2-deoxy D-glucose treatment does not elicit a hair growth response in alopecia areata.

2-deoxy D-glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T-cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell-mediated autoimmune mouse disease, alopecia areata.

Inhibition of Tissue-Nonspecific Alkaline Phosphatase Attenuates Ectopic Mineralization in the Abcc6-/- Mouse Model of PXE but Not in the Enpp1 Mutant Mouse Models of GACI.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.

Skin Diseases in Laboratory Mice: Approaches to Drug Target Identification and Efficacy Screening.

A large variety of mouse models for human skin, hair, and nail diseases are readily available from investigators and vendors worldwide. Mouse skin is a simple organ to observe lesions and their response to therapy, but identifying and monitoring the progress of treatments of mouse skin diseases can still be challenging. This chapter provides an overview on how to use the laboratory mouse as a preclinical tool to evaluate efficacy of new compounds or test potential new uses for compounds approved for use for treating an unrelated disease. Basic approaches to handling mice, applying compounds, and quantifying effects of the treatment are presented.

The effects of bisphosphonates on ectopic soft tissue mineralization caused by mutations in the ABCC6 gene.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by ABCC6 mutations by feeding Abcc6(-/-) mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the ABCC6 gene, when combined with careful monitoring of efficacy and potential side-effects.

Genetic modulation of nephrocalcinosis in mouse models of ectopic mineralization: the Abcc6(tm1Jfk) and Enpp1(asj) mutant mice.

Ectopic mineralization of renal tissues in nephrocalcinosis is a complex, multifactorial process. The purpose of this study was to examine the role of genetic modulation and the role of diet in nephrocalcinosis using two established mouse models of ectopic mineralization, Abcc6(tm1Jfk) and Enpp1(asj) mice, which serve as models for pseudoxanthoma elasticum and generalized arterial calcification of infancy, two heritable disorders, respectively. These mutant mice, when on standard rodent diet, develop nephrocalcinosis only at a very late age. In contrast, when placed on an 'acceleration diet' composed of increased phosphate and reduced magnesium content, they showed extensive mineralization of the kidneys affecting primarily the medullary tubules as well as arcuate and renal arteries, as examined by histopathology and quantitated by chemical assay for calcium. Mineralization could also be detected noninvasively by micro computed tomography. Whereas the heterozygous mice did not develop nephrocalcinosis, compound heterozygous mice carrying both mutant alleles, Abcc6(tm1Jfk/+) and Enpp1(+/asj), developed ectopic mineralization similar to that noted in homozygous mice for either gene, indicating that deletion of one Abcc6 allele along with Enpp1 haploinsufficiency resulted in renal mineralization. Thus, synergistic genetic defects in the complex mineralization/antimineralization network can profoundly modulate the degree of ectopic mineralization in nephrocalcinosis.

Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

Skin diseases in laboratory mice: approaches to drug target identification and efficacy screening.

A large variety of mouse models for human skin and adnexa diseases are readily available from investigators and vendors worldwide. While the skin is an obvious organ to observe lesions and their response to therapy, actually treating and monitoring progress in mice can be challenging. This chapter provides an overview on how to use the laboratory mouse as a preclinical tool to evaluate efficacy of a new compound or test potential new uses for a compound approved for use for treating an unrelated disease. Basic approaches to handling mice, applying compounds, and quantifying effects of the treatment are presented.

The C3H/HeJ mouse and DEBR rat models for alopecia areata: review of preclinical drug screening approaches and results.

The C3H/HeJ inbred mouse strain and the Dundee Experimental Bald Rat (DEBR) strain spontaneously develop adult onset alopecia areata (AA), a cell-mediated disease directed against actively growing hair follicles. The low frequency of AA and the inability to predict the stage of AA as it evolves in the naturally occuring C3H/HeJ model of AA can be converted into a highly predictable system by grafting full thickness skin from AA-affected mice to normal haired mice of the same strain. The rat DEBR model develops spontaneous AA at a higher frequency than in the mouse model but they are more expensive to use in drug studies owing to their larger size. Regardless of the shortcomings of either model, these rodent models can be used succesfully to screen novel or approved drugs for efficacy to treat human AA. As the pathogenesis of AA follows the canonical lymphocytic co-stimulatory cascade in the mouse AA model, it can be used to screen compounds potentially useful to treat a variety of cell-mediated diseases. Efficacy of various agents can easily be screened by simply observing the presence, rate, and cosmetic acceptability of hair regrowth. More sophisticated assays can refine how the drugs induce hair regrowth and evaluate the underlying pathogenesis of AA. Some drugs commonly used to treat human AA patients work equally as well in both rodent models validating their usefulness as models for drug efficacy and safety for humanAA.