Toxicologic Pathology Forum: Opinion on Performing Good Laboratory Practice Histopathology Evaluation for Nonclinical Toxicity Studies in a Remote Location.

Toxicologic/veterinary pathologists are working remotely from Good Laboratory Practice (GLP) test facilities (TFs) in increasing numbers, most commonly in home-office settings. A study pathologist (SP) generating data on GLP-compliant nonclinical studies must be keenly aware of applicable national GLP regulations and comply with TF and protocol requirements. This Toxicological Pathology Forum Opinion Piece will summarize primary areas of emphasis for the SP generating GLP data using glass slides. Peer review and digital review of whole slide images are out of scope for this opinion piece. Key GLP considerations for primary pathology on glass slides are discussed with respect to SP location and employment status, including pathologist qualifications, specimen management, facilities, equipment, archive, and quality assurance. Notable differences between national GLP regulations of the United States, the United Kingdom, Germany, the Netherlands, France, Ireland, Switzerland, Italy, and Israel are presented. With the understanding that each combination of location and employment is unique, the authors provide a general overview of considerations for successful remote GLP work.

ND0701, A Novel Formulation of Apomorphine for Subcutaneous Infusion, in Comparison to a Commercial Apomorphine Formulation: 28-Day Pharmacokinetic Study in Minipigs and a Phase I Study in Healthy Volunteers to Assess the Safety, Tolerability, Pharmacokinetics and Relative Bioavailability.

BACKGROUND: Subcutaneous apomorphine is used for the treatment of Parkinson's disease (PD); however, infusion site reactions are a common adverse event (AE), which can lead to treatment discontinuation. Apomorphine formulations that are more tolerable and convenient for use are needed. OBJECTIVE: Our aim was to compare the toxicity and bioavailability of ND0701, a new concentrated formulation of apomorphine free base, with one of the commercially available apomorphine HCl formulations (APO-go®, Britannia Pharmaceuticals Ltd). METHODS: (1) Preclinical study: 16 minipigs were randomly assigned to placebo, APO-go®, and ND0701 groups, and treated for 28 days. Pharmacokinetic, clinical, and pathological assessments were performed. (2) Phase I study: 18 healthy volunteers participated in an open-label, two-sequence, randomized, three single-dose, partial crossover study to compare the pharmacokinetics, safety, and tolerability of ND0701 with APO-go® (1%). RESULTS: (1) Preclinical study: No systemic toxicity was observed in apomorphine-treated minipigs, but local skin reactions were observed at the infusion sites. These effects were less frequent and less severe and recovery was more rapid for ND0701 compared with APO-go®. (2) Phase I study: Both formulations were safe and well tolerated under the conditions of the study and no severe or serious treatment-emergent AEs were reported. Infusion site nodules were reported more frequently, with higher severity, and recovered slower at APO-go®-treated sites compared with ND0701-treated sites. Bioavailability of apomorphine was comparable between the two formulations. CONCLUSION: Based on these pilot studies, ND0701 appears to be superior to APO-go® in terms of tolerability and safety, while maintaining comparable bioavailability with APO-go®, and shows promise as a future treatment for PD.

Ninety-day Local Tolerability and Toxicity Study of ND0612, a Novel Formulation of Levodopa/Carbidopa, Administered by Subcutaneous Continuous Infusion in Minipigs.

A 90-day study in Göttingen minipigs was conducted to test the local tolerability and systemic toxicity of ND0612, a novel aqueous solution of carbidopa (CD)/levodopa (LD) intended for the treatment of Parkinson's disease by continuous subcutaneous administration using a discrete infusion pump. To evaluate tissue site reactions, we used a unique study design involving multiple infusion sites to evaluate the effect of dose per site (270/63, 360/45, and 360/84 mg LD/CD), volume of infusion per site (4.5 and 6 ml per site), formulation concentration (60/14 and 60/7.5 mg/ml LD/CD), daily rate of infusion per site (240 µl/hr for16 hr and 80 µl/hr for 8 hr, 320 µl/hr for 16 hr and 100 µl/hr for 8 hr, or 750 µl/hr for 8 hr), frequency (once every 5, 10, 15, or 20 days), and number of infusions (4, 6, or 9) to the same infusion site. No systemic adverse effects were observed. Histopathological changes at infusion sites started with localized minimal necrosis and acute inflammation that progressed to subacute and chronic inflammatory and reparative changes with evidence of progressive recovery following the final infusion. None of the infusion site effects were judged to be adverse, and clinical exposures to ND0612 are not expected to result in adverse responses.

The minipig as a new model for the evaluation of doxorubicin-induced chronic toxicity.

Doxorubicin can cause life-threatening toxic effects in several organs, with cardiotoxicity being the major concern. Although a large number of animal models have been utilized to study doxorubicin toxicity, several restrictions limit their use. Since the Göttingen minipig is an accepted species for non-clinical safety assessment and translation to man, we aimed at exploring its use as a non-rodent animal model for safety assessment and regulatory toxicity studies using doxorubicin. Three groups of three males and three females adult Göttingen minipigs received 1.5 mg kg(-1) , 3/2.3 mg kg(-1) or vehicle at intervals of 3 weeks for 7 cycles. Doxorubicin treatment resulted in a dose-related decrease in the erythrocytes, hemoglobin and hematocrit count, accompanied by leukopenia and thrombocytopenia. Bone marrow smears revealed dose-related hypocellularity. Urea and creatinine levels were elevated in treated animals, associated with proteinuria and hematuria. Histopathological evaluation detected nephropathy and atrophy of hematopoietic tissues/organs, mucosa of the intestinal tract and male genital tract. Cardiac lesions including chronic inflammation, endocardial hyperplasia, hemorrhage and myxomatous changes were evident in hematoxylin and eosin stains, and evaluation of semi-thin sections showed the presence of dose-related vacuolation in the atrial and ventricular cardiomyocytes. Cardiac troponin levels were increased in the high-dose group, but there was no direct correlation to the severity of the histopathological lesions. This study confirms that the Göttingen minipig has a comparable toxicity profile to humans and considering its anatomical, physiological, genetic and biochemical resemblance to humans, it should be considered as the non-rodent species of choice for studies on doxorubicin toxicity. Copyright © 2015 John Wiley & Sons, Ltd.

Aryl hydrocarbon receptor knockout rats are insensitive to the pathological effects of repeated oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Sustained activation of the aryl hydrocarbon receptor (AHR) is believed to be the initial key event in AHR receptor-mediated tumorigenesis in the rat liver. The role of AHR in mediating pathological changes in the liver prior to tumor formation was investigated in a 4-week, repeated-dose study using adult female wild-type (WT) and AHR knockout (AHR-KO) rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Beginning at 8 weeks of age, AHR-KO and WT rats were dosed by oral gavage with varying concentrations of TCDD (0, 3, 22, 100, 300 and 1000 ng kg(-1) day(-1) ). Lung, liver and thymus histopathology, hematology, serum chemistry and the distribution of TCDD in liver and adipose tissue were examined. Treatment-related increases in the severity of liver and thymus pathology were observed in WT, but not AHR-KO rats. In the liver, these included hepatocellular hypertrophy, bile duct hyperplasia, multinucleated hepatocytes and inflammatory cell foci. A loss of cellularity in the thymic cortex and thymic atrophy was observed. Treatment-related changes in serum chemistry parameters were also observed in WT, but not AHR-KO rats. Finally, dose-dependent accumulation of TCDD was observed primarily in the liver of WT rats and primarily in the adipose tissue of AHR-KO rats. The results suggest that AHR activation is the initial key event underlying the progression of histological effects leading to liver tumorigenesis following TCDD treatment. Copyright © 2015 John Wiley & Sons, Ltd.

Spontaneous glomerulonephritis in Göttingen minipigs.

The Göttingen minipig is one of the nonrodent species recommended by various regulatory authorities for safety assessment of drugs in preclinical studies. In such studies, knowledge of background pathology is critical in order to evaluate the potential renal toxicity. In the present study, the authors report 4 cases of glomerulonephritis out of 154 microbiologically defined Göttingen minipigs microscopically evaluated in preclinical studies. One animal required early sacrifice because of general poor health, and an additional animal died spontaneously. Histopathological evaluation revealed renal lesions in all 4 animals, exhibiting membranous or membranoproliferative glomerulonephritis at different stages, accompanied by secondary tubulo-interstitial damage. The renal changes observed were considered spontaneous in origin and of unknown etiology. Development of this condition in this strain should be considered in future studies.