- Email: [email protected]
The minipig in toxicology
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 57 (2006) 335–339
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
3RD EUROPEAN CONGRESS OF TOXICOLOGIC PATHOLOGY, 2005, COPENHAGEN, DENMARK
The minipig in toxicology Ove Svendsen Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Ridebanevej 9, DK-1870 Frederiksberg C, Copenhagen, Denmark Received 23 February 2006; accepted 30 March 2006
Abstract The use of pigs (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species have been found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. Because of their well-accepted physiological and other similarities to humans, minipigs are becoming increasingly attractive toxicological and pharmacological models. There are several strains of minipigs (Go¨ttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Go¨ttingen minipigs. In toxicology in Europe minipigs have become very popular for pharmaceutical studies in place of dogs and primates. Minipigs have been shown to be sensitive to a wide variety of drugs and chemicals. It has become obvious that minipigs can be used for all routes of administration, and in many cases are preferable to dogs or primates for metabolic or pharmacological reasons. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been wellestablished allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature. Now there is good supply of high-quality minipigs of known disease status. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. Consequently, there are scientific, economic and sociological reasons that make minipigs good toxicological and pharmacological models. The principal disadvantage is that toxicity testing in minipigs may require more test compound than the traditional species. r 2006 Elsevier GmbH. All rights reserved. Keywords: Minipig; Toxicology; Toxicology testing; General toxicology; Reproductive toxicology; Dosing routes; Sampling techniques; Necropsy; Toxicopathology
Introduction The pig was domesticated from the wild swine by the Chinese more than 7000 years ago. Within the last Tel.: +45 35 28 31 65; fax: +45 35 35 35 14.
E-mail address: [email protected]. 0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.03.003
centuries the pig has increasingly been used in biomedical research. Today in the EU more than 60,000 pigs are used a year for scientific procedures. The number of minipigs used for such purposes is relatively low. The minipig was introduced in Europe in the early 1970s for scientific purposes. In the mid-1980s the minipig was introduced in toxicology as an alternative non-rodent
ARTICLE IN PRESS 336
O. Svendsen / Experimental and Toxicologic Pathology 57 (2006) 335–339
species. The basic reason for this introduction was the realization of the many biochemical, anatomical and physiologically similarities with humans relative to other non-rodent species (Swindle and Smith, 1998). Extensive use since then has highlighted in details the areas where the minipig has become a particularly useful animal species for toxicological studies and toxicity testing.
pig and then with German Landrace to obtain pale skin. It is now a white non-pigmented and small-size minipig with an adult body weight of 30–40 kg if held on restricted diet. They have been purpose bred in Europe for years and now also in the US and are readily available. They are of very high microbiological and parasitic quality. There is a full genetic background available and background toxicology control data have accumulated in the scientific literature.
Domestic pigs and minipigs The use of the domestic pig (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species were found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. There are several strains of minipigs (Go¨ttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Go¨ttingen minipig. There is an increasing interest in minipigs because they share many similarities with humans anatomically and physiologically relative to other non-rodent species; in particular the skin, heart and kidney are very similar to those of humans (Montiero-Riviere and Riviere, 1996; Mortensen et al., 1998; Swindle, 1998). They are available as purpose-bred SPF high-quality animals. In addition, there is a growing amount of background data and growing regulatory acceptability. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. The minipig has been shown to be sensitive to a wide variety of drugs and chemicals. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. It has been demonstrated that minipigs can be used for all routes of administration such as orally or parenterally, and in many cases are preferable for dogs or primates for metabolic or pharmacological reasons. Consequently, there are scientific, economic and sociological reasons that make minipigs good animal models in toxicology and other scientific disciplines. The principal disadvantage is that toxicity testing in the minipig may require more test compound than the traditional species.
Go¨ttingen minipigs There are several benefits related to the Go¨ttingen strain of minipigs. This strain was developed by Go¨ttingen University in the 1960s by cross-breeding of the Minnesota minipig first with Vietnamese put belly
Pharmacokinetics and metabolism Pharmacokinetic studies are easily performed in the minipig with repeated blood sampling or sampling of other body fluids or tissues as demonstrated by Anzenbacherova´ et al. (2003) and Witkamp and Monshouwer (1998). The cytochrome P450 liver metabolic pattern has been extensively studied by Skaanild and Friis (1997, 1999, 2005) and compared to that of humans. It is beyond the scope of this paper to describe in detail the similarities and dissimilarities with that of humans.
Minipigs in repeat dose toxicology Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been well established allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature.
Routes of dose administration The most common route of dosing is the oral route either by gavage or dietary dosing with medicated diet. Dosing by oral gavage in conscious animals may be stressing for the animals. Because of anatomically and physiologically similarities with skin of humans the minipig is particularly useful for dermal studies. This route has been used for acute and repeat dose dermal toxicology studies, dermal absorption studies, phototoxicity studies and photosensitization studies. Parenteral dosing can be applied by intramuscular, subcutaneous, intradermal and intravenous injection. The latter can be applied either by bolus injection or as continuous intravenous infusion. The details of continuous intravenous infusion in the minipig have been described by Brinck (2000a) who also has described the
ARTICLE IN PRESS O. Svendsen / Experimental and Toxicologic Pathology 57 (2006) 335–339
surgical preparation for the application of continuous intravenous infusion (Brinck, 2000b). Other routes as nasal dosing or inhalation dosing (Koch et al., 2001) have also been successfully applied in minipigs.
337
rhagic syndrome and probably associated with an immunocomplex mediated condition and marked degenerative/ regenerative megakaryocytic changes (Carrasco et al., 2003).
Observations and sampling techniques The observations required in toxicity testing such as clinical signs, body weight, ophthalmoscopy and electrocardiography (Nahas et al., 2002) are routine procedures in toxicity testing with the minipig. In addition, control hematology and clinical chemistry data are available in the scientific literature (Ellegaard et al., 1995; Damm Jørgensen et al., 1998a). Parameters of immunotoxicity could be included (Hinton, 2000). Blood sampling techniques have been described in detail by Bollen et al. (2000) and Swindle (1998).
Necropsy procedure While conducting post-mortem procedures in minipigs some major differences to other non-rodent species have to be taken into consideration. This includes the gastrointestinal tract, thymus, thyroid and parathyroid. In minipigs the cecum is very large as is the colon. In addition, the colon is formed as a spiral. The majority of the thymus is located in the cervical region and a minor part in the thoracic cavity. However, microscopic examination is usually performed on the thoracic part as in other species. The thyroid is located on the ventral part of the trachea just in front of thoracic curvature. As another variation the two lobes of the thyroid are merged into one organ. The parathyroids are not embedded or attached to the thyroids, but are instead located in the cranial part of the thymus in 90% of the cases. It is not unusual that the parathyroids are placed in the very most cranial part of the thymus just behind the hyoid bone. It requires good skills to be able to find both glands routinely.
Histopathology The Go¨ttingen minipig is free from any porcine infectious bacterial or viral disease and free from any porcine parasitic disease. Histopathology findings of spontaneous nature are few and in general focal and mild (Svendsen et al., 1998; Dincer and Svendsen, 2006). The most recent and most extensive collection of control data is reported by Skydsgaard (2006 in prep.). There are four significant spontaneous findings: (1) Arteritis (scattered, focal, necrotic/fibrinoid), (2) tubular atrophy/hypoplasia in testis (focal), (3) serous atrophy (bone marrow fat cells, mainly femur/tibia) and (4) hemorrhagic syndrome. The etiology of all four features is unknown. Thrombocytopenia is extensive in the hemor-
Reproductive toxicology In testing for reproductive toxicity in general two animal species have to be used, a rodent and a non-rodent species. The two species routinely used are the rat and the rabbit. The minipig may be an alternative species in teratogenicity and reproductive studies where there is a lack of suitability of traditional species such as mice, rats or rabbits. Nonhuman primates have supply and conservation considerations and the majority have single offspring. Minipigs are purpose-bred to SPF standards, relatively inexpensive, and have a number of similarities with humans. The reproductive characteristics of the Go¨ttingen minipig have been described by Damm Jørgensen (1998a) together with a teratogenicity testing protocol and historical control data. The testing protocol can be summarized as follows. Sexual maturity of Go¨ttingen minipigs is reached before the animals are 6 months of age. In general minipig gilts are 5–10 months of age when included in studies. In teratogenicity studies the treatment period extends from start of implantation (day 11) to the closure of the hard palate (day 35) inclusive of gestation. Different routes of administration can be used. While the oral route is the most commonly used, continuous intravenous infusion can also be applied (McAnulty, 2000). Pregnancy status can be controlled by ultrasonography in week 4 or 5 of gestation. The sows are killed on day 110–112 of gestation. As the fetus weight is 350–400 g, it is possible to perform full necropsy on the fetuses. In general the protocol includes fetal examinations after removal from uterus, alizarin staining of skeleton and free-hand sectioning of Bouin fixed heads. For skeletal examination X-rays may be added because the bones of the skull and bone densities are better visualized than with alizarin staining (Damm Jørgensen, 1998a). The historical data include skeletal diagnoses from 220 fetuses and spontaneous malformation data from more than 3700 newborn or stillborn fetuses. By this protocol it has been shown that the Go¨ttingen minipig is highly susceptible to the teratogenic effects of tretinoin with malformations resembling retinoic acid teratogenicity in humans (Damm Jørgensen K, 1998b). Hematological and clinical chemical control data of pregnant Go¨ttingen minipigs have been reported by Damm Jørgensen et al. (1998a). The Go¨ttingen minipig has also proven useful as a model for studying effects on male fertility (Damm Jørgensen et al., 1998b). The reported data support the assumption that the male minipig may prove to be more susceptible to chemicals with adverse effect on fertility
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than the male rat. The minipig has closer similarities to man with respect to fertility rate, percentage morphologically abnormal sperm, percentage of sperm with progressive motility and incidence of cryptorchidism.
Juvenile studies So-called juvenile studies were introduced into nonclinical testing programs because it was realized that several drugs were applied in pediatric clinics to children without testing data justifying safe use of the drugs in this particular age group. Standard non-clinical studies using adult animals, or safety information from adult humans, cannot always adequately predict these differences in safety profiles for all pediatric age groups, especially reaction of immature systems such as the developing brain, pulmonary system, kidneys and reproductive and immune systems. This motivated the US Food and Drug Administration and the European Medicines Agency to issue draft guidelines for non-clinical testing in juvenile animals on human pharmaceuticals for pediatric indications. Traditionally, rats and dogs have been the species of choice for such testing, but other species may be more appropriate in some circumstances. The appropriateness of the minipig in this type of testing has been evaluated by scientists at Scantox in Denmark and the results presented at various scientific conferences (Harling and Makin, personal communication, 2005). The optimal protocols for the conduct of juvenile studies includes cross-fostering in order to handle all animal allocated to one mother as one study group and thus avoiding litter effect. Cross-fostering of juvenile minipigs for performance of juvenile toxicology studies is a realistic practical proposition using estrus synchronization and mating in breeding facilities with subsequent housing in the testing laboratory. Properly controlled synchronization results in delivery of all mimipigs within few days allowing for cross-fostering. Various standard techniques such as oral or parenteral dosing, ophthalmoscopy, ECG and repeated collection of blood samples for clinical pathology and toxicokinetics are practically feasible. Implantation of vascular access ports was successful from day 7 and onwards for daily intravenous dosing. Background clinical pathology data are important because many of the standard parameters change rapidly with increasing age (Damm Jørgensen et al., 1998a).
Regulatory acceptance The pig and minipig as a model in toxicity testing of pharmaceuticals and other chemicals has now been well accepted by Japan, EU and USA (Ikeda et al., 1998). The pig and minipig is specifically mentioned as a
potential non-rodent species in guidelines of Japan and Canada. The OECD 409 guideline lists pig and minipig as optional species. However, evidence should be provided that it is a suitable species and there is still some regulatory resistance or unfamiliarity related to issues with background data and spontaneous and toxicologic pathology.
Conclusions The minipig is a useful non-rodent animal species in investigations of safety issues in drug development. However, selection of the most suitable non-rodent species is a complex issue. Many pharmaceutical industries still hold the dog as the number one choice and move only from the dog where there is a justification. The minipig may be disregarded merely from the basis of body weight because they require larger quantities of test article. Quality of minipigs in Europe is generally high. However, housing the Go¨ttingen minipig in other laboratories requires closed barrier systems because this strain is raised under conditions that result in some immunological naivity. There is still some regulatory resistance or unfamiliarity related to issues with background data and spontaneous and toxicologic pathology. The key area with true benefit for the pharmaceutical industry is that there always is a good reason for selecting the minipig when developing dermally applied drugs.
Acknowledgments The contributions of Robert Harling and Andrew Makin, Scantox A/S, DK- 4623 Lille Skensved, Denmark, are highly appreciated.
References Anzenbacherova´ E, Anzenbacher P, Svoboda Z, Ulrichova´ J, Kvetina J, Zoulova´ J, et al. Minipig as a model for drug metabolism in man: comparison of in vitro and in vivo metabolism of propafenone. Biomed Papers 2003;147: 155–9. Bollen PJA, Hansen AK, Rasmussen HJ. The laboratory swine. London: CRC Press; 2000. Brinck P. Multidose infusion toxicity studies in the minipig. In: Healing G, Smith D, editors. Handbook of pre-clinical continuous intravenous infusion. London: Taylor & Francis; 2000a. p. 233–40. Brinck P. Surgical preparation of the minipig. In: Healing G, Smith D, editors. Handbook of pre-clinical continuous intravenous infusion. London: Taylor & Francis; 2000b. p. 225–32.
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Carrasco L, Madsen LW, Salguero FJ, Nunez A, SanchezCordon P, Bollen P. Immune complex-associated thrombocytopenic purpura syndrome in sexually mature Go¨ttingen minipigs. J Comp Pathol 2003;128:25–32s. Damm Jørgensen K. Minipig in reproduction toxicology. In: Svendsen O, editor. The minipig in toxicology. Scand J Lab Anim Sci 1998a; 25(suppl. 1):63–75. Damm Jørgensen K. Teratogenic activity of tretionoin in the Go¨ttingen minipig. In: Svendsen O, editor. The minipig in toxicology, Scand J Lab Anim Sci 1998b;25 (suppl. 1): 235-43. Damm Jørgensen K, Ellegaard L, Klastrup S, Svendsen O. Haematological and clinical chemical values in pregnant and juvenile Go¨ttingen minipigs. In: Svendsen O, editor, The minipig in toxicology, Scand J Lab Anim Sci 1998a; 25 (suppl. 1):181–90. Damm Jørgensen K, Kledal TSA, Svendsen O, Skakkebæk NE. The Go¨ttingen minipig as a model for studying effects on male fertility. In: Svendsen O, editor. The minipig in toxicology, Scand J Lab Anim Sci 1998b;25(suppl. 1): 161–9. Dincer Z, Svendsen O. Pathology of Go¨ttingen minipig: background and incidentally occurring changes. In: Gad SC, editor. Animal models in toxicology, 2nd ed., Marcel Dekker, New York, 2006, in press 2006. Ellegaard L, Dam Jørgensen K, Klastrup S, Kornerup Hansen A, Svendsen O. Haematologic and clinical chemical values in 3 and 6 months old Go¨ttingen minipigs. Scand J Lab Anim Sci 1995;22:239–48. Hinton DM. US FDA ‘‘Redbook II’’ immunotoxicity testing guidelines and research in immunotoxicity evaluations of food chemicals and new food proteins. Toxicol Pathol 2000;28:467–78. Ikeda GJ, Friedman L, Hattan DG. The minipig as a model in regulatory toxicity testing. In: Svendsen O, editor. The minipig in toxicology, Scand J Lab Anim Sci 1998; 25(suppl. 1):99–105. Koch W, Windt H, Walles M, Borlak J, Clausing P. Inhalation studies with the Go¨ttingen minipig. Inhalation Toxicol 2001;13:249–59.
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McAnulty P. Reproductive infusion toxicity studies in the minipig. In: Healing G, Smith D, editors. Handbook of preclinical continuous intravenous infusion. London: Taylor & Francis; 2000. p. 241–50. Montiero-Riviere NA, Riviere J. The pig as a model for cutaneous pharmacology and toxicology research. In: Tumbleson ME, Schook LB, editors. Advances in swine in biomedical research, vol. 2. New York: Plenum Press; 1996. p. 425–58. Mortensen JT, Brinck P, Lichtenberg J. The minipig in dermal toxicology. A literature review. In: Svendsen O, editor. The minipig in toxicology. Scand J Lab Anim Sci 1998;25(suppl. 1):77–83. Nahas K, Baneux P, Detweiler D. Electrocardiographic monitoring in the Go¨ttingen minipig. Comp Med 2002;52: 258–64. Skaanild MT, Friis C. Characterization of the P450 system in Go¨ttingen minipigs. Pharmacol Toxicol 1997;80(suppl. 11): 28–33. Skaanild MT, Friis C. Cytochrome P450 sex differences in minipigs and conventional pigs. Pharmacol Toxicol 1999;85:174–80. Skaanild MT, Friis C. Porcine CYP2A polymorphisms and activity. Basic Clin Pharmacol Toxicol 2005;97:115–21. Skydsgaard M, Harling SM, Nielsen G. Background pathology in the Go¨ttingen minipig. Exp Toxicol Pathol 2006, (in prep.). Svendsen O, Skydsgaard M, Aarup V, Klastrup S. Spontaneously occurring microscopic lesions in selected organs of the Go¨ttingen minipig. In: Svendsen O, editor. The minipig in toxicology. Scand J Lab Anim Sci 1998;25(suppl. 1): 230–4. Swindle MM. Surgery, anaesthesia and experimental techniques in swine. Ames: Iowa State University Press; 1998. Swindle M, Smith AC. Comparative anatomy and physiology of the pig. In: Svendsen O, editor. The minipig in toxicology. Scand J Lab Anim Sci 1998;25(suppl. 1):10–21. Witkamp RF, Monshouwer M. Pharmacokinetics in vivo and in vitro in swine. In: Svendsen O, editor. The minipig in toxicology. Scand J Lab Anim Sci 1998;25(suppl. 1):45–56.
Experimental and Toxicologic Pathology 57 (2006) 335–339
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
3RD EUROPEAN CONGRESS OF TOXICOLOGIC PATHOLOGY, 2005, COPENHAGEN, DENMARK
The minipig in toxicology Ove Svendsen Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Ridebanevej 9, DK-1870 Frederiksberg C, Copenhagen, Denmark Received 23 February 2006; accepted 30 March 2006
Abstract The use of pigs (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species have been found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. Because of their well-accepted physiological and other similarities to humans, minipigs are becoming increasingly attractive toxicological and pharmacological models. There are several strains of minipigs (Go¨ttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Go¨ttingen minipigs. In toxicology in Europe minipigs have become very popular for pharmaceutical studies in place of dogs and primates. Minipigs have been shown to be sensitive to a wide variety of drugs and chemicals. It has become obvious that minipigs can be used for all routes of administration, and in many cases are preferable to dogs or primates for metabolic or pharmacological reasons. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been wellestablished allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature. Now there is good supply of high-quality minipigs of known disease status. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. Consequently, there are scientific, economic and sociological reasons that make minipigs good toxicological and pharmacological models. The principal disadvantage is that toxicity testing in minipigs may require more test compound than the traditional species. r 2006 Elsevier GmbH. All rights reserved. Keywords: Minipig; Toxicology; Toxicology testing; General toxicology; Reproductive toxicology; Dosing routes; Sampling techniques; Necropsy; Toxicopathology
Introduction The pig was domesticated from the wild swine by the Chinese more than 7000 years ago. Within the last Tel.: +45 35 28 31 65; fax: +45 35 35 35 14.
E-mail address: [email protected]. 0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.03.003
centuries the pig has increasingly been used in biomedical research. Today in the EU more than 60,000 pigs are used a year for scientific procedures. The number of minipigs used for such purposes is relatively low. The minipig was introduced in Europe in the early 1970s for scientific purposes. In the mid-1980s the minipig was introduced in toxicology as an alternative non-rodent
ARTICLE IN PRESS 336
O. Svendsen / Experimental and Toxicologic Pathology 57 (2006) 335–339
species. The basic reason for this introduction was the realization of the many biochemical, anatomical and physiologically similarities with humans relative to other non-rodent species (Swindle and Smith, 1998). Extensive use since then has highlighted in details the areas where the minipig has become a particularly useful animal species for toxicological studies and toxicity testing.
pig and then with German Landrace to obtain pale skin. It is now a white non-pigmented and small-size minipig with an adult body weight of 30–40 kg if held on restricted diet. They have been purpose bred in Europe for years and now also in the US and are readily available. They are of very high microbiological and parasitic quality. There is a full genetic background available and background toxicology control data have accumulated in the scientific literature.
Domestic pigs and minipigs The use of the domestic pig (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species were found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. There are several strains of minipigs (Go¨ttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Go¨ttingen minipig. There is an increasing interest in minipigs because they share many similarities with humans anatomically and physiologically relative to other non-rodent species; in particular the skin, heart and kidney are very similar to those of humans (Montiero-Riviere and Riviere, 1996; Mortensen et al., 1998; Swindle, 1998). They are available as purpose-bred SPF high-quality animals. In addition, there is a growing amount of background data and growing regulatory acceptability. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. The minipig has been shown to be sensitive to a wide variety of drugs and chemicals. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. It has been demonstrated that minipigs can be used for all routes of administration such as orally or parenterally, and in many cases are preferable for dogs or primates for metabolic or pharmacological reasons. Consequently, there are scientific, economic and sociological reasons that make minipigs good animal models in toxicology and other scientific disciplines. The principal disadvantage is that toxicity testing in the minipig may require more test compound than the traditional species.
Go¨ttingen minipigs There are several benefits related to the Go¨ttingen strain of minipigs. This strain was developed by Go¨ttingen University in the 1960s by cross-breeding of the Minnesota minipig first with Vietnamese put belly
Pharmacokinetics and metabolism Pharmacokinetic studies are easily performed in the minipig with repeated blood sampling or sampling of other body fluids or tissues as demonstrated by Anzenbacherova´ et al. (2003) and Witkamp and Monshouwer (1998). The cytochrome P450 liver metabolic pattern has been extensively studied by Skaanild and Friis (1997, 1999, 2005) and compared to that of humans. It is beyond the scope of this paper to describe in detail the similarities and dissimilarities with that of humans.
Minipigs in repeat dose toxicology Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been well established allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature.
Routes of dose administration The most common route of dosing is the oral route either by gavage or dietary dosing with medicated diet. Dosing by oral gavage in conscious animals may be stressing for the animals. Because of anatomically and physiologically similarities with skin of humans the minipig is particularly useful for dermal studies. This route has been used for acute and repeat dose dermal toxicology studies, dermal absorption studies, phototoxicity studies and photosensitization studies. Parenteral dosing can be applied by intramuscular, subcutaneous, intradermal and intravenous injection. The latter can be applied either by bolus injection or as continuous intravenous infusion. The details of continuous intravenous infusion in the minipig have been described by Brinck (2000a) who also has described the
ARTICLE IN PRESS O. Svendsen / Experimental and Toxicologic Pathology 57 (2006) 335–339
surgical preparation for the application of continuous intravenous infusion (Brinck, 2000b). Other routes as nasal dosing or inhalation dosing (Koch et al., 2001) have also been successfully applied in minipigs.
337
rhagic syndrome and probably associated with an immunocomplex mediated condition and marked degenerative/ regenerative megakaryocytic changes (Carrasco et al., 2003).
Observations and sampling techniques The observations required in toxicity testing such as clinical signs, body weight, ophthalmoscopy and electrocardiography (Nahas et al., 2002) are routine procedures in toxicity testing with the minipig. In addition, control hematology and clinical chemistry data are available in the scientific literature (Ellegaard et al., 1995; Damm Jørgensen et al., 1998a). Parameters of immunotoxicity could be included (Hinton, 2000). Blood sampling techniques have been described in detail by Bollen et al. (2000) and Swindle (1998).
Necropsy procedure While conducting post-mortem procedures in minipigs some major differences to other non-rodent species have to be taken into consideration. This includes the gastrointestinal tract, thymus, thyroid and parathyroid. In minipigs the cecum is very large as is the colon. In addition, the colon is formed as a spiral. The majority of the thymus is located in the cervical region and a minor part in the thoracic cavity. However, microscopic examination is usually performed on the thoracic part as in other species. The thyroid is located on the ventral part of the trachea just in front of thoracic curvature. As another variation the two lobes of the thyroid are merged into one organ. The parathyroids are not embedded or attached to the thyroids, but are instead located in the cranial part of the thymus in 90% of the cases. It is not unusual that the parathyroids are placed in the very most cranial part of the thymus just behind the hyoid bone. It requires good skills to be able to find both glands routinely.
Histopathology The Go¨ttingen minipig is free from any porcine infectious bacterial or viral disease and free from any porcine parasitic disease. Histopathology findings of spontaneous nature are few and in general focal and mild (Svendsen et al., 1998; Dincer and Svendsen, 2006). The most recent and most extensive collection of control data is reported by Skydsgaard (2006 in prep.). There are four significant spontaneous findings: (1) Arteritis (scattered, focal, necrotic/fibrinoid), (2) tubular atrophy/hypoplasia in testis (focal), (3) serous atrophy (bone marrow fat cells, mainly femur/tibia) and (4) hemorrhagic syndrome. The etiology of all four features is unknown. Thrombocytopenia is extensive in the hemor-
Reproductive toxicology In testing for reproductive toxicity in general two animal species have to be used, a rodent and a non-rodent species. The two species routinely used are the rat and the rabbit. The minipig may be an alternative species in teratogenicity and reproductive studies where there is a lack of suitability of traditional species such as mice, rats or rabbits. Nonhuman primates have supply and conservation considerations and the majority have single offspring. Minipigs are purpose-bred to SPF standards, relatively inexpensive, and have a number of similarities with humans. The reproductive characteristics of the Go¨ttingen minipig have been described by Damm Jørgensen (1998a) together with a teratogenicity testing protocol and historical control data. The testing protocol can be summarized as follows. Sexual maturity of Go¨ttingen minipigs is reached before the animals are 6 months of age. In general minipig gilts are 5–10 months of age when included in studies. In teratogenicity studies the treatment period extends from start of implantation (day 11) to the closure of the hard palate (day 35) inclusive of gestation. Different routes of administration can be used. While the oral route is the most commonly used, continuous intravenous infusion can also be applied (McAnulty, 2000). Pregnancy status can be controlled by ultrasonography in week 4 or 5 of gestation. The sows are killed on day 110–112 of gestation. As the fetus weight is 350–400 g, it is possible to perform full necropsy on the fetuses. In general the protocol includes fetal examinations after removal from uterus, alizarin staining of skeleton and free-hand sectioning of Bouin fixed heads. For skeletal examination X-rays may be added because the bones of the skull and bone densities are better visualized than with alizarin staining (Damm Jørgensen, 1998a). The historical data include skeletal diagnoses from 220 fetuses and spontaneous malformation data from more than 3700 newborn or stillborn fetuses. By this protocol it has been shown that the Go¨ttingen minipig is highly susceptible to the teratogenic effects of tretinoin with malformations resembling retinoic acid teratogenicity in humans (Damm Jørgensen K, 1998b). Hematological and clinical chemical control data of pregnant Go¨ttingen minipigs have been reported by Damm Jørgensen et al. (1998a). The Go¨ttingen minipig has also proven useful as a model for studying effects on male fertility (Damm Jørgensen et al., 1998b). The reported data support the assumption that the male minipig may prove to be more susceptible to chemicals with adverse effect on fertility
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than the male rat. The minipig has closer similarities to man with respect to fertility rate, percentage morphologically abnormal sperm, percentage of sperm with progressive motility and incidence of cryptorchidism.
Juvenile studies So-called juvenile studies were introduced into nonclinical testing programs because it was realized that several drugs were applied in pediatric clinics to children without testing data justifying safe use of the drugs in this particular age group. Standard non-clinical studies using adult animals, or safety information from adult humans, cannot always adequately predict these differences in safety profiles for all pediatric age groups, especially reaction of immature systems such as the developing brain, pulmonary system, kidneys and reproductive and immune systems. This motivated the US Food and Drug Administration and the European Medicines Agency to issue draft guidelines for non-clinical testing in juvenile animals on human pharmaceuticals for pediatric indications. Traditionally, rats and dogs have been the species of choice for such testing, but other species may be more appropriate in some circumstances. The appropriateness of the minipig in this type of testing has been evaluated by scientists at Scantox in Denmark and the results presented at various scientific conferences (Harling and Makin, personal communication, 2005). The optimal protocols for the conduct of juvenile studies includes cross-fostering in order to handle all animal allocated to one mother as one study group and thus avoiding litter effect. Cross-fostering of juvenile minipigs for performance of juvenile toxicology studies is a realistic practical proposition using estrus synchronization and mating in breeding facilities with subsequent housing in the testing laboratory. Properly controlled synchronization results in delivery of all mimipigs within few days allowing for cross-fostering. Various standard techniques such as oral or parenteral dosing, ophthalmoscopy, ECG and repeated collection of blood samples for clinical pathology and toxicokinetics are practically feasible. Implantation of vascular access ports was successful from day 7 and onwards for daily intravenous dosing. Background clinical pathology data are important because many of the standard parameters change rapidly with increasing age (Damm Jørgensen et al., 1998a).
Regulatory acceptance The pig and minipig as a model in toxicity testing of pharmaceuticals and other chemicals has now been well accepted by Japan, EU and USA (Ikeda et al., 1998). The pig and minipig is specifically mentioned as a
potential non-rodent species in guidelines of Japan and Canada. The OECD 409 guideline lists pig and minipig as optional species. However, evidence should be provided that it is a suitable species and there is still some regulatory resistance or unfamiliarity related to issues with background data and spontaneous and toxicologic pathology.
Conclusions The minipig is a useful non-rodent animal species in investigations of safety issues in drug development. However, selection of the most suitable non-rodent species is a complex issue. Many pharmaceutical industries still hold the dog as the number one choice and move only from the dog where there is a justification. The minipig may be disregarded merely from the basis of body weight because they require larger quantities of test article. Quality of minipigs in Europe is generally high. However, housing the Go¨ttingen minipig in other laboratories requires closed barrier systems because this strain is raised under conditions that result in some immunological naivity. There is still some regulatory resistance or unfamiliarity related to issues with background data and spontaneous and toxicologic pathology. The key area with true benefit for the pharmaceutical industry is that there always is a good reason for selecting the minipig when developing dermally applied drugs.
Acknowledgments The contributions of Robert Harling and Andrew Makin, Scantox A/S, DK- 4623 Lille Skensved, Denmark, are highly appreciated.
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