The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are widely acknowledged as important and rate-limiting to the hepatic uptake of many drugs in clinical use. Accordingly, to better understand the in vivo relevance of OATP1B transporters, targeted disruption of murine Slco1b2 gene was carried out. It is noteworthy that Slco1b2(Ϫ/Ϫ) mice were fertile, developed normally, and exhibited no overt phenotypic abnormalities. We confirmed the loss of Oatp1b2 expression in liver using real-time polymerase chain reaction, Western Blot analysis, and immunohistochemistry. Expression of Oatp1a4 and Oatp2b1 but not Oatp1a1 was greater in female Slco1b2(Ϫ/Ϫ) mice, but expression of other non-OATP transporters did not significantly differ between wild-type and Slco1b2(Ϫ/Ϫ) male mice. Total bilirubin level was elevated by 2-fold in the Slco1b2(Ϫ/Ϫ) mice despite the fact that liver enzymes ALT and AST were normal. Pharmacological characterization was carried out using two prototypical substrates of human OATP1B1 and -1B3, rifampin and pravastatin. After a single intravenous dose of rifampin (1 mg/kg), a 1.7-fold increase in plasma area under the concentration-time curve (AUC) was observed, whereas the liver-to-plasma ratio was reduced by 5-fold, and nearly 8-fold when assessed at steady-state conditions after 24 h of continuous subcutaneous infusion in Slco1b2(Ϫ/Ϫ) mice. Likewise, continuous subcutaneous infusion at low (8 g/h) or high (32 g/h) dose rates of pravastatin resulted in a 4-fold lower liver-plasma ratio in the in Slco1b2(Ϫ/Ϫ) mice. This is the first report of altered drug disposition profile in the Slco1b2 knockout mice and suggests the utility of this model for understanding the in vivo role of hepatic OATP transporters in drug disposition.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14 ؉ CD16 ؉ monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1 op /Csf1 op ) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Development of feline immunodeficiency virus (FIV) infection in cats as a small animal model for lentiviral immunodeficiency disease has been hampered by the prolonged and variable disease course following experimental infection. To address this issue, we generated high-titer, unselected FIV stocks by pooling plasma from cats acutely infected with a subgroup C FIV isolate designated CABCpadyOOC (FIV-C-PGammer). Subsequent infection with this virus pool resulted in rapidly progressive, fatal disease in greater than 50% of infected cats. Accelerated FIV disease was characterized by rapid and progressive CD4 ؉ T-cell loss, lymphadenopathy, weight loss, lymphoid depletion, and severe thymic atrophy. Mortality and rate of disease progression were affected by the age of each cat at infection and whether the virus source animal was in the acute or chronic stage of infection. The rapid FIV disease syndrome was consistently associated with systemic lymphoid depletion, clinical disease, and susceptibility to opportunistic infections, analogous to accelerated and/or terminal HIV-1 infection. The results of this study demonstrate that FIV infection is a valid small animal model for lentiviral immunodeficiency disease.

Malignant histiocytosis (MH) was diagnosed in a 13-year-old neutered male Domestic Shorthair cat on the basis of light microscopic and immunohistochemical findings. Thoracic fluid analysis showed a modified transudate which contained a very few atypical discrete cells. Cytologic and histologic evaluation of mediastinal and splenic masses revealed a pleomorphic population of large, discrete, round cells 10 to 30 micrometers in diameter with marked cellular atypia. Nuclei were oval to reniform, often with prominent, bizarre nucleoli. Multinucleated cells and mitotic figures were commonly seen. Erythro- and leucocytophagia were noted. Immunohistochemistry indicated a scattered positive staining pattern with the histiocytic antigenic marker Mac387 and a minor population of cells showing positive reactivity for lysozyme. This report describes the characterization of MH in a cat and emphasizes that MH should be considered as a differential diagnosis in proliferative disorders of discrete-cells in this species.

To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV ؉ cells were of either of two morphological types, large stellate or small round. Those FIV RNA ؉ cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses.

Mucosal infection by feline immunodeficiency virus (FIV) was assessed via a single exposure of the vaginal or rectal mucosa to either infectious peripheral blood mononuclear cells (PBMCs), infectious plasma, or cell-free cultured virus. All cats inoculated with cell-free cultured virus (100 or 400 TCID) and 9 of 10 cats inoculated with infected PBMCs (2 x 10(7) or 2 x 10(5)) became persistently viremic within 3 weeks. Neither cat inoculated with 2 x 10(3) PBMCs became viremic. Rectal and vaginal exposure were equally effective routes to induce viremia. CD4+ T cells and mitogen-stimulated PBMC proliferation declined in all infected cats. However, a transient PBMC proliferative response to FIV p24gag occurred in most virus-exposed cats, especially those that did not develop detectable infection. FIV was not transmitted by mucosal exposure to infectious virus in plasma (100 TCID), a dose > 10-fold that needed for infection by parental injection. In vitro studies suggested that a plasma heat-stable virus-neutralizing factor may be associated with failure of plasma virus to establish infection via the mucosal route. Mucosal FIV infection provides a new model with which to study early stages of infection and intervention in transmucosal lentivirus infections.