivo monitoring of odanacatib (ODN) treatment efficacy was compared to alendronate (ALN) in ovariectomized (OVX) non-human primates (NHPs) using high-resolution peripheral computed tomography (HR-pQCT). Treatment effects were evaluated using several determinants of bone strength, density and quality, including volumetric bone mineral density (vBMD), three-dimensional structure, finite element analysis (FEA) estimated peak force and biomechanical properties at the ultradistal (UD) radius at baseline, 3, 6, 9, 12, and 18 months of dosing in three treatment groups: vehicle (VEH), low ODN (2 mg/kg/day, L-ODN), and ALN (30 µg/kg/week). Biomechanical axial compression tests were performed at the end of the study. Bone strength estimates using FEA were validated by ex-vivo mechanical compression testing experiments. After 18months of dosing, L-ODN demonstrated significant increases from baseline in integral vBMD (13.5%), cortical thickness (24.4%), total bone volume fraction BV/TV (13.5%), FEA-estimated peak force (26.6%) and peak stress (17.1%), respectively. Increases from baseline for L-ODN at 18 months were significantly higher than that for ALN in DXA-based aBMD (7.6%), cortical thickness (22.9%), integral vBMD (12.2%), total BV/TV (10.1%), FEA peak force (17.7%) and FEA peak stress (11.5%), respectively. These results demonstrate a superior efficacy of ODN treatment compared to ALN at the UD radii in ovariectomized NHPs.

Effect of odanacatib on bone turnover markers, bone density and geometry of the spine and hip of ovariectomized monkeys: a head-to-head comparison with alendronate.

Odanacatib (ODN) is a selective and reversible Cathepsin K (CatK) inhibitor currently being developed as a once weekly treatment for osteoporosis. Here, effects of ODN compared to alendronate (ALN) on bone turnover, DXA-based areal bone mineral density (aBMD), QCT-based volumetric BMD (vBMD) and geometric parameters were studied in ovariectomized (OVX) rhesus monkeys. Treatment was initiated 10 days after ovariectomy and continued for 20 months. The study consisted of four groups: L-ODN (2 mg/kg, daily p.o.), H-ODN (8/4 mg/kg daily p.o.), ALN (15 µg/kg, twice weekly, s.c.), and VEH (vehicle, daily, p.o.). L-ODN and ALN doses were selected to approximate the clinical exposures of the ODN 50-mg and ALN 70-mg once-weekly, respectively. L-ODN and ALN effectively reduced bone resorption markers uNTx and sCTx compared to VEH. There was no additional efficacy with these markers achieved with H-ODN. Conversely, ODN displayed inversely dose-dependent reduction of bone formation markers, sP1NP and sBSAP, and L-ODN reduced formation to a lesser degree than ALN. At month 18 post-OVX, L-ODN showed robust increases in lumbar spine aBMD (11.4%, p<0.001), spine trabecular vBMD (13.7%, p<0.001), femoral neck (FN) integral (int) vBMD (9.0%, p<0.001) and sub-trochanteric proximal femur (SubTrPF) int vBMD, (6.4%, p<0.001) compared to baseline. L-ODN significantly increased FN cortical thickness (Ct.Th) and cortical bone mineral content (Ct.BMC) by 22.5% (p<0.001) and 21.8% (p<0.001), respectively, and SubTrPF Ct.Th and Ct.BMC by 10.9% (p<0.001) and 11.3% (p<0.001) respectively. Compared to ALN, L-ODN significantly increased FN Ct. BMC by 8.7% (p<0.05), and SubTrPF Ct.Th by 7.6% (p<0.05) and Ct.BMC by 6.2% (p<0.05). H-ODN showed no additional efficacy compared to L-ODN in OVX-monkeys in prevention mode. Taken together, the results from this study have demonstrated that administration of ODN at levels which approximate clinical exposure in OVX-monkeys had comparable efficacy to ALN in DXA-based aBMD and QCT-based vBMD. However, FN cortical mineral content clearly demonstrated superior efficacy of ODN versus ALN in this model of estrogen-deficient non-human primates.

Isolation and characterization of a novel helicobacter species, "Helicobacter macacae," from rhesus monkeys with and without chronic idiopathic colitis.

Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, "Helicobacter macacae" (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

PrimaTB STAT-PAK assay, a novel, rapid lateral-flow test for tuberculosis in nonhuman primates.

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.

Medical records for animals used in research, teaching, and testing: public statement from the American College of Laboratory Animal Medicine.

Medical records are considered to be a key element of a program of adequate veterinary care for animals used in research, teaching, and testing. However, prior to the release of the public statement on medical records by the American College of Laboratory Animal Medicine (ACLAM), the guidance that was available on the form and content of medical records used for the research setting was not consistent and, in some cases, was considered to be too rigid. To address this concern, ACLAM convened an ad hoc Medical Records Committee and charged the Committee with the task of developing a medical record guideline that was based on both professional judgment and performance standards. The Committee provided ACLAM with a guidance document titled Public Statements: Medical Records for Animals Used in Research, Teaching, and Testing, which was approved by ACLAM in late 2004. The ACLAM public statement on medical records provides guidance on the definition and content of medical records, and clearly identifies the Attending Veterinarian as the individual who is charged with authority and responsibility for oversight of the institution's medical records program. The document offers latitude to institutions in the precise form and process used for medical records but identifies typical information to be included in such records. As a result, the ACLAM public statement on medical records provides practical yet flexible guidelines to assure that documentation of animal health is performed in research, teaching, and testing situations.

Integrase inhibitors and cellular immunity suppress retroviral replication in rhesus macaques.

We describe the efficacy of L-870812, an inhibitor of HIV-1 and SIV integrase, in rhesus macaques infected with the simian-human immunodeficiency virus (SHIV) 89.6P. When initiated before CD4 cell depletion, L-870812 therapy mediated a sustained suppression of viremia, preserving CD4 levels and permitting the induction of virus-specific cellular immunity. L-870812 was also active in chronic infection; however, the magnitude and durability of the effect varied in conjunction with the pretreatment immune response and viral load. These studies demonstrate integrase inhibitor activity in vivo and suggest that cellular immunity facilitates chemotherapeutic efficacy in retroviral infections.

Sub-chronic administration of zolpidem affects modifications to rat sleep architecture.

Gamma-aminobutyric acidA receptor (GABAAR) modulators constitute the majority of clinically relevant sedative-hypnotics. Animal studies have clearly demonstrated sedative efficacy for these compounds in acute studies. However, relatively less is known regarding their efficacy under brief periods of repeat administration or following intermittent dosing. Therefore zolpidem, a short-acting GABAAR modulator with selectivity for the type-I (omega1) benzodiazepine receptor, was studied for efficacy in altering rat sleep architecture as determined by electrocorticogram (ECoG) and electromyogram (EMG) activity over a 7-day sub-chronic administration period. Zolpidem caused significant reductions in wakefulness entries and rapid eye movement (REM) sleep entries and duration, with increases in Delta sleep duration throughout the administration period. Examination of sleep architecture 24 h after cessation of sub-chronic zolpidem administration revealed a decrease in Delta sleep, suggesting that repeated zolpidem administration might elicit enduring modifications to sleep organization. This was not seen following similar dosing of diazepam. The efficacy of sub-chronic administration of zolpidem to alter sleep architecture was enhanced when the administration regimen was repeated following a 7-day hiatus. Significant increases in Delta sleep duration, with significant decreases in light sleep and wakefulness were observed during the repeated exposure to zolpidem. Therefore, sub-chronic administration of zolpidem affected lasting modifications in sleep organization that appeared both 1 day following administration and during reiterated administration without eliciting tolerance.

Postoperative pain management using fentanyl patches in dogs.

To the ability of the fentanyl patch to control pain in a postoperative canine model, we provided two male beagles with 25-mg/h patches and two with 50-mg/h patches 24 h prior to surgery. Each animal underwent a major abdominal surgical procedure to place three separate catheters with associated vascular access ports. Serum plasma levels of fentanyl were analyzed at multiple time points throughout the study period. Animals were subjectively assessed for postoperative pain by using a Simple Descriptive Scale at regular intervals postoperatively. Other parameters observed and recorded included heart and respiration rates, rectal temperature, appetite, and activity. The fentanyl patch appeared to adequately control postoperative pain in our canine abdominal surgical model. Three animals demonstrated mild pain 1 to 2 h postoperatively. Two animals, one from each dose group, showed mild pain 8 h postoperatively. Mild pain is commensurate with USDA category C, which encompasses procedures that do not result in more than momentary or slight pain or distress and do not require intervention. At no other time points were any of the animals considered to be in pain. Our study also suggested that increased subcutaneous fat delayed the rate of absorption of fentanyl. The lower body-weight beagles, which had the 25-mg/h patches, reached reported human serum analgesic levels within 8 h after placement, whereas the heavier beagles with the 50-mg/h patches reached human serum analgesic levels 12 h after placement. Fentanyl concentrations remained at the reported human analgesic levels in all animals between 2 to 4 h after the patches were removed. Regardless of the dose, decreases in heart rate, respiration rate, and temperature were observed in all four animals 12 h after placing the patches. Issues regarding the regulatory requirements to prevent drug abuse, the side effects and potency of fentanyl, and the prolonged duration of action as a transdermal system should be addressed by the veterinarian when considering usage of this analgesic method.

An alternative method of chronic cerebrospinal fluid collection via the cisterna magna in conscious rhesus monkeys.

Models of chronic cerebrospinal fluid (CSF) collection previously have been established for nonhuman primates and canines; many of these methods implement stainless-steel cannulas into the lateral or 4th ventricles or catheters into the cerebral or spinal subarachnoid space. These models have proved successful and reliable but unfortunately require invasive techniques to pass through the skull or require a laminectomy to enter the spinal subarachnoid space, involve the use of expensive and highly specialized stereotaxic equipment for the precise placement of the implants, and may require exteriorized hardware which is cumbersome to maintain and unaesthetic. The model we developed for the rhesus monkey allows for direct access to CSF outflow from the cisterna magna by using a 3.5-French fenestrated silicone catheter which was placed 1.0 cm into the cisterna. The catheter was attached to a titanium port placed subcutaneously between the scapulae to permit easy access for sampling CSF in a conscious, chaired rhesus monkey. We currently have instrumented animals from which we have consistently collected CSF for over 18 months. This novel, economical, less-invasive method permits chronic, reliable collection of CSF in conscious rhesus monkeys and has the additional advantages that the model is easier to maintain and more aesthetic.

Antigen recognition by serum antibodies in non-human primates experimentally infected with Mycobacterium tuberculosis.

Tuberculosis is a significant threat to non-human primates and their caretakers. The diagnosis of tuberculosis in living non-human primates is currently based on the tuberculin skin test, which is cumbersome and sometimes inaccurate. Development of an accurate serodiagnostic test requires identification of the key antigens of Mycobacterium tuberculosis involved in antibody production. When sequential serum samples obtained from 17 cynomolgus, rhesus, and African green monkeys up to seven months since experimental infection with M. tuberculosis Erdman were screened for antibody against purified proteins of M. tuberculosis, three highly seroreactive antigens were identified. One protein, ESAT-6, reacted with sera from all infected animals. Two additional proteins, alpha-crystallin and MTSA-10, were recognized by sera from approximately 90% of infected animals. Time course analysis of antibody production indicated that the earliest response was usually to ESAT-6 alone or to ESAT-6 and other antigen(s). These results provide experimental evidence of the potential value of ESAT-6 as an antigen for use in serodiagnosis of tuberculosis in non-human primates.

Clinical and microbiologic characterization of hemorrhagic pneumonia due to extraintestinal pathogenic Escherichia coli in four young dogs.

Over a 21-month period, three Beagle dogs and one mixed-breed dog at our facility developed fatal pneumonia. The four dogs, all purpose bred, came from three vendors and had received the standard canine vaccines prior to shipment. In each instance, the affected dog had been shipped to our facility within the past 10 days. Three cases presented as a peracute clinical syndrome, and all had gross and microscopic findings consistent with hemorrhagic pneumonia. Escherichia coli was isolated from the lungs of all four dogs. Results of testing of lung tissue for canine parainfluenza virus and canine adenovirus were negative. Escherichia coli was also isolated from blood of three of the four dogs. Serotyping of the E. coli isolates indicated that two were serotype 06 and two were 04. Isolates from all four dogs were positive for the virulence factors alpha hemolysin and cytotoxic necrotizing factor 1 and for the adhesin factor class-III papG allele. These traits place the isolates in the class of extraintestinal pathogenic E. coli, which is being increasingly implicated as a cause of extraintestinal infections in animals and humans and may represent a zoonotic risk to humans working with research dogs.

Preclinical and clinical pharmacodynamic assessment of L-778,123, a dual inhibitor of farnesyl:protein transferase and geranylgeranyl:protein transferase type-I.

Farnesyl:protein transferase (FPTase) inhibitors were developed as anti-Ras drugs, but they fail to inhibit Ki-Ras activity because Ki-Ras can be modified by geranylgeranyl:protein transferase type-I (GGPTase-I). L-778,123, an inhibitor of FPTase and GGPTase-I, was developed in part because it can completely inhibit Ki-Ras prenylation. To support the clinical development of L-778,123, we developed pharmacodynamic assays using peripheral blood mononuclear cells (PBMCs) to measure the inhibition of prenylation of HDJ2 and Rap1A, proteins that are FPTase- and GGPTase-I substrates, respectively. We validated these assays in animal models and show that inhibition of HDJ2 prenylation in mouse PBMCs correlates with the concentration of FPTase inhibitors in blood. In dogs, continuous infusion of L-778,123 inhibited both HDJ2 and Rap1A prenylation in PBMCs, but we did not detect inhibition of Ki-Ras prenylation. We reported previously results from the first L-778,123 Phase I trial that showed a dose-dependent inhibition of HDJ2 farnesylation in PBMCs. In this report, we present additional analysis of patient samples from this trial and a second Phase I trial of L-778,123, and demonstrate the inhibition of both HDJ2 and Rap1A prenylation in PBMC samples. This study represents the first demonstration of GGPTase-I inhibition in humans. However, no inhibition of Ki-Ras prenylation by L-778,123 was detected in patient samples. These results confirm the pharmacologic profile of L-778,123 in humans as a dual inhibitor of FPTase and GGPTase-I, but indicate that the intended target of the drug, Ki-Ras, was not inhibited.

A prostate-specific antigen (PSA)-activated vinblastine prodrug selectively kills PSA-secreting cells in vivo.

Currently, there is no therapy for men with androgen-refractory prostate cancer that substantially extends survival. This report characterizes by in vitro and in vivo techniques a new chemotherapeutic that is composed of desacetyl-vinblastine covalently linked to a peptide that contains a peptide bond that can be hydrolyzed by prostate-specific antigen (PSA). This compound (referred to as vinblastine-conjugate) is minimally toxic to cells in culture which do not express PSA. In the presence of PSA, the peptide moiety is hydrolyzed, generating several highly toxic metabolites that contain vinblastine. Animals bearing PSA-positive human prostate tumors that were treated with the vinblastine-conjugate experienced a >99% reduction in PSA serum level. In contrast, animals bearing PSA-positive human prostate tumors treated with the cytotoxic metabolites derived from the PSA hydrolysis of the vinblastine-conjugate showed a nonsignificant change in both PSA and tumor weight values. The cell killing activity of the vinblastine-conjugate is PSA dependent because animals bearing non-PSA-producing human tumor xenografts had a nonsignificant increase in tumor weight after vinblastine-conjugate treatment. Exploratory efficacy/toxicity studies in LNCaP tumor-bearing nude mice were conducted with animals treated for 5 consecutive days with various doses of either the vinblastine-conjugate or a PSA-generated toxic metabolite (desacetyl-vinblastine). The desacetyl-vinblastine treatment resulted in 10-70% mortality with a very slight effect on tumor growth. In contrast, vinblastine-conjugate treatments resulted in no mortality, good to excellent antitumor efficacy, very slight to slight peripheral neuropathy and myelopathy, and slight to severe testicular degeneration. Similar treatment of beagle dogs with the vinblastine-conjugate showed even less toxicity. These data support the use of the PSA-hydrolyzable vinblastine-conjugate as an experimental therapy for prostate cancer in man.

Use of three infusion pumps for postoperative administration of buprenorphine or morphine in dogs.

Results of using an implantable osmotic pump, a preset disposable infusion pump, or a reusable programmable infusion pump for postoperative administration of buprenorphine or morphine in dogs undergoing abdominal surgery are described. Ten dogs underwent abdominal surgery for implantation of vascular access ports. Dogs were given buprenorphine s.c. by use of an implantable osmotic pump (4 dogs), morphine s.c. by use of a preset infusion pump (4), or buprenorphine intra-arterially by use of a programmable infusion pump (2). Dogs were monitored, and serum buprenorphine or morphine concentration was measured for 72 hours after surgery; pumps were removed 48 hours after surgery. Severity of pain was determined by assigning a pain score. The preset infusion pump and the programmable infusion pump resulted in comparable pain relief and sustained serum analgesic concentrations throughout the recovery period. However, the cost of the pumps and other associated factors may limit their use to dogs undergoing invasive surgical procedures expected to result in substantial postoperative pain. The level of analgesia obtained with the implantable osmotic pumps was inconsistent.

Novel Helicobacter species isolated from rhesus monkeys with chronic idiopathic colitis.

Chronic, idiopathic diffuse colitis is a well recognised clinical and pathological entity in captive rhesus monkeys. Six rhesus monkeys were diagnosed with clinically debilitating, chronic diarrhoea. Histologically, colonic tissues were characterised as chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia and multifocal micro-abscesses. Colonic tissues were cultured for Salmonella spp. and Shigella spp.; all results were negative. Samples were negative for Clostridium difficile A and B toxins, and special stains of colonic tissue for acid-fast bacteria were also negative. The six diarrhoeic monkeys tested gave negative results for serum IgG antibodies to herpes B virus, STLV, SRV and SIV. Colonic tissue from the six diarrhoeic and two clinically normal monkeys with histologically confirmed colitis from the same colony were also subjected to micro-aerobic culture. Micro-aerobic cultures from all eight monkeys incubated at 37 degrees C and 42 degrees C revealed pinpoint or spreading colonies on antibiotic-containing media. Bacteria were identified as gram-negative, oxidase positive and urease negative. Of the nine strains characterised biochemically, two separate biotypes (corresponding to different species by 16S rRNA analysis) were identified. One biotype (type 1), from non-diarrhoeic monkeys and the second biotype (type 2) from diarrhoeic animals with subclinical chronic colonic inflammation, differed by catalase activity, ability to reduce nitrate to nitrite and sensitivity to cephalothin. Complete 16S rRNA analysis of five of the nine strains characterised biochemically indicated that the organisms isolated were two novel Helicobacter spp. By electron microscopy, these novel helicobacters had spiral morphology with bipolar sheathed flagella. This is the first report describing the isolation of novel Helicobacter spp. from inflamed colons of rhesus monkeys. Studies are needed to determine whether these novel Helicobacter spp. play a causal role in the initiation and progression of chronic colitis in macaques. Further microbiological and histological analysis of this chronic idiopathic colitis syndrome in macaques may prove useful in understanding the aetiology and pathogenesis of inflammatory bowel disease in man.

Influence of Blood Collection Sites on Plasma Glucose and Insulin Concentration in Conscious C57BL/6 Mice.

Blood collection from conscious, unanesthetized mice is often performed during the drug development process. The site of collection may influence the parameter(s) of interest. To investigate the potential influence of collection site on plasma glucose and insulin, a study was conducted to compare plasma glucose and insulin concentrations in blood samples collected without anesthesia from the retroorbital sinus versus the tail vein in 10- to 12-week-old female C57BL/6 mice. Two experiments were performed. In the first experiment, mice were randomized to be bled from the tail vein then the retroorbital sinus or vice-versa in a balanced two-period crossover design. In this experiment, the retroorbital and tail vein bleeds were performed a few minutes apart. The second experiment was similar to the first, except the bleeds were performed 1 week apart. Overall, retroorbital collection yielded lower glucose levels (p, 0.001) and higher insulin levels (p, 0.001) than did tail vein collection. The minimum difference in measured glucose in a retroorbital collection versus a tail vein collection, after adjusting for the effect of sequential bleeding was -97 mg/dl; the maximum was 98 mg/dl. We estimate that about 98% of observations taken under similar conditions would fall in this interval. The minimum difference in measured insulin in a retroorbital collection versus a tail vein collection was -0.6 ng/ml; the maximum was 7.3 ng/ml. We estimate that about 98% of observations taken under similar conditions would fall in this interval.