Pharmacokinetics of Buprenorphine and Sustained-release Buprenorphine in Common Marmosets (Callithrix jacchus)

Buprenorphine is an essential component of analgesic protocols in common marmosets (Callithrix jacchus). The use of buprenorphine HCl (BUP) and sustained-release buprenorphine (BSR) formulations has become commonplace in this species, but the pharmacokinetics have not been evaluated. Healthy adult (age, 2.4 to 6.8 y; 6 female and 6 male) common marmosets were enrolled in this study to determine the pharmacokinetic parameters, plasma concentration–time curves, and any apparent adverse effects of these compounds. Equal numbers of each sex were randomly assigned to receive BUP (0.02 mg/kg IM) or BSR (0.2 mg/kg SC), resulting in peak plasma concentrations (mean ± 1 SD) of 15.2 ± 8.1 and 2.8 ± 1.2 ng/mL, terminal phase t_1/2 of 2.2 ± 1.0 and 32.6 ± 9.6 h, and AUC_0-last of 16.1 ± 3.7 and 98.6 ± 42.7 ng×h/mL. The plasma concentrations of buprenorphine exceeded the proposed minimal therapeutic threshold (0.1 ng/mL) at 5 and 15 min after BUP and BSR administration, showing that both compounds are rapid-acting, and remained above that threshold through the final time points of 8 and 72 h. Extrapolation of the terminal elimination phase of the mean concentration–time curves was used to develop the clinical dosing frequencies of 6 to 8 h for BUP and 3.0 to 3.5 d for BSR. Some adverse effects were observed after the administration of BUP to common marmosets in this study, thus mandating judicious use in clinical practice. BSR provided a safe, long-acting option for analgesia and therefore can be used to refine analgesic protocols in this species.

Renewed interest in common marmosets (Callithrix jacchus) as models for biomedical research is being driven by the fields of gene-editing technology, neuroscience, and infectious disease.^{7,19,23,28,32,34} Research study aims as well as clinical interventions necessitate appropriate pain management protocols for these animals. Current recommendations for analgesia in common marmosets are extrapolated from other species or are based on anecdotal evidence. Dosage, duration of action, and potential adverse effects of analgesics used in this species require evaluation to refine guidelines for their use in clinical practice.Buprenorphine is the most commonly used opioid analgesic in many NHP species, including common marmosets.^6,24,33 It is a partial µ-opioid receptor agonist used for the treatment of moderate to severe pain, when included as part of a multimodal pain management approach. Buprenorphine''s widespread use in laboratory animal medicine is attributed to a relatively long duration of action and favorable safety profile when compared with other available opioid agents. A considerable amount of data on the efficacy and recommended dosage of buprenorphine is available for various laboratory species, including mice, rats, rabbits, cats, dogs, and pigs.^{2,10,14,17,20,35,41}Formulations of sustained-release buprenorphine (BSR) have been developed and have become commercially available, providing a longer acting option than the standard buprenorphine HCl (BUP) formulation. Longer-acting compounds are preferable, because they have the potential to improve animal welfare by reducing handling and the number of injections per animal, reducing adverse effects associated with peak plasma buprenorphine concentrations, and avoiding repeated trough plasma buprenorphine concentrations, which may result in inadequate analgesia. The uses of both formulations of buprenorphine in several common laboratory animal species, including mice, rats, guinea pigs, dogs, cats, and macaques, have been described.^{5,9,10,16,30,39}The pharmacokinetics of BUP and BSR have already been described in both cynomolgus and rhesus macaques, the most commonly used NHP in biomedical research. The widely accepted dosage range for BUP in macaques is 0.01 to 0.03 mg/kg administered either intramuscularly or intravenously. Dosing recommendations provided in one study, using a plasma threshold of 0.1 ng/mL, suggest that for macaques, BUP at 0.01 mg/kg IM should be given every 6 to 8 h and at 0.03 mg/kg IM should be given every 12 h.³⁰ The same study showed that a single subcutaneous dose of BSR at 0.2 mg/kg can be given every 5 d. A similar study demonstrated that BUP given to rhesus macaques at 0.03 mg/kg either intravenously or intramuscularly maintains plasma concentrations above 0.1 ng/mL for 24 and 12 h, respectively.¹⁸BUP dosing recommendations for New World NHP, such as common marmosets, tend to be lower than those of Old World species, such as macaques. These recommendations are a result of the more profound adverse effects (for example, respiratory depression, apnea, and death) seen in common marmosets when higher doses of BUP are used or when BUP is used in combination with anesthetic agents, such as alfaxalone or isoflurane.^1,4 Guidelines for BUP dosing in common marmosets range from 0.005 to 0.02 mg/kg IM.^4,21,24The purpose of the current study was to investigate the plasma concentration of buprenorphine over time after the administration of BUP and BSR in common marmosets. Observations were made to determine whether adverse effects occurred after the administration of BUP and BSR. We hypothesized that BUP dosed at 0.02 mg/kg IM would remain above a plasma threshold of 0.1 ng/mL for 6 to 8 h and that BSR dosed at 0.2 mg/kg SC would remain above the same threshold for 72 h.     

Factors Affecting Hematologic and Serum Biochemical Parameters in Healthy Common Marmosets (Callithrix jacchus)

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.     

Pharmacokinetics of Single-Dose Intramuscular and Subcutaneous Injections of Buprenorphine in Common Marmosets (Callithrix jacchus)

Although buprenorphine is the most frequently used opioid analgesic in common marmosets (Callithrix jacchus), there is limited information in the literature supporting current dosing regimens used for this species. The purpose of this study was to determine the pharmacokinetic profiles of single-dose buprenorphine HCl administered intramuscularly (IM) at 0.01 mg/kg in 6 adult marmosets (1.8 to 12.8 y old; 2 males, 4 females) and subcutaneously (SQ) at 0.01 mg/kg in 6 adult marmosets (2.3-4.4 y old; 3 males, 3 females) by mass spectrometry. Blood was collected at multiple time points from 0.25 to 24 h from unsedated animals following a hybrid sparse-serial sampling design. The maximal observed plasma concentration of buprenorphine (C_max) administered IM (2.57 ± 0.95 ng/mL) was significantly higher than administered SQ (1.47 ± 0.61 ng/mL). However, the time to C_max (T_max) was not statistically different between routes (17.4 ± 6 min for IM and 19.8 ± 7.8 min for SQ). The time of the last quantifiable concentration of buprenorphine was 5 ± 1.67 h for IM compared with 6.33 ± 1.51 h for SQ, which was not statistically different. The mean buprenorphine plasma concentration-time curves were used to propose a dosing frequency of 4 to 6 h for buprenorphine at 0.01 mg/kg IM or SQ based on a theoretical therapeutic plasma concentration threshold of 0.1 ng/mL. Based on the mean pharmacokinetic parameters and plasma-concentration time curves_, both IM and SQ routes of buprenorphine at this dose provide a rapid increase in the plasma concentration of buprenorphine above the therapeutic threshold, and may be more effective for acute rather than long-lasting analgesia. Further studies are needed to examine repeated dosing regimens and the efficacy of buprenorphine in common marmosets.

The common marmoset (Callithrix jacchus), a New World (platyrrhine) monkey belonging to the Callitrichidae family, is an important experimental model used in diverse areas of study, including neuroscience, behavior, drug metabolism and toxicology, infectious diseases, and in the development of genetically modified NHP models for biomedical research.^11,28,32,33 Marmosets often undergo minor and major survival surgical procedures as part of experimental protocols, including implantations, laparotomies, and obstetric procedures. However, biomedical research and veterinary procedures performed on marmosets have the potential to cause pain and distress, requiring effective analgesic regimens.⁵ The provision of appropriate analgesia to counteract painful stimuli is a humane necessity fundamental to multiple animal research regulatory policies.^3,14,24Buprenorphine, a Schedule III opioid analgesic, is a partial μ-agonist, δ- and κ-antagonist, and nociceptin receptor agonist.^2,8,48 This drug is the most commonly used opioid analgesic in NHPs, including laboratory marmosets.^{7,18,31,33,35} Dosages recommended for marmosets range from 0.005 to 0.02 mg/kg, either alone for mild to moderate pain or as part of a multimodal analgesic plan for moderate-to-severe pain.^6,18,30,33 These dosage recommendations have been primarily based on anecdotal information and limited evidence related to use and efficacy present in the literature. However, a recent study by Fitz and colleagues evaluated the pharmacokinetics of single-dose intramuscular (IM) buprenorphine at 0.02 mg/kg and subcutaneous (SQ) sustained-release buprenorphine (Bup SR) at 0.2 mg/kg in marmosets. Based on a therapeutic plasma concentration threshold of 0.1 ng/mL, the authors recommended dosing frequencies of 6 to 8 h for buprenorphine and 3.0 to 3.5 d for Bup SR.¹⁸New World monkey species are more sensitive to opioid-induced adverse side effects such as profound respiratory depression at doses considered safe in other NHPs, rodents, and other laboratory animal species.^{6,10,18,27,35,46} For example, in one study, buprenorphine, administered to C. jacchus at 0.02 mg/kg IM as a premedication before alfaxalone induction, resulted in apnea in 8 out of 9 study animals.⁶ In unsedated marmosets, administration of buprenorphine at 0.02 mg/kg IM has resulted in mild to moderate ataxia and moderate sedation.¹⁸At the Massachusetts Institute of Technology (MIT, Cambridge, MA), buprenorphine HCl is administered to marmosets at 0.005–0.01 mg/kg IM and SQ, and rarely elicits adverse effects. However, analgesic efficacy at these doses is unknown and redosing schedules have been empirically based. In addition, absorption of SQ buprenorphine in some species such as cats can be highly variable and unreliable.⁴³ Although IM and SQ routes are commonly used in marmosets, how the route of administration affects buprenorphine absorption and plasma clearance is unknown. These issues underscore the importance of experimentally determining PK data and developing optimal dosing strategies for the use of this drug in marmosets.We performed a study using healthy adult marmosets to evaluate and compare the IM and SQ pharmacokinetics of single-dose buprenorphine HCl, administered at a clinically relevant dosage of 0.01 mg/kg. Liquid chromatography–electrospray ionization–tandem mass spectrometry was used to demonstrate that buprenorphine reached quantifiable plasma concentrations after administration, and to determine the interval for which buprenorphine was quantifiable in the plasma. We hypothesized that a single-dose of buprenorphine administered SQ would have slower absorption (longer T_max) and longer duration (longer T_last), when compared with IM administration.     

Twinning and Survivorship of Captive Common Marmosets (Callithrix jacchus) and Cotton-Top Tamarins (Saguinus oedipus)

Here we present the results of a demographic analysis of 25 y (1985 to 2010) of common marmoset (Callithrix jacchus) and cotton-top tamarin (Saguinus oedipus) records from the New England Primate Research Center. Summaries of longevity and survivorship are analyzed by birth-type category (including singletons, twins, triplets, and quadruplets) and sex. In addition, a brief evolutionary review is presented. Surrogates of hematopoietic chimerism, twinning, and reproductive output are explored in a large number of animals to help decipher the potential effects of chimerism on life history in marmosets and tamarins. In addition to exploring chimerism through demographic data, multiple-birth frequency and survivorship are compared between species. New World primates can make ideal translational models for disease and behavioral research across multiple disciplines. A better understanding of their reproductive success and survivorship in captivity helps develop these nonhuman primate models, their role in aging research, and understanding of their behavioral ecology. This mission is likely to only increase in its importance to biomedical research due to both the sequencing of the marmoset genome and the growing demand for alternatives to Old World primate models.Here we review demographic and life history data collected on common marmoset (Callithrix jacchus) and cotton-top tamarin (Saguinus oedipus) colonies housed at the New England Primate Research Center between 1985 and 2010. Information was gathered from a large database of 2753 C. jacchus and 2212 S. oedipus whose births were recorded over the 25 y. The cotton-top tamarin colony was established coincident with the founding of the center in the mid1960s. Initially used as a model system for biomedical research, the cotton-top tamarin was placed on the endangered species list in 1977, and invasive research was ended. The marmoset colony at our institution was established in late 1976 as an alternative model system, and attention in the tamarin colony turned to developing a better understanding of cotton-top tamarin health and reproduction. Several studies reflecting on this time discussed the factors influencing infant mortality and the veterinary practices to best minimize rejection.^11,12 This current work extends these studies to include animals born during the subsequent years and the entirety of the life histories of both marmosets and tamarins.     

Using Snacks High in Fat and Protein to Improve Glucoregulatory Function in Adolescent Male Marmosets (Callithrix jacchus)

The common marmoset (Callithrix jacchus), a laboratory nonhuman primate, is a well-known model of several human diseases and conditions, but the nutritional needs of these animals are not fully understood. Here we describe a 4-mo controlled study in which we increased the dietary fat and protein of subadult male common marmosets by using healthy snacks. Six male marmosets received their normal diet (control), and an additional 6 were given their normal diet supplemented daily with a 14-kcal snack. Cashews and waxworms were used as the snack, given their high-fat content. Although body weight did not differ between the 2 groups, only control male marmosets showed increased chest circumferences over the course of the study. Glucoregulatory function remained consistent in the snack-fed marmosets, whereas control animals had progressed toward higher insulin. Other indices of glucoregulation indicated significant differences in adiponectin and the cortisol:cortisone ratio between the 2 groups, but no differences in lipid concentration were detected. Therefore, the most notable difference attributable to the snack feeding was improved glucoregulation. Because the snacks we used had a high proportion of unsaturated compared with saturated fat, we suggest that these healthy high-fat–high-protein snacks provide an important contribution to the nutrition of this laboratory species. This study also demonstrates the utility of marmosets as a model for understanding the implications of dietary fats in humans.Abbreviation: HMW, high molecular weightThe common marmoset (Callithrix jacchus) has proven to be an excellent model for biomedical and behavioral research in both the United States and Europe for many years (see reference 15 for review). Recently, a particular interest has been in the use of marmosets for modeling human obesity and metabolic syndrome.²⁷ There are many advantages to using marmosets in understanding metabolic diseases. As a primate species, marmosets are similar to humans in fat cell function.³³ The marmoset pancreas exhibits the same structure, marker genes, and the presence of the Glut 5 and 9 transporters as does human pancreas.²² In addition, lipogenesis occurs in the adipose tissues of marmosets, as in all primates, rather than in the liver, as in rodents.² Marmosets are cooperative breeders, showing the family arrangement most often seen in humans. Because marmosets have a short lifespan of approximately 16 y and achieve reproductive maturity by 2 y of age, many studies can be performed in a relatively short time. Furthermore, marmosets generally produce twins, thus affording the opportunity to select subjects for different treatments within the same study. Indicating similar dietary needs to those of humans, the natural diet of marmosets is omnivorous and consists of fruits, plant exudates, nectar, invertebrates, and small vertebrates.²⁶ In captivity, marmosets show diet-dependent early-onset weight gain and obesity.²⁷ Dietary changes can have a pronounced influence on their weight and health.^4,30,33There is a lack of consensus regarding appropriate diets for laboratory-housed marmosets. Little is known about the optimal energy and nutrient requirements of marmosets.¹⁸ Commercially available diets for marmosets differ in nutrients and makeup. There is limited knowledge of the fat requirements or the effects of different fat composition on the glucoregulatory system. Only one study has examined the effect of a high-fat diet on regulatory functioning. In that study,³³ 8 adult marmosets fed a high-fat diet (that is, 20% added saturated fats) for 1 y showed no adverse effects on the levels of glycosylated hemoglobin as an index of blood glucose use but manifested atherosclerosis. Whether the same effects occur with a combination of unsaturated and saturated fats is unknown.Many teenagers receiving a Western diet consume high amounts of sugars and fats in the form of snacks. Teen obesity has grown to epidemic proportions in the United States, with as many as 17% of 12- to 19-y-olds considered obese.³ Fat consumption and adiposity are significantly associated,^4,8,21 but the relationship between BMI and high fat consumption is unclear.¹ Fats are important for brain and body functions, including the regulation of circulating lipid levels, neuronal development, cardiovascular health and immune, insulin, and visual functions.^7,12,24,28 The discrepancy in dietary fat''s influence on body function may be due to the type of fats consumed. The current study aims to provide evidence that increased fats, providing that they comprise appropriately balanced fatty acids (saturated and unsaturated), may be beneficial in adolescents. We monitored the metabolic effects of providing high-fat–high-protein snacks to male marmosets for 3 mo. We hypothesized that this dietary scheme would not lead to detrimental changes.     

Advantages and Risks of Husbandry and Housing Changes to Improve Animal Wellbeing in a Breeding Colony of Common Marmosets (Callithrix jacchus)

Between 1975 and 2014, housing conditions for laboratory-housed marmosets changed dramatically after the introduction of new guidelines designed to improve their care and wellbeing. According to these guidelines, our facility provided marmosets with outside enclosures, switched to deep litter as bedding material, and discontinued the use of disinfectant agents in animal enclosures. However, both deep litter and access to outside enclosures hypothetically increase the risk of potential exposure to pathogenic microorganisms. We evaluated whether these housing and husbandry modifications constituted an increased veterinary risk for laboratory-housed common marmosets (Callithrix jacchus). After the animals had been exposed to these new housing conditions for 2.5 y, we examined their intestinal bacterial flora and feces, the deep litter, and insects present in the housing. In addition, we assessed the marmosets’ general health and the effect of outdoor housing on, for example, vitamin D levels. Although numerous bacterial strains—from nonpathogenic to potentially pathogenic—were cultured, we noted no increase in illness, mortality, or breeding problems related to this environmental microflora. Housing laboratory marmosets in large enriched cages, with both indoor and outdoor enclosures, providing them with deep litter, and eliminating the use of disinfectants present an increased veterinary risk. However, after evaluating all of the collected data, we estimate that the veterinary risk of the new housing conditions is minimal to none in terms of clinical disease, disease outbreaks, abnormal behavior, and negative effects on reproduction.The Biomedical Primate Research Centre (Rijswijk, The Netherlands) houses a self-sustaining breeding colony of common marmosets (Callithrix jacchus) for the purpose of conducting biomedical research on life-threatening human diseases. The marmoset colony was formed in 1975 and has been used mainly for research on autoimmune diseases, neurodegenerative disorders, and comparative genetics.^{3,5,6,14,21,29}After the introduction of new European and Dutch guidelines regarding animal care and welfare, animal housing conditions changed markedly between 1975 and 2014. Our facility responded promptly to these new guidelines, by providing larger and more complex cages comprising outdoor enclosures, each with an attached heated indoor enclosure, where the animals are housed in family groups to improve animal wellbeing.^22,35 The concept of environmental enrichment continued to be developed and optimized over the years.Potential benefits of outdoor enclosures are exposure to seasonal fluctuations in light and climate and increased sensory stimulation. These enclosures provide opportunities for exploration and manipulation that are considered to contribute positively to the animals’ wellbeing. Furthermore, marmosets housed indoors with no access to UV light are susceptible to metabolic bone diseases. Marmosets cannot synthesize vitamin D3 (cholecalciferol) from the plant form of the vitamin (ergocalciferol, vitamin D2). Without access to UVB radiation, they cannot form vitamin D3 from 7-dehydroxycholesterol in the skin.³¹ In addition to dietary supplementation with vitamin D3, we surmised that access to unfiltered sunlight in outside enclosures would limit or prevent vitamin D deficiency.Another change initiated in response to BPRC''s new housing guidelines was the cleaning of the housing facilities. Scent marking is an important aspect of the natural behavior of marmosets. In laboratory settings, marmosets scent-mark their cages constantly.¹¹ To minimize the removal of scents, disinfectants are no longer used to clean the enclosures. In addition to effects on scent, limiting disinfectant use could have other beneficial effects. For example, the chemical disinfection of their environment was suggested to be one cause of chromosomal disorders in marmosets.¹³A third important housing-related change was the provision of deep litter in the outdoor and indoor enclosures. Deep litter is a floor covering, preferably of organic origin, that promotes activities including locomotion, foraging, and playing. In general, the changes associated with providing deep litter typically involved a shift in the animals’ behavioral profiles toward those that might be observed in their wild counterparts; therefore, the provision of deep litter is seen as environmental enrichment.^{7,9,10,24,30,32}Although some of these changes have been implemented in zoos, primate centers that breed marmosets for research purposes have been more reticent because of potential health issues. To evaluate whether the new housing conditions enhance the animals’ wellbeing, we studied their benefits and potential threats to the animals, the practical consequences for personnel and management, and the influence on experimental results. In particular, the health risks for the marmosets due to increased microbiologic exposure because of the new housing conditions were examined. The aim of the study was to determine whether these changes in their housing constituted not only an improvement in their wellbeing but also a possible increased veterinary risk for laboratory-housed common marmosets.     

Bioavailability and Efficacy of Levofloxacin against Francisella tularensis in the Common Marmoset (Callithrix jacchus)

Pharmacokinetic and efficacy studies with levofloxacin were performed in the common marmoset (Callithrix jacchus) model of inhalational tularemia. Plasma levofloxacin pharmacokinetics were determined in six animals in separate single-dose and multidose studies. Plasma drug concentrations were analyzed using liquid chromatography-tandem mass spectrometry-electrospray ionization. On day 7 of a twice-daily dosing regimen of 40 mg/kg, the levofloxacin half-life, maximum concentration, and area under the curve in marmoset plasma were 2.3 h, 20.9 μg/ml, and 81.4 μg/liter/h, respectively. An efficacy study was undertaken using eight treated and two untreated control animals. Marmosets were challenged with a mean of 1.5 × 10² CFU of Francisella tularensis by the airborne route. Treated animals were administered 16.5 mg/kg levofloxacin by mouth twice daily, based on the pharmacokinetic parameters, beginning 24 h after challenge. Control animals had a raised core body temperature by 57 h postchallenge and died from infection by day 5. All of the other animals survived, remained afebrile, and lacked overt clinical signs. No bacteria were recovered from the organs of these animals at postmortem after culling at day 24 postchallenge. In conclusion, postexposure prophylaxis with orally administered levofloxacin was efficacious against acute inhalational tularemia in the common marmoset. The marmoset appears to be an appropriate animal model for the evaluation of postexposure therapies.Francisella tularensis is a Gram-negative intracellular pathogen that is the causative agent of tularemia. The disease is prevalent in many countries in the northern hemisphere, including the United States (8), where the reported number of cases averages 100 to 200 annually (11). The pathogen is infectious by a number of routes, has a low infectious dose, and is readily transmitted by the airborne route and so is included on the CDC list of select agents that have potential to pose a major threat to public health and safety (http://emergency.cdc.gov/agent/agentlist-category.asp).Currently, the recommended preferred treatment for adults with tularemia is systemic gentamicin or streptomycin or, alternatively, doxycycline or ciprofloxacin for 14 to 21 days (5; http://www.hpa.org.uk/deliberate_accidental_releases/biological). In the case of a mass bioweapon attack, prophylaxis with orally administered ciprofloxacin or doxycycline twice daily is suggested (5; http://www.hpa.org.uk/deliberate_accidental_releases/biological). Fluoroquinolones such as ciprofloxacin and levofloxacin are attractive alternative antibiotics for tularemia, as they have good in vitro activity, have bactericidal effects, and can be administered orally (13). Levofloxacin has a broad spectrum of activity, a good safety record, and an oral bioavailability of over 99% (10), while single daily dosing gives it a further advantage over the more commonly used antibiotic ciprofloxacin. Murine studies indicate that intraperitoneal administration of levofloxacin is 100% effective against intranasal tularemia challenge, even when administration is delayed up to 72 h postchallenge (17), although demonstration of efficacy following inhaled challenge in other models is lacking. Intravenous levofloxacin has been successful in treating human tularemia without relapse (1, 20). Levofloxacin has already proven utility in the biodefense arena and has been approved for the treatment of inhalational anthrax in both adults and children (6, 19), thus reducing the number of antibiotics required in the arsenal.In order to determine the efficacy of pre- or postexposure therapies, there is a need to develop and characterize animal models of inhalational tularemia. To date, most research on tularemia has focused on developing murine models of infection (4). This has increased the understanding of disease progression and has identified important immunological responses to infection. However, in order to undertake the pivotal studies required for the licensure of any therapy, nonhuman primate models of infection are required. Previous work has demonstrated that the common marmoset (Callithrix jacchus) is susceptible to experimental inhalational tularemia and develops a lethal infection representative of human disease (21).The purpose of this work was to investigate the use of orally administered levofloxacin as a postexposure therapy for tularemia using the common marmoset. Initially, this involved establishing the pharmacokinetic (PK) parameters in the marmoset and, using this data, testing the efficacy of levofloxacin against inhalational tularemia.     

Population-averaged standard template brain atlas for the common marmoset (Callithrix jacchus)

Advanced magnetic resonance (MR) neuroimaging analysis techniques based on voxel-wise statistics, such as voxel-based morphometry (VBM) and functional MRI, are widely applied to cognitive brain research in both human subjects and in non-human primates. Recent developments in imaging have enabled the evaluation of smaller animal models with sufficient spatial resolution. The common marmoset (Callithrix jacchus), a small New World primate species, has been widely used in neuroscience research, to which voxel-wise statistics could be extended with a species-specific brain template. Here, we report, for the first time, a tissue-segmented, population-averaged standard template of the common marmoset brain. This template was created by using anatomical T(1)-weighted images from 22 adult marmosets with a high-resolution isotropic voxel size of (0.2 mm)(3) at 7-Tesla and DARTEL algorithm in SPM8. Whole brain templates are available at International Neuroinformatics Japan Node website, http://brainatlas.brain.riken.jp/marmoset/.     

Orientation selectivity in the common marmoset (Callithrix jacchus): the periodicity of orientation columns in V1 and V2

Orientation selectivity is a ubiquitous property of the primary visual cortex of mammals. Within the primate, orientation selectivity is arranged into vertical columns that are organized into a regular patchy pattern. Previous studies, in old world primates, have noted an anisotropy in this arrangement that appears to be due to the presence of ocular dominance columns within the same tissue. In addition, orientation selective responses appear to be arranged into bands of activity within the adjoining extrastriate region V2. Little is known about the precise arrangement of orientation columns within V2. In this study, we examined the layout of orientation columns within both V1 and V2 of a new world primate, the common marmoset, using optical imaging. New world primates have the advantage that, unlike the macaque, V2 exists on the cortical surface, a requirement for this form of optical mapping. We found the arrangement of orientation columns to be isotropic within marmoset V1 with an average repeat distance of around 575 mum, smaller than the repeat distance previously reported for the macaque. We found no evidence of ocular dominance within the animals tested supporting the claim that ocular dominance columns when present distort the mapping of orientation in V1. In V2 we found that orientation columns were larger and as in other primates were represented in discrete bands throughout V2. Orientation columns were spaced on average around 1 mm apart. This suggests that, at least in the marmoset, the visual system maps orientation at a different scale within V1 and V2.     

Natural antibodies to EBV-VCA antigens in common marmosets (Callithrix jacchus) and response after EBV inoculation

Healthy common marmosets from two separate colonies, one in England and one in France, were found to have antibodies cross-reacting with Epstein-Barr virus structural antigens (EBV-VCA). All seropositive animals were at least 2 years old. Experimental EBV infection of marmosets of different ages led to seroconversion of inoculated weanlings. Adult animals either developed antibody for the first time or showed an increase in their existing titers. Both control and infected animals developed a progressive interstitial nephritis. The lymphocytic infiltration was more extensive, diffuse and immature in appearance in inoculated animals, but no definite evidence of lymphoproliferative disease or lymphoma was found.     

Septicemia and peritonitis in a colony of common marmosets (Callithrix jacchus) secondary to Klebsiella pneumoniae infection.

Six common marmosets from a colony of 50 died over a period of 3 weeks, with the predominant finding of gram-negative bacterial septicemia. Four of these animals died peracutely; the other two were found when they were moribund, and they subsequently died despite clinical intervention. Gram-negative bacterial rods were present in the blood vessels of stained tissues from five of the six marmosets. Three marmosets also had severe fibrinopurulent peritonitis. In addition, one of the marmosets with peritonitis also had purulent mesenteric lymphadenitis with large colonies of gram-negative bacterial rods within dialated colonic crypts. Klebsiella pneumoniae was isolated from multiple organs in three of the marmosets. Clinical evaluation of the entire colony identified four marmosets with anorexia, nasopharyngeal discharge and diarrhea. These marmosets were treated with enrofloxacin immediately, and they responded well. K. pneumonia could not be cultured from nasal or fecal samples obtained from the colony animals. Because of the peracute nature of the disease, animals often die before exhibiting clinical symptoms, and antibiotics are seldom helpful. In this outbreak we saw both of the major forms of Klebsiella infection in common marmosets: the peracute form with bacteremia and minimal inflammatory reaction around blood vessels, and the chronic form with bacteremia, fibrinopurulent peritonitis, and mesenteric lymphadenitis.     

Bites by the King Cobra (Ophiophagus hannah) in Myanmar: Successful Treatment of Severe Neurotoxic Envenoming

Three patients bitten by the world's largest species of venomoussnake, the king cobra (Ophiophagus hannah), were observed inMyanmar (Burma). All three were involved in the famous snakedance in Yangon (Rangoon) Zoological Gardens. One patient showedno signs of envenoming despite a sustained bite, another developedonly signs of local envenoming, but in a third there was severeneurotoxic envenoming requiring mechanical ventilation for 64hours, episodes of hypotension and massive swelling of the bittenlimb. This patient showed some signs of recovery before delayedtreatment with specific antivenom. It is possible that all threepatients had some immunity to king cobra venom resulting fromtraditional ‘immunization’ achieved by seratchingvenom into the skin. The literature on king cobra bites is reviewedand recommendations given for antivenom and ancillary treatments.     

Successful Treatment of Amoxapine-induced Refractory Status Epilepticus with Propofol (Diprivan)

Tonic-clonic seizure activity is a recognized complication of amoxapine overdose. Refractory drug-induced status epilepticus is associated with significant morbidity and mortality. Standard regimens for controlling status epilepticus may be ineffective for aborting drug-induced seizures. The authors report the case of a 30-year-old woman who presented with an amoxapine overdose that deteriorated into status epilepticus refractory to conventional therapy. Propofol given by intravenous bolus and maintenance infusion successfully halted the patient's seizure activity. This case suggests that propofol may be effective as an anticonvulsant in refractory drug-induced status epilepticus.     

Successful treatment of enterovirus-infected mice by 2-(alpha- hydroxybenzyl)-benzimidazole and guanidine

Echo virus 9- or Coxsackie A 9-infected newborn mice are protected from paralysis and death by combined treatment with nontoxic concentrations of HBB plus guanidine. HBB alone also protects Coxsackie A 9, but not echo virus 9-infected animals, whereas guanidine alone is ineffective in either case. Protection is due to inhibition of virus multiplication via the antiviral activity of these selective inhibitors. Treatment must be begun at the latest 48 h after virus inoculation. 3 days of treatment are sufficient if started at the time of virus inoculation. Failure of protection after treatment with one compound alone is not due to rapid development of drug-resistant virus mutants. Infected, successfully treated mice may develop a solid immunity.     

Successful Treatment of Lumbar Discogenic Pain Using Intradiscal Biacuplasty in Previously Discectomized Disc

Discogenic back pain is frequently present in patients after discectomy. Here, we describe a case of young woman, previously discectomized, who was treated by a novel annuloplasty procedure, intradiscal biacuplasty (IDB). The improvement in functional capacity and pain scores were profound 12 months after the IDB. Visual analog pain scores (0–10) changed from 5 to 3. Oswestry scale showed functional improvement from 52%, or severe disability, to 14%, or minimal disability, and the SF-36 physical function scale score changed from 55 to 95. IDB may be an effective treatment for patients with discogenic pain from previously discectomized discs.     

Successful Treatment of Ventricular Tachycardia by Physiological Pacing

The case is presented of a young patient with atrioventricular (AV) block but no evidence of other disease; in this patient exercise or stress-related syncope continued after implantation of a ventricular inhibited (VVI) pacemaker. Investigation revealed exercise-induced limited rapid multiform ventricular tachycardia (VT) which was associated with faintness or syncope. Temporary atrial triggered ventricular inhibited ventricular (VDD) pacing resulted in enhanced exercise tolerance with no significant arrhythmia. A permanent full function dual chamber [DDD] pacemaker was implanted and prevented the VT. There have been no further exercise-related symptoms during two years 0f follow up.     

Embryonic stem cells of the non‐human primate Callithrix jacchus can be differentiated into definitive endoderm by Activin‐A but not IDE‐1/2

Pluripotent stem cells hold great promise for regenerative medicine, due to their unlimited self‐renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non‐human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed, get relatively old (≤ 13 years), are small in size, are relatively cost‐effective and have a high genetic proximity to the human. In particular, diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane‐permeable, chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However, the potent small molecules IDE‐1 and IDE‐2 were not able to induce definitive endoderm‐like cells when ESCs from the common marmoset were treated with these compounds, whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus, differences between the species of human ESCs and ESCs of this non‐human primate might be a useful model to further evaluate the exact mode of action of these compounds. Copyright © 2013 John Wiley & Sons, Ltd.     

复方连蒲颗粒治疗感冒(风热证)的随机对照临床试验

目的 评价复方连蒲颗粒治疗感冒(风热证)的疗效和安全性.方法在药品临床试验管理规范(GCP)的指导下,进行Ⅱ、Ⅲ期临床试验.Ⅱ期临床试验采用多中心随机双盲双模拟平行对照的方法,本中心选择感冒(风热证)患者48例,采用数字随机法随机分为复方连蒲颗粒组(试验组)24例,双黄连口服液组(对照组)24例;Ⅲ期临床试验采用多中心随机平行对照的方法,本中心选择感冒(风热证)患者80例,采用数字随机法随机分为试验组60例,对照组20例.未执行分配隐藏.试验组给予复方连蒲颗粒,每日3次,每次8 g;对照组给予双黄连口服液,每日3次,每次10 ml.疗程均为3 d.统计分析采用符合方案数据分析(per-protocol population,PP)和Stata 7.0统计软件.结果试验组Ⅱ、Ⅲ期临床试验的临床综合疗效显效率分别为90.00(18/20)和85.00％(51/60),总有效率分别为95.00％(19/20)和98 33％(59/60);中医症状疗效显效率分别为85.00％(17/20)和85 00％(51/60),总有效率分别为95.00％(19/20)和98.33％(59/60);体温疗效显效率分别为85 00％17/20)和86.05％(37/43),总有效率分别为95.00％(19/20)和95.35％(41/43).对照组的临床综合疗效显效率分别为85.71％(18/21)和80.00％(16/20),80.00％(16/20)总有效率分别95.24％(20/21)和95.00％(19/20);中医症状疗效显效率分别为85.71％(18/21)和80.00％(16/20),总有效率分别为95.24％(20/21)和95.00％(19/20);体温疗效显效率分别为85.71％(18/21)和80.00％(12/15),总有效率分别为90.48％(19/21)和93.33％(14/15).两组比较其差异均无统计学意义(P＞0.05).在临床试验中,未发现复方连蒲颗粒明显的不良反应.结论复方连蒲颗粒8 g tid的临床疗效与双黄连口服液10 ml tid相当,未发现明显毒副作用.