Hemochromatosis in Two Female Olive Baboons (Papio anubis).

This report describes hemochromatosis associated with chronic parenteral iron dextran administration in 2 female olive baboons (Papio anubis). These baboons were enrolled on an experimental protocol that induced and maintained anemia by periodic phlebotomy for use in studying potential treatments for sickle cell anemia. The 2 baboons both presented with clinical signs consistent with iron overload, including decreased appetite, weight loss, elevated liver enzymes, and hepatosplenomegaly. Histopathologic findings supported a morphologic diagnosis of systemic hemosiderosis, as evidenced by the overwhelming presence of iron in the reticuloendothelial system and liver after the application of Prussian blue stain. This finding, combined with the clinical presentation, lead to a final diagnosis of hemochromatosis. This case report suggests that providing anemic patients with chronic parenteral iron supplementation in the absence of iron deficiency can result in iatrogenic iron overload and subsequent systemic toxicity. Furthermore, these subjects may present with hemochromatosis and its associated clinical signs many years after cessation of iron supplementation.

Metal Protein-Attenuating Compound for PET Neuroimaging: Synthesis and Preclinical Evaluation of [11C]PBT2.

Dyshomeostasis or abnormal accumulation of metal ions such as copper, zinc, and iron have been linked to the pathogenesis of multiple neurodegenerative disorders including Alzheimer's disease (AD) and Huntington's disease (HD). 5,7-Dichloro-2-((dimethylamino)methyl)quinolin-8-ol, PBT2, is a second generation metal protein-attenuating compound that has recently advanced in Phase II clinical trials for the treatment of AD and HD based on promising preclinical efficacy data. Herein, we report the first radiosynthesis and preclinical positron emission tomography (PET) neuroimaging evaluation of [11C]PBT2 in rodents and nonhuman primates. Carbon-11 labeled PBT2 was synthesized in 4.8 ± 0.5% (nondecay corrected) radiochemical yield (RCY) at end-of-synthesis, based upon [11C]CH3I (n = 6), with >99% radiochemical purity and 80-90 GBq/µmol molar activity (Am) from the corresponding normethyl precursor. In the nonhuman primate brain, [11C]PBT2 uptake was extensive with peak concentration SUVpeak of 3.2-5.2 within 2.5-4.5 min postinjection in all cortical and subcortical gray matter regions (putamen > caudate > cortex ≫ white matter) followed by rapid washout from normal brain tissues. Furthermore, it is shown that [11C]PBT2 binds specifically in AD human brain tissue in vitro. The results presented here, combined with the clinical data available for PBT2, warrant the evaluation of [11C]PBT2 as an exploratory PET radiotracer in humans.

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28.

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.

Preclinical drug evaluation system in the Plasmodium knowlesi baboon model of malaria: the methotrexate study.

BACKGROUND: Drug resistance against first-line antimalarials warrants search for new lead compounds and repurposing of drugs such as methotrexate. Animal models are required for preclinical drug development before clinical testing. This study aimed to develop a preclinical drug development system in baboons infected with Plasmodium knowlesi. METHODS: Protocols for drug administration, pharmacokinetics, clinical chemistry and haematology were developed in the baboon model. Baboons were infected with P. knowlesi and methotrexate administered orally for 5 days. Clinical signs, parasitaemia, gross and histopathology examinations were conducted to determine effect of methotrexate in baboons. RESULTS: No major clinical chemistry, haematology and pathological changes attributable to methotrexate were observed. Parasitaemia suppression of 77.67% was achieved at a methotrexate dose of 3.0 mg/kg. CONCLUSIONS: A protocol for preclinical drug development in the baboon was optimized. Methotrexate suppressed P. knowlesi malaria in baboons. These findings warrant further characterization of methotrexate for use in combination therapy.

Short-term effects of high-dose khat on sperm parameters and reproductive hormonal levels in olive baboons (Papio anubis).

The biological effects of khat (Catha edulis) on reproduction and fertility are inadequately investigated and controversial, hence we determined the effects of oral administration of high-dose khat on sperm parameters and male hormonal levels in olive baboons. In this study, 6 male baboons received a high dose of khat (500 g/week) during 1 month. Electroejaculation for sperm studies (concentration, motility and chromatin integrity) and plasma collection for hormonal analysis (testosterone, prolactin and cortisol) were done weekly during 1 month before and 1 month during khat administration as well as 2 weeks after the last dose of khat administration. Administration of khat extract induced a significant reduction in sperm motility (p = 0.008), sperm count (p = 0.041), sperm chromatin integrity (p = 0.0003), testosterone levels (p = 0.035) and prolactin levels (p = 0.0115), but not in cortisol levels and sperm volume (p > 0.05). The results suggest that high-dose khat decreases sperm quality and testosterone and hence may contribute to male infertility.

LRP5 sequence and polymorphisms in the baboon.

BACKGROUND: LRP5 is known to have an important relationship with bone density and a variety of other biological processes. Mapping to human chromosome 11q13.2, LRP5 shows considerable evolutionary conservation. Orthologs of this gene exist in many species, although comparison of human LRP5 with other non-human primates has not been performed until now. METHODS: We reported the complementary DNA (cDNA) sequence and deduced amino acid sequence for baboon LRP5, and compared the baboon and human sequences. cDNA sequences for 21 baboons were examined to identify single-nucleotide polymorphisms (SNPs). RESULTS: Sequences of coding regions in human and baboon LRP5 showed 97- 99% homology. Twenty-five SNPs were identified in the coding region of baboon LRP5. CONCLUSION: The observed degree of coding sequence homology in LRP5 led us to expect that the baboon may serve as a useful model for future research into the role(s) of this gene in primate metabolic diseases.

An alternate pathway to long-chain polyunsaturates: the FADS2 gene product Delta8-desaturates 20:2n-6 and 20:3n-3.

The mammalian Delta6-desaturase coded by fatty acid desaturase 2 (FADS2; HSA11q12-q13.1) catalyzes the first and rate-limiting step for the biosynthesis of long-chain polyunsaturated fatty acids. FADS2 is known to act on at least five substrates, and we hypothesized that the FADS2 gene product would have Delta8-desaturase activity. Saccharomyces cerevisiae transformed with a FADS2 construct from baboon neonate liver cDNA gained the function to desaturate 11,14-eicosadienoic acid (20:2n-6) and 11,14,17-eicosatrienoic acid (20:3n-3) to yield 20:3n-6 and 20:4n-3, respectively. Competition experiments indicate that Delta8-desaturation favors activity toward 20:3n-3 over 20:2n-6 by 3-fold. Similar experiments show that Delta6-desaturase activity is favored over Delta8-desaturase activity by 7-fold and 23-fold for n-6 (18:2n-6 vs 20:2n-6) and n-3 (18:3n-3 vs 20:3n-3), respectively. In mammals, 20:3n-6 is the immediate precursor of prostaglandin E1 and thromboxane B1. 20:3n-6 and 20:4n-3 are also immediate precursors of long-chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid, respectively. These findings provide unequivocal molecular evidence for a novel alternative biosynthetic route to long-chain polyunsaturated fatty acids in mammals from substrates previously considered to be dead-end products.

The hormonal effects of Tribulus terrestris and its role in the management of male erectile dysfunction--an evaluation using primates, rabbit and rat.

Hormonal effects of Tribulus terrestris (TT) were evaluated in primates, rabbit and rat to identify its usefulness in the management of erectile dysfunction (ED). TT extract was administered intravenously, as a bolus dose of 7.5, 15 and 30 mg/kg, in primates for acute study. Rabbits and normal rats were treated with 2.5, 5 and 10mg/kg of TT extract orally for 8 weeks, for chronic study. In addition, castrated rats were treated either with testosterone cypionate (10mg/kg, subcutaneously; biweekly for 8 weeks) or TT orally (5mg/kg daily for 8 weeks). Blood samples were analyzed for testosterone (T), dihydrotestosterone (DHT) and dehydroepiandrosterone sulphate (DHEAS) levels using radioimmunoassay. In primates, the increases in T (52%), DHT (31%) and DHEAS (29%) at 7.5mg/kg were statistically significant. In rabbits, both T and DHT were increased compared to control, however, only the increases in DHT (by 30% and 32% at 5 and 10mg/kg) were statistically significant. In castrated rats, increases in T levels by 51% and 25% were observed with T and TT extract respectively that were statistically significant. TT increases some of the sex hormones, possibly due to the presence of protodioscin in the extract. TT may be useful in mild to moderate cases of ED.

Pre-clinical evaluation of an sLe x-glycosylated complement inhibitory protein in a non-human primate model of reperfused stroke.

BACKGROUND: Soluble complement receptor-1 (sCR1), a potent complement inhibitor, confers neuroprotection in a murine stroke model. Additional neuroprotective benefit is achieved by sLe x-glycosylation of sCR1. In an effort to translate sCR1-sLe x to clinical trials, we evaluated this agent in a primate stroke model. METHODS: Adult male baboons randomly received either sCR1-sLe x or vehicle. Stroke volume was assessed on day 3, and neurological examinations were conducted daily. Complement activity (CH50) was measured at 30 minute, 2, 6, 12 hour, 3, and 10 days post-ischemia. RESULTS: The experiment was terminated prematurely following an interim analysis. In a preliminary cohort (n = 3 per arm), infarct volume was greater in the treated animals. No difference in neurological score was found between groups. CH50 levels were significantly reduced in the sCR1sLe x-treated groups. A hypotensive response was also observed in animals treated with sCR1-sLe x. Conclusions Further work is necessary to explain the hypotensive response observed in primates prior to further clinical development of sCR1-sLe x.

Extrapolating from animal studies to the efficacy in humans of a pretreatment combination against organophosphate poisoning.

The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60-100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6-1.8 LD(50)). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70-100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.

[11C]LBT-999: a suitable radioligand for investigation of extra-striatal dopamine transporter with PET.

A new tropane derivative, (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999), was evaluated in baboons as a carbon-11 radioligand for studies of the dopamine transporter (DAT) using positron emission tomography (PET). Brain uptake was high in the striatum (17 and 13% ID/100 mL tissue in the putamen and the caudate, respectively), moderate in the midbrain and thalamus (5 and 3% ID/100 mL tissue, respectively), and low in the cortex and cerebellum (2% ID/100 mL tissue) at 30 min post injection. The striatum-to-cerebellum ratio was high (30 at 110 min post injection). Specific binding was completely blocked following pretreatment with the DAT antagonists GBR12909 (5 mg/kg i.v.) or PE2I (1 mg/kg i.v.). The [(11)C]LBT-999 uptake was decreased by these antagonists in the putamen (-79 and -92%, respectively), caudate (-80 and -91%, respectively), midbrain (-73 and -78%, respectively), and thalamus (-34 and -46%, respectively). The serotonin transporter (SERT) antagonist citalopram (5 mg/kg i.v.) or the norepinephrine transporter antagonist maprotiline (5 mg/kg i.v.) had no effect on LBT specific binding. Pharmacological challenge with PE2I (1 mg/kg i.v.) induced a rapid and almost complete decrease of the specific binding in the putamen (-97%), caudate (-96%), midbrain (-96%), and thalamus (-81%), confirming the reversibility of [(11)C]LBT-999 binding. The high brain uptake of [(11)C]LBT-999 together with its low nonspecific binding (reflected by the very high brain structure-to-cerebellum ratio) indicate that this radiotracer is an excellent candidate for in vivo quantification of the DAT, especially in extrastriatal structures, such as the midbrain.

Preclinical evaluation of the neuroprotective effect of soluble complement receptor type 1 in a nonhuman primate model of reperfused stroke.

OBJECT: Postischemic cerebral inflammatory injury has been extensively investigated in an effort to develop effective neuroprotective agents. The complement cascade has emerged as an important contributor to postischemic neuronal injury. Soluble complement receptor Type 1 (sCR1), a potent inhibitor of complement activation, has been shown to reduce infarct volume and improve functional outcome after murine stroke. Given numerous high-profile failures to translate promising antiinflammatory strategies from the laboratory to the clinic and given the known species-specificity of the complement cascade, the authors sought to evaluate the neuroprotective effect of sCR1 in a nonhuman primate model of stroke. METHODS: A total of 48 adult male baboons (Papio anubis) were randomly assigned to receive 15 mg/kg of sCR1 or vehicle. The animals were subjected to 75 minutes of middle cerebral artery occlusion/reperfusion. Perioperative blood samples were analyzed for total complement activity by using a CH50 assay. Infarct volume and neurological scores were assessed at the time the animals were killed, and immunohistochemistry was used to determine cerebral drug penetration and C1q deposition. An interim futility analysis led to termination of the trial after study of 12 animals. Total serum complement activity was significantly depressed in the sCR1-treated animals compared with the controls. Immunostaining also demonstrated sCR1 deposition in the ischemic hemispheres of treated animals. Despite these findings, there were no significant differences in infarct volume or neurological score between the sCR1--and vehicle-treated cohorts. CONCLUSIONS: A preischemic bolus infusion of sCR1, the most effective means of administration in mice, was not neuroprotective in a primate model. This study illustrates the utility of a translational primate model of stroke in the assessment of promising antiischemic agents prior to implementation of large-scale clinical trials.