key: cord-0927145-l9wcgpez
authors: Orsi, Antonia; Rees, Daryl; Andreini, Isabella; Venturella, Silvana; Cinelli, Serena; Oberto, Germano
title: Overview of the marmoset as a model in nonclinical development of pharmaceutical products
date: 2010-12-13
journal: Regul Toxicol Pharmacol
DOI: 10.1016/j.yrtph.2010.12.003
sha: 25fe2b79784b021edf5282bb99f82d3208cc3d46
doc_id: 927145
cord_uid: l9wcgpez

Callithrix jacchus (common marmoset) is one of the more primitive non-human primate species and is used widely in fundamental biology, pharmacology and toxicology studies. Marmosets breed well in captivity with good reproductive efficiencies and their sexual maturity is reached within 18 months of age allowing for rapid expansion of colonies and early availability of sexually mature animals permitting an earlier assessment of product candidates in the adult. Their relatively small size allows a reduction in material requirements leading to a reduction in development time and cost. Fewer animals are also required due to their ability to be used in both pharmacology and toxicology (nonclinical) studies. These factors, alongside a better understanding of their optimal nutrient and welfare requirements over recent years, facilitate the generation of a more cohesive and robust dataset. With the growth of biotechnology-derived pharmaceuticals, non-human primate use has, by necessity, also increased; nevertheless, there is also a growing public call for minimizing their use. Utilizing, the more primitive marmoset species may provide the optimal compromise and once the scientific rationale has been carefully considered and their use justified, there are several advantages to using the marmoset as a model in nonclinical development of pharmaceutical products.

Before evaluation in human volunteer or patient clinical studies, all investigational medicinal products (IMPs, e.g. pharmaceuticals including biotechnology derived products, gene therapies and vaccines) require evaluation in nonclinical toxicology and safety studies, generally in a rodent and a non-rodent ('second') species; (ICH-Topic-M3(R2), 2009). These nonclinical toxicology and safety studies allow a risk assessment to be performed to act as a safeguard for human subjects. The use of a non-rodent species in addition to a rodent species aims at limiting the uncertainty in the extrapolation process from animal toxicity and safety data to the human situation. Dogs, and in specific circumstances minipigs, are the more frequently used non-rodent species (ICH-Topic-M3(R2), 2009). However, when it is scientifically justified that these are inappropriate, regulatory authorities worldwide accept the use of non-human primates. Non-human primates are also used when they represent a well-established model of a compound class or when they are the relevant species (especially for biotechnology-derived products) for detecting known side effects or upon specific recommendations from regulatory authorities. ''New World monkeys'' such as Callithrix jacchus (the common marmoset) and the larger ''Old World monkeys'' such as Macaca Fascicularis (cynomolgus monkeys) and Macaca Mulatta (rhesus monkeys) are typically used (Weatherall, 2006; SCHER, 2009) . This paper provides a general overview of the marmoset as model in nonclinical development of pharmaceutical products taking into account the recent regulatory changes in husbandry, new micro sampling technologies, the requirements for reducing primate use alongside the contrasting need for relevant species selection in biotechnology derived product development.

Regulatory authorities worldwide recognize the use of marmosets in nonclinical development and as they are one of the more primitive, non-human primate species and the most phylogenetically distant from humans, their use requires less ethical justification than the larger ''Old World monkeys'' (Home-Office, 2000) .

The marmoset is a relatively small animal, approximately the size of an adult rat (up to 500 g; Table 1 ). Their small size has several advantages during the early phases of IMP development, as 0273-2300 Ó 2010 Elsevier Inc. doi:10.1016/j.yrtph.2010.12.003 material production is usually sub-optimal and therefore sufficient quantities can be rate limiting for studies. Since the amount of material required is roughly proportional to the size of the animals there are significant savings, both in cost and time, in the use of smaller species such as marmosets. The reduction in material requirement has been estimated to be 10-15-fold when comparing a 500 g marmoset to a 5-10 kg macaque (Weatherall, 2006; Zuhlke and Weinbauer, 2003; Smith et al., 2001) . Shorter synthesis time offers the potential for earlier administration to human subjects which in turn may lead to substantial cost savings (estimates range from 12 to 18 months' reduction in development time). The reduction in the amount of material required for early development studies may also have a corresponding benefit in allowing more time for large scale synthesis optimization, with further opportunities for benefit in time and costs later in development (Smith et al., 2001) . The marmoset also requires smaller cages than those for larger non-human primates and many methodologies and technologies used for the rat can also be used for the marmoset such as metabolic cages.

In the past, in certain circumstances, their small size was considered a potential hurdle as larger volumes of blood were needed for pharmacokinetic and toxicokinetic investigations, resulting in an increased number of animals required with a consequent increase in material requirement and cost. Nowadays, the need for large blood volumes has diminished with the availability of more sensitive analytical techniques such as ''dried spot detection'', newer clinical pathology techniques and enzyme linked immuno assay (ELISA) kits (Pastori et al., 2010; Dunbar, 2006; Hu and Wang, 2007; http://www.sfbr.org/SNPRC/detail.aspx?r=60, 2010) .

The marmoset breeds well in captivity and nowadays nearly all marmosets used are captive bred and have been for sometime (F 4/ 5 generation), providing a more reproducible dataset (SCHER, 2009) . The marmoset has a high reproductive efficiency, typically producing twins twice a year with sexual maturity reached within 18 months of age (compared to approximately 4 years for ''Old World monkeys''). This aspect is particularly important, as the issue of toxicity studies conducted in non-rodent species where the animals are not sexually mature has been of considerable concern. The high reproductive efficiency also allows for the rapid establishment of an in-house breeding colony, with easier availability of animals and more reliable results due to reduced stress from transportation and colony alterations. A secondary benefit is a reduction in overall cost. Furthermore, the life cycle of the marmoset is physiologically faster (7-20 years) than that of the ''Old World monkeys'' resulting in greater availability of aged animals, an important aspect for studies involving aging and bone disease.

Marmosets are born as naturally occurring chimeras, so although the animals are genetically distinct, they share and are tolerant to each other cells (Niblack et al., 1977; Ross et al., 2007) . This natural chimerism enables the study of cell transfer without initiating an alloresponse (Mansfield et al., 2004) .

The marmoset is an arboreal, diurnal species originating from the South American forests and as such a suitable environment and diet is required to reflect this natural habitat.

The natural behavior of marmosets indicates that the captive environment should have a degree of complexity and stimulation (OJ-L-197-(vol-50), 2007) and due to their arboreal nature the volume of available space and the vertical height of the enclosure appear to be more important than floor area (Badihi et al., 2007) .

The European guidelines for their housing recommend the following:

The marmosets should be housed in groups and during studies in pairs. Cages should be constructed with a height of at least 1.5 m (with the top of the cage at least 1.8 m from the floor) and a minimum floor area of 0.55 m 2 for a breeding pair and 2.0 m 2 for family groups (OJ-L-197-(vol-50), 2007) . Rich and complex environments, including opportunities for climbing, foraging (e.g. via puzzle feeders high up in cages), gnawing, swinging, and social play, as well as rest places should be provided. The cages should be equipped with wooden bars and hiding provisions and should take into account that marmosets are used to an arboreal habitat. It is important to provide a certain degree of variability in orientation, diameter and firmness to allow the animals to perform appropriate locomotor and jumping behaviors (Weatherall, 2006; OJ-L-197-(vol-50) , 2007) ( Fig. 1 and Fig. 2 ). Marmosets frequently scent-mark their environment and the total removal of familiar scent may cause behavioral problems. Alternate cleaning and sanitation of the enclosure and the enrichment devices retains some of the territorial scent-marking. Regular handling and human contact are beneficial for improving the animals' habituation to monitoring and experimental conditions and facilitate training to co-operate with some procedures. Since the marmosets originate from tropical rain forests, they should be kept at a relatively high temperature of 23-28°C with a relative humidity of between 45 and 70%. A light and dark cycle of 12 h is recommended. The lighting source should illuminate uniformly the holding room. However, within the animal enclosures, a shaded area should always be provided. Special consideration should be given to minimize exposure to ultrasound and any sudden unexpected noise, within the hearing range of marmosets (OJ-L-197-(vol-50), 2007) .

The United States National Research Council's ''Guide for the Care and Use of Laboratory Animals'' integrates recently published data, scientific principles and expert opinion to recommend practices for the humane care and use of animals in research, testing, and teaching (National Research Council, 2010) . The ''Guide'' is an internationally accepted primary reference on animal care for the scientific community and previous editions have served as the basis for accreditation of institutions worldwide by the Associ- Minimum space based on the needs of pair or group housed marmosets; however the group composition should be critically considered.

Cages with a minimum height of at least 76.2 cm and a minimum floor area of 0.20 m 2 for a breeding pair. However this recommendation highlights that in light of the marmosets arboreal nature, taller cages should be utilized, with species-specific environmental and psychological enrichments (National Research Council, 2010).

The marmosets' climate tolerance, monogamy and preference for living in small family groups make it comparatively straightfor-ward to provide housing and husbandry that meets the animals' needs and promotes their well-being. This is a significant advantage over the larger non-human primates where it is considerably more difficult to provide laboratory housing, due not only to their larger physical size, but also their greater natural troop, home range sizes, and social characteristics, including complex dominance relationships and aggressive tendencies (Baskerville, 1999) .

This, coupled with the physical and microbiological health hazards that the larger non-human primates pose to humans (as well as climate requirements of cynomolgus monkeys) suggests that husbandry of marmosets, is one of the major advantages in providing an acceptable quality of life for them as laboratory animals. From an ethical perspective there are several benefits in using marmosets, as their characteristics facilitate the provision of a more complex and enriched environments, being less destructive than larger non-human primates. Their greater wellbeing from this environment should also translate into more consistent and reliable results and over the last few decades extensive databases have also been generated within companies, toxicology service providers and breeding establishments providing essential background data to support this (Abbott, 2003; Smith et al., 2001) .

Marmosets eat fresh fruit, bread loaf, eggs, nuts high protein intake. A commercial supplement is also available (e.g. ''New World monkey'' prepared diet by Harlan Teklad). Biscuits and condensed milk are also typically used as remuneration food (RTC S.p.A.; internal procedures). The marmoset has a high requirement for protein and since they are unable to synthesize vitamin D3 without access to UV-B radiation, the diet must also be supplemented with adequate levels of vitamin D3 (OJ-L-197-(vol-50), 2007) . Continual improvements in diet as well as husbandry and environmental enrichments are helping to dramatically reduce the incidence of the marmoset 'wasting syndrome' (Tucker, 1984) resulting in the generation of more consistent and reproducible datasets.

All clinical examinations in vivo as laid out in the regulatory guidelines are feasible, established and reproducible in the marmoset. Once the marmoset is gently restrained, it is relatively easy for experienced staff to take blood samples from the animals or to give injections. However, the marmoset is too small to allow a large number of samples to be taken from the same individual for pharmacokinetic/toxicokinetic or clinical pathology studies. Blood samples are usually taken from the femoral vein, although the tail vein can be used for very small toxicokinetic samples. Typically only 15% of the circulating blood volume can be removed within one month, requiring more sensitive analytical methods to be developed for a small sample size, similar to that for rodents (Smith et al., 2001; Zuhlke and Weinbauer, 2003) , consequently the marmosets' small size does not pose a problem if analytical methodologies are already in place for rodents. Furthermore, the volume of blood required for pharmacokinetic/toxicokinetic analysis is now significantly reduced with the development of ''dried spot blood detection'' (in some circumstances down to 30 ll) (Pastori et al., 2010) .

The small size of marmosets allows for the collection of urine using metabolism cages similar to those used for rodents, eliminating additional difficulties associated with the use of larger non-human primates.

Examination of the eye may be performed with or without sedation and abnormalities are few with only the anticipated routine finding of hyaloid artery remnants in young animals. Screening compounds for ototoxicity can be performed in sedated marmosets using the Brainstem Auditory Evoked Response to monitor hearing loss (e.g. loop diuretics and aminoglycoside antibiotics; (Kuzel et al., 1990) . Screening potential effects on the cardiovascular system is an important component of both safety pharmacology and repeat dose toxicology studies (ICH-Topic-S7A, 2000; ICH-Topic-S7B, 2005; ICH-Topic-M3(R2), 2009). These can be determined in restrained or sedated marmosets, as in other species, but more informative data can be obtained by remote telemetry (Schnell and Wood, 1995) which can also monitor motor activity and body temperature.

The marmoset is a well-established laboratory species and many reviews of pathological changes have been reported as long ago as the early 1980s with database collection and marmoset pathology experience continuing within a number of establishments. The marmoset has its own range of background pathology and, as with any species, experience is needed in the interpretation of any lesions found during the course of a toxicology study (Broadmeadow, 2005; Zuhlke and Weinbauer, 2003; Smith et al., 2001) .

The proposed dosing route in humans determines the dosing route in the test species and oral, intravenous, intramuscular, subcutaneous, intranasal, intradermal and intraperitoneal are all feasible in the marmoset. Oral gavage is the most common method of dosing by way of a rubber catheter. Intravenous dosing is commonly performed using the saphenous vein (although some laboratories have successfully employed the cephalic vein). With experienced technicians, daily intravenous dosing can be performed relatively easily as well as continuous intravenous infusion (Ruiz de Elvira and Abbott, 1986 ) and repeated intravenous dosing, the latter using subcutaneously implanted vascular access ports (Smith et al., 2001; Zuhlke and Weinbauer, 2003) . Intramuscular injections are usually given in the thigh muscle while subcutane-ous injections are made into the upper back or abdomen in combination with lightweight backpack systems (Smith et al., 2001; Zuhlke and Weinbauer, 2003) . Dietary administration of non-palatable products and inhalation are the only dosing methods that present challenges. For inhalation studies it is possible to provide masks for the marmoset in a similar fashion to those used for macaques; however, their small size makes this more difficult. A one month repeated inhalation study has been described using whole body exposure where the animals were exposed for 6 h per day to a vapor (Kurata et al., 1997) .

The marmoset has been used successfully by a number of pharmaceutical and biotechnology companies to support clinical trials and for product registration with the United States (US) Food and Drug Administration (FDA) and the European Medicines Agency (EMA) (Zuhlke and Weinbauer, 2003; Smith et al., 2001) .

The European Union distinguishes six nonclinical categories where experiments using marmosets may be conducted (Commision of the European Communities, 2007) as follows:

(1) Biological studies of a fundamental nature (2) Research, development and quality control of products and devices for human medicine, dentistry and for veterinary medicine (3) Toxicological and other safety evaluations (4) Diagnosis of disease (5) Education and training (6) Other

The distribution of testing according to these categories is outlined in Table 2 ; (Commision of the European Communities, 2007).

The ability of the marmoset to be utilized in fundamental biology, pharmacology (pharmacodynamics and pharmacokinetics) and toxicology studies allows for a strong interconnected relationship between these disciplines, leading to a reduced number of animals used with a more complete data set at a lower cost (e.g. there is no need to repeat dose ranging studies, there are less pharmacokinetic/toxicokinetic animals required, less material is required and development time is reduced).

Marmosets are used widely in the fields of neural and cognitive sciences, immunology, infectious disease, reproductive biology and stem cell research due to their close genetic, physiological and metabolic similarity to humans (Mansfield et al., 2004) . These activities are centered on understanding fundamental elements of human biology as well as the pathogenesis of disease syndromes of humans and providing relevant translational research for potential new medicines. Recently, several international activities have been implemented to further support the use of the marmoset in nonclinical development of pharmaceuticals including biotechnology derived products, gene therapies and vaccines. These include the following: the significant progress in the marmoset genome sequencing (Mansfield et al., 2004; http://ucscbrowse, 2010) , the successful creation of a marmoset specific DNA microarray , the public availability of a large collection (3215) of express sequence tags (ESTs;) of marmoset origin [GenBank: EF214838 -EF215447, EH380242 -EH382846] (http://www.biomedcentral.com/content/supplementary/1471- 2164-8-190-S2.xls, 2007) and the creation of transgenic marmosets (Cyranoski, 2009; Sasaki et al., 2009 ).

Marmosets are key pharmacodynamic models in the fields of neuroscience, immunology, and infectious disease and also serve as useful models in other disease areas (Table 3) .

Marmosets are widely used models for a number of neurological disorders including multiple sclerosis, Parkinson's disease and Huntington's disease (Mansfield et al., 2004) as well as for studies on normal neurophysiology (Brysch et al., 1990; Hornung et al., 1990; Guldin et al., 1993; Peretta, 2009) .

Multiple sclerosis is a chronic progressive autoimmune disease of the brain and spinal cord (Rumrill, 2009 ). The experimental autoimmune encephalomyelitis (EAE) model in marmosets represents a chronic relapsing multiple sclerosis (Genain and Hauser, 1997; t Hart et al., 2000; Merkler et al., 2006) whereas the EAE model, in rodents and in rhesus monkeys, is more reminiscent of acute disseminated encephalomyelitis. Several nonclinical studies have been successfully conducted in the EAE marmoset model using in vivo magnetic resonance imaging (MRI), visual evoke potentials and neuropathology as measures of efficacy (Boretius et al., 2006; Diem et al., 2008; t Hart and Massacesi, 2009 ). New potential therapeutic antibodies against human cluster of differentiation (CD) 40 or the shared interleukin-12p40 (IL-12p40) subunit of interleukins 12 and 23 have been successfully tested in the EAE marmoset model (Boon et al., 2001; t Hart et al., 2005) .

Parkinson's disease (PD) is a neurodegenerative disease characterized by insufficient production of dopamine from the substantia nigra area of the brain which leads to significant impairment of movement (Dawson and Dawson, 2002) . The marmoset is a recognized model of Parkinson's disease (Owen et al., 1997) using selective neurotoxins to destroy the dopamine neurons. The toxins most commonly used are 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA). More recently, transgenic models of the disorder have been developed in marmosets leading to over-expression of proteins such as a-synuclein using viral vectors (Kirik et al., 2003; Eslamboli et al., 2007) . The experimental use of marmosets is an important intermediary step between rodent studies and controlled clinical trials when the phylogenetic similarity to humans is pivotal for assessing and optimizing the efficacy of candidate therapies. However, the marmoset is not the most suitable non-human primate species to use when specific behavioral tests are required (e.g. cognitive testing, tremor rating) (Eslamboli, 2005) .

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder with most animal models falling into two broad categories, non-genetic (e.g. quinolinic acid; QA) and transgenic. Historically, the former have dominated the field of HD research (Ramaswamy et al., 2007) . The unilateral QA putamen lesion in the marmoset provides a useful and recognized tool for the investigation of potential treatments (Kendall et al., 1998) , however this model does not replicate the pathology or the symptoms of HD but rather develops a consistently reproducible lesion with motor impairments (Kendall et al., 1998 (Kendall et al., , 2000a . Recently the marmoset huntingtin (Htt) gene has been isolated and identified, resulting in the potential to prepare a transgenic HD marmoset model, that could provide further information on the underlying pathology and the ability to test therapeutic candidates and gene therapies (Hohjoh et al., 2009; Ramaswamy et al., 2007) .

For the development of IMPs it is of critical importance that potential effects on the immune system (either intended pharmacodynamic or resultant toxicological effects) are investigated in appropriate models. Non-human primates are typically considered superior to other models to investigate immunological, pharmacodynamic and potential immune toxic effects on IMPs (Weatherall, 2006) .

The marmoset is a key species for several reasons.

The marmoset represents a unique species for the study of T and B cell-mediated diseases, because they are born as naturally occurring bone marrow chimeras (Niblack et al., 1977) . A large variety of surface receptors on lymphocytes and other blood cells have been found to cross-react with monoclonal antibodies against the corresponding human epitopes, indicating a high degree of similarity between these species (Barton et al., 1984; Neubert et al., 1996; Riecke et al., 2000; Kireta et al., 2005; Kametani et al., 2009) .

Several studies have been performed resulting in the generation of a data bank from more than 400 animals, the largest data bank available for surface receptors (Neubert et al., 1993) . However, in both marmosets and humans, some qualitative and quantitative differences exist with respect to surface markers on lymphocytes relevant for studies on immunotoxicity: the marmoset lacks natural killer cells with typical markers found in humans (CD2 -CD56 + ), conversely cytotoxic killer effector CD4 + cells (CD4 + CD56 + ) found in marmosets are not found in humans (Neubert et al., 1993) .

Marmosets have been used extensively in infectious disease research due, in large part, to their unique sensitivity to a number of viruses, bacteria and parasites responsible for human infectious disease (Mansfield et al., 2004) . Furthermore, in an era where bioterrorism has become a reality, there is an increased demand for non-human primate models and regulatory authorities have established new guidelines for testing vaccines and therapeutics for pathogens that governments have determined are potential biological weapons.

The marmoset is also used to investigate the absorption, distribution, metabolism and excretion of new IMPs. In addition, techniques that are not practical in larger non-human primates such as whole-body autoradiography can be performed in the marmoset (Broadmeadow, 2005) . Furthermore, the marmoset represents a key model for IMPs that are substrates of cytochrome P450 (CYP450) such as CYP1A2, CYP3A4 and for investigating glucuronidation in vitro.

CYP1A2 is constitutively expressed at a high level in the human liver and plays an important role in the mutagenic activation of heterocyclic amines (Butler et al., 1989; Shimada et al., 1994) . CYP1A2 is also constitutively expressed in the liver of marmosets (Edwards et al., 1994; Bullock et al., 1995) but not in cynomolgus monkeys. Furthermore, the marmoset and human CYP1A2 respond to similar inducers, and appear to be under the same or similar control mechanisms (Sakuma et al., 1997) .

CYP3A4 constitutes the largest fraction of human hepatic CYP450 and is involved in the metabolism of 45-60% of medicines currently in use (Koehler et al., 2006) . This broad substrate specificity represents the basis for many clinically relevant ''medicine-medicine'' interactions and the marmoset is a key model for studying these metabolic interactions as there is a strong similarity between marmoset and human genes, proteins and their functions (McArthur et al., 2003; Nelson et al., 2004; Williams et al., 2004; Koehler et al., 2006) (Table 4) .

Uridine diphosphate glucuronosyltransferases (UGT) are a large family of membrane bound enzymes responsible for the detoxification of a wide range of xenobiotics and endogenous compounds. The large number of products glucuronidated, whether directly or following the Phase I step, necessitates a thorough understanding of glucuronidation for efficient pharmaceutical development (Dutton, 1980) . Typically, rodents are used as the primary animal model and the dog commonly used as the second animal model for pharmacokinetic studies. However a wide range of products are more extensively glucuronidated by dog liver microsomes when compared with human hepatic microsomes (Soars et al., 2001) . Furthermore, qualitative differences in glucurondation are observed between dogs and humans with extrahepatic glucuronidation being markedly reduced in dogs (Soars et al., 2001) . By contrast, there are quantitative and qualitative similarities between human and marmoset glucuronidation in vitro and as such the marmoset is a useful model especially when the rate and pathway of glucuronidation of a medicine are known to differ significantly between rodents, dogs and humans (Soars et al., 2001) . The significant extrahepatic (e.g. kidney) glucuronidation observed in marmosets in comparison with other animals merits its use for preclinical pharmacokinetic and safety evaluation studies. Nevertheless, further work should also be performed to confirm that observations in different species in vitro accurately represent the situation in vivo (Soars et al., 2001) .

Toxicological and other safety evaluations, represent the largest use of marmosets in nonclinical development with typical use being for acute, sub-acute, chronic and toxicokinetic studies (Commision of the European Communities, 2007). Interestingly, long term studies of up to 6.5-7 years duration have also been reported (Smith et al., 2001) (Table 5) .

The marmoset can be used for pharmacology as well as for toxicology studies. In addition, there are well-established classes of compounds where the marmoset may well offer an alternative species to the dog that shows hypersensitivity or idiosyncrasy (Smith et al., 2001; Gaudy et al., 1987) (Table 6) .

Marmosets represent a useful model to investigate the teratologic potential of pharmaceuticals and to investigate aspects of reproductive physiology and immune-contraception due to their early sexual maturity, high reproduction efficiency and similarities in placentation to humans (Mansfield et al., 2004) (Table 7) .

By contrast, there are several aspects of the reproductive process in marmosets that exhibit discrepancies to those described for humans (Cline, 2007; Luetjens et al., 2005; Zuhlke and Weinbauer, 2003) . The differences for females include multi-ovulation, postpartum conception, the lack of external signs of menstruation and for males include alterations or even absence of genes known to be essential for reproductive development. The duration of the menstrual cycle in marmosets varies, on average, from 16 to 28 days and unlike women, the luteal phase is comparatively long at the expense of the follicular phase with 1 and 4 eggs ovulated. A mid-phase follicle stimulating hormone (FSH) peak and the presence of multiple dominant follicles in marmosets represents another difference to humans (Zuhlke and Weinbauer, 2003) .

For juvenile toxicology studies, useful background data has been generated in the marmoset over the years examining differences in clinical pathology endpoints and organ weights between young and adult marmosets (Wadsworth et al., 1981; Davy et al., 1984) . Table 4 Summary of marmoset CYP3A characteristics.

CYP3A characteristics CYP3A21 is the dominant hepatic CYP3A protein in marmosets The sequence similarity between human CYP3A4 and CYP3A21 in marmoset across the first 7.5 kb of the cloned CYP3A21 promoter is 88%. The marmoset CYP3A21 gene is clustered with the human CYP3A genes, while rodent CYP3A genes form several separate clusters (McArthur et al., 2003; Nelson et al., 2004; Williams et al., 2004) . The marmoset CYP3A21 cDNA exhibits high similarity to human CYP3A genes (Williams et al., 2004) . The enzymatic activities of CYP3A21 are similar to those of CYP3A4 (Koehler et al., 2006) .

Once the scientific rationale has been carefully considered and its use as the non-rodent species justified, there are considerable advantages in using the marmoset as a model in pharmaceutical product development. These range from reduced material requirement due to their small size (up to15 fold less), a predicted acceleration of development (up to 18 months faster), an earlier assessment of product candidates in the adult due to their early sexual maturation and reduced numbers of animals required due to their ability to be used in both pharmacology and toxicology studies. These factors, alongside a better understanding of their optimal nutrient and welfare requirements over recent years, facilitate the generation of a more cohesive and robust dataset. With the growth of biotechnology-derived products and gene therapies, the use of non-human primates has, by necessity, also increased since these are often the only relevant species that provide an adequate safety assessment. Nevertheless, there is a growing public call for minimizing their use now and in the future. Utilizing, the more primitive marmoset species may provide the optimal compromise. Table 6 Compound classes where marmosets may be more suitable as a model than dogs.

Class of compounds where marmosets may be a more suitable model than dogs Vomiting with morphine compounds Atrio-ventricular block with calcium antagonists Reflex tachycardia with anti-hypertensives Allergic reactions with vehicles such as cremophor Table 7 Examples of marmoset use in teratology and reproductive studies.

Higher non-human primates, such as the cynomolgus and rhesus monkeys, have generation times of about 5 years, making multigenerational studies impractical.

However, multigenerational studies are feasible in the marmoset, which has a comparatively short generation time (14-18 months) (Marshall et al., 2003b) To investigate the mechanism of thalidomide teratogenesis: metabolites of thalidomide may cause the down-regulation of surface adhesion receptors thereby altering cell to cell and cell to extracellular matrix interactions within the developing limb bud (Heger et al., 1988 (Heger et al., , 1994 Klug et al., 1994; Neubert et al., 1996) To examine the effect of luteinizing hormone and follicular stimulating hormone on granulosa cell development and steroidogenesis (Barrett, 2007; Millar et al., 2000; Hillier et al., 1997) To define the mechanisms of estradiol inactivation, in vitro fertilization, embryo transfer and parthenogenesis (Husen et al., 2001; Summers et al., 1987; Marshall et al., 1997 Marshall et al., , 1998 Marshall et al., , 2003a Luetjens and Wesselmann, 2008) To determine whether infant feeding with soya formula milk, which contains high levels of plant estrogens, poses any immediate or longer-term health risk to the developing testis and reproductive system of the male. An initial study found that testosterone levels were suppressed in animals fed with soya formula milk. A longer term follow-up analysis of these animals indicated that infant feeding with soya formula milk had no gross reproductive effects in male marmosets, but that it does alter testis size and cell composition. The authors concluded that similar changes are likely to occur in adult men fed soya formula milk as infants (Sharpe et al., 2002; Tan et al., 2006) Matsumoto et al. (1987) Lipid lowering agent Fibrates (e.g. ciprofibrate and clofibrate)

Oral: several studies up to 7 years

Fibrates are hypolipidaemic agents which, in chronic studies in rats or mice, have produced tumors or pre-neoplastic changes not predictable for humans. Clofibrate and ciprofibrate toxicity studies up to 7 years duration conducted by two laboratories have shown that the marmoset is predictive for humans and in the marmoset studies, unlike rodents, there is no induction of liver tumors Eason et al. (1988) , Spencer et al. (1989) , Smith et al. (2001) , Reddy et al. (1983) , Makowska et al. (1992) , Graham et al. (1994) Non steroidal antiinflammatory Indomethacin Oral: 4 weeks Non-steroidal anti-inflammatory to reduce fever, pain stiffness and swelling. It inhibits prostaglandin release Oberto et al. (1990) Parkinson's disease Ropinirole Oral-safety studies Ropinirole at microgram/kg doses produced significant decreases in the number of postures and significant increases in the time spent at the cage front in the ''human threat'' test. In rodents the effects were seen at mg/kg Eden et al. (1991) Psychotic disorders including schizophrenia Clozapine Oral, intramuscularsafety studies

Clozapine over a wide dose range, unlike other antipsychotics, increased the motivational state of the marmoset suggesting a unique clinical profile Cilia et al. (2001) Varies Thalidomide The marmoset has been used to comprehensively investigate the mechanism of thalidomide teratogenesis Heger et al. (1994) ; Neubert et al. (1996) 

Aspects of common marmoset basic biology and life history important for biomedical research

The effects of increased space, complexity, and choice, together with their loss, on the behavior of a family group of Callithrix jacchus: a case study

The testosterone test: phthalate inhibits leydig cell aggregation

Binding of human lymphocytespecific monoclonal antibodies to common marmoset lymphoid cells

UFAW Handbook on the Care and Management of Laboratory Animals

Prevention of experimental autoimmune encephalomyelitis in the common marmoset (Callithrix jacchus) using a chimeric antagonist monoclonal antibody against human CD40 is associated with altered B cell responses

Monitoring of EAE onset and progression in the common marmoset monkey by sequential high-resolution 3D MRI

The common marmoset (Callithrix jacchus) in preclinical research and development of new pharmaceutical products

The topology of the thalamo-cortical projections in the marmoset monkey (Callithrix jacchus)

Induction of liver microsomal cytochrome P450 in cynomolgus monkeys

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4 0 -methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases

Clozapine enhances breakpoint in common marmosets responding on a progressive ratio schedule

Assessing the mammary gland of nonhuman primates: effects of endogenous hormones and exogenous hormonal agents and growth factors

Fifth Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member States of the European Union

Development of the first marmoset-specific DNA microarray (EUMAMA): a new genetic tool for large-scale expression profiling in a non-human primate

A molecular blueprint of gene expression in hippocampal subregions CA1, CA3, and DG is conserved in the brain of the common marmoset

Reference intervals for some clinical chemical parameters in the marmoset (Callithrix jacchus): effect of age and sex

Neuroprotective and neurorestorative strategies for Parkinson's disease

Autoimmune optic neuritis in the common marmoset monkey: comparison of visual evoked potentials with MRI and histopathology

Applications of Luminex xMAP technology for rapid, highthroughput multiplexed nucleic acid detection

Glucuronidation of Drugs and Other Compounds

Species variation in gastric toxicity following chronic administration of ciprofibrate to rat, mouse, and marmoset

Preclinical pharmacology of ropinirole (SK&F 101468-A) a novel dopamine D2 agonist

Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human

Marmoset monkey models of Parkinson's disease: which model, when and why?

Long-term consequences of human alpha-synuclein overexpression in the primate ventral midbrain

The effects of cremophor EL in the anaesthetized dog

Creation of a model for multiple sclerosis in Callithrix jacchus marmosets

Lack of peroxisome proliferation in marmoset liver following treatment with ciprofibrate for 3 years

Connections from the neocortex to the vestibular brain stem nuclei in the common marmoset

Embryotoxic effects of thalidomide derivatives on the non-human primate Callithrix jacchus; 3. Teratogenic potency of the EM 12 enantiomers

Embryotoxic effects of thalidomide derivatives in the non-human primate Callithrix jacchus. IV. Teratogenicity of micrograms/kg doses of the EM12 enantiomers

Location and developmental regulation of androgen receptor in primate ovary

Product monograph: Inhibace Ò (cilazapril)

Product monograph: Tamiflu Ò (oseltamivir phosphate capsule)

Product monograph: Invirase Ò (Saquinavir mesylate)

Molecular cloning and characterization of the common marmoset Huntington gene

Statistics of Scientific Procedures on Living Animals Great Britain

Distribution of two morphologically distinct subsets of serotoninergic axons in the cerebral cortex of the marmoset

Updated Marmoset Genome Browser Available

Overview of 3215 sequences submitted to GenBank and dbEST

A rapid, multiplexed new technology xMAP liquid chip for detection and identification of pathogens

Mechanisms of estradiol inactivation in primate endometrium

Guideline on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals

The Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals

Development of monoclonal antibodies for analyzing immune and hematopoietic systems of common marmoset

The influence of excitotoxic basal ganglia lesions on motor performance in the common marmoset

Striatal tissue transplantation in nonhuman primates

Functional integration of striatal allografts in a primate model of Huntington's disease

Cross-reactivity of anti-human chemokine receptor and anti-TNF family antibodies with common marmoset (Callithrix jacchus) leukocytes

Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease

Embryotoxic effects of thalidomide derivatives in the non-human primate Callithrix jacchus 5 Lack of teratogenic effects of phthalimidophthalmide

Marmoset CYP3A21, a model for human CYP3A4: protein expression and functional characterization of the promoter

Four-week repeated inhalation study of HCFC 225ca and HCFC 225cb in the common marmoset

Screening procedure for assessment of ototoxicity in the common marmoset

Primate spermatogenesis: new insights into comparative testicular organisation, spermatogenic efficiency and endocrine control

The fate of paternal mitochondria in marmoset pre-implantation embryos

Species differences in ciprofibrate induction of hepatic cytochrome P450 4A1 and peroxisome proliferation

White Paper for Complete sequencing of the common marmoset (Callithrix jacchus) genome

Functional and histological evidence for the protective effect of NXY-059 in a primate model of stroke when given 4 h after occlusion

Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone

Nonsurgical embryo transfer in the common marmoset monkey

Parthenogenetic activation of marmoset (Callithrix jacchus) oocytes and the development of marmoset parthenogenones in vitro and in vivo

Six-month toxicity study with mitomycin C in marmosets

Phylogenetic analysis of the cytochrome P450 3 (CYP3) gene family

Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in the common marmoset reflects the immunopathology of pattern II multiple sclerosis lesions

Marmoset spermatogenesis: organizational similarities to the human

Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council

Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants

Cross-reactivity of antihuman monoclonal antibodies with cell surface receptors in the common marmoset

Down-regulation of adhesion receptors on cells of primate embryos as a probable mechanism of the teratogenic action of thalidomide

Effects of small doses of dioxins on the immune system of marmosets and rats

T-and B-lymphocyte chimerism in the marmoset

Evaluation of the toxicity of indomethacin in a 4-week study, by oral route, in the marmoset (Callithrix jacchus)

on guidelines for the accommodation and care of animals used for experimental and other scientific purposes

Report on primate supply for biomedical scientific work in the UK. EUPREN UK working party

Comparison and validation of Dried Blood Spot (DBS) and Solid Phase Extraction (SPE) techniques

Non-human primate models in neuroscience research

Animal models of Huntington's disease

Chemical carcinogens without mutagenic activity: peroxisome proliferators as a prototype

Cross-reactivity of antibodies on thymic epithelial cells from humans and marmosets by flowcytometry

Germ-line chimerism and paternal care in marmosets (Callithrix kuhlii)

A backpack system for long-term osmotic minipump infusions into unrestrained marmoset monkeys

Multiple sclerosis: medical and psychosocial aspects, etiology, incidence, and prevalence

Marmoset CYP1A2: primary structure and constitutive expression in livers

Generation of transgenic non-human primates with germline transmission

The need for non human primates for biomedical research, production and testing of products and devices. European Commission

Measurement of blood pressure and heart rate by telemetry in conscious unrestrained marmosets

Infant feeding with soy formula milk: effects on the testis and on blood testosterone levels in marmoset monkeys during the period of neonatal testicular activity

Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians

The selection of marmoset monkeys (Callithrix jacchus) in pharmaceutical toxicology

Evaluation of the marmoset as a model species for drug glucuronidation

Gastric morphological changes including carcinoid tumors in animals treated with a potent hypolipidemic agent, ciprofibrate

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Suppression of ongoing disease in a nonhuman primate model of multiple sclerosis by a human-anti-human IL-12p40 antibody

Clinical, pathological, and immunologic aspects of the multiple sclerosis model in common marmosets (Callithrix jacchus)

A new primate model for multiple sclerosis in the common marmoset

Infant feeding with soy formula milk: effects on puberty progression, reproductive function and testicular cell numbers in marmoset monkeys in adulthood

A survey of the pathology of marmosets (Callithrix jacchus) under experiment

Organ weight data in juvenile and adult marmosets (Callithrix jacchus)

The Use of Non-Human Primates in Research

Defining relationships between the known members of the cytochrome P450 3A subfamily, including five putative chimpanzee members

The common marmoset (Callithrix jacchus) as a model in toxicology

The authors declare that there are no conflicts of interest. The manuscript was sponsored by Research Toxicology Center S.p.A., a contract research organization specialized in nonclinical safety studies for product registration.