Sarcocystis Infection in Laboratory Rabbits.

Sarcocystosis, presumably caused by Sarcocystis cuniculi, was diagnosed in 2 purpose-bred, SPF Dutch belted laboratory rabbits from a class A breeder. The rabbits were purchased by a research facility and conventionally housed individually in stainless-steel suspended caging. At necropsy and tissue harvest, gross lesions were not observed in the muscles. Upon histologic examination, sarcocysts were found in the eyelid of one rabbit and the tongue of the other. To our knowledge, this report is the first description of infection by Sarcocystis spp. in laboratory rabbits.

Nonclinical Safety and Toxicokinetics of MnTE-2-PyP (BMX-010), a Topical Agent in Phase 2 Trials for Psoriasis and Atopic Dermatitis.

Since our earlier publication (Gad et al, 2013), BioMimetix has advanced BMX-010 (Manganese (III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin or MnTE020PyP; CASRN 219818-60-7) into clinical development as a topical agent for the treatment of psoriasis, atopic dermatitis, and pruritus (idiopathic nonspecific itch). A multiple dose phase I study has been completed in 64 patients without any serious adverse effects. During the course of development, the formulation was initially a gel but has been modified to a cream formulation. The nonclinical safety program has been carried onward to assess preclinical risk to patients. Additional studies completed and reported here include dermal sensitization in a Guinea Pig maximization test study, 2 rabbit phototoxicity studies, a 28-day oral toxicity study in juvenile mice, a 28-day topical systemic toxicity study in Gottingen minipigs, range-finding studies, and complete embryo-fetal developmental toxicity (Segment II) studies in mice and rabbits, an ICH M7 compliant qualification of impurities using 2 (Q)SAR in silico methods, and a 14-day subcutaneous toxicity study of mice to qualify an impurity. All studies (except the (Q)SAR evaluations) were performed in accordance with Good Laboratory Practices (GLP) using Good Manufacturing Practices (GMP) drug substance. The systemic toxicity studies, with the exception of the juvenile toxicity study, included toxicokinetic evaluations, which are reported here. The phase I clinical study had 67 patient participants who received topically applied BMX-010, and there were no notable safety findings and included pharmacokinetic determinations on these patients which are also reported here. Chronic GLP toxicity studies have been initiated in the mouse (6-month oral) and minipig (9-month dermal). To date, the only observed nonclinical toxicity remains a reversible hypertension seen in mice in response to Cmax levels with a no effect threshold, and there have been no drug-related adverse effects.

Nonclinical Safety and Toxicokinetics of MnTnBuOE-2-PyP5+ (BMX-001).

BMX-001, a manganese porphyrin that has anti-inflammatory, antioxidant, and antitumor properties, is being developed as a potential therapeutic for high-grade glioma (HGG) and head and neck (H&N) cancer. An IND has been opened for BMX-001 in the treatment of HGG (NCT02655601) and another is in preparation for H&N. The safety of BMX-001 has been evaluated in a battery of nonclinical Good Laboratory Practice (GLP)-compliant studies. Systemic toxicity has been evaluated using the intended cGMP product administered subcutaneously for periods of up to 5 weeks in both the mouse and the monkey and included toxicokinetic evaluations to characterize systemic exposure and tissue distribution and clearance of BMX-001. In additional GLP studies, BMX-001 was not irritating to the skin or eye and caused no changes in cardiac rate or rhythm or blood pressure. Mixed results for genotoxicity were seen with the weight of evidence indicating that BMX-001 poses no genotoxic risk in humans. In systemic mouse and monkey studies, loading/maintenance dose no observed adverse effect levels were 12/2 mg/kg/dose and 6/2 mg/kg/dose, respectively, with maintenance doses administered every 3 days after the initial loading dose. Systemic data were used to determine a Food and Drug Administration-approved safe starting dose for the initial clinical study in patients with HGG. BMX-001 was detected in analyzed tissues, including the brain, persisting well past the short plasma clearance period. The highest levels of BMX-001 were seen in the liver and kidneys, with amounts in these tissues returning to close to undetectable levels after a 2-week cessation of dosing.

Tolerable Levels of Nonclinical Vehicles and Formulations Used in Studies by Multiple Routes in Multiple Species With Notes on Methods to Improve Utility.

Formulation of nonclinical evaluations is a challenge, with the fundamental need to achieve multiples of the clinical exposure complicated by differences in species and routes of administration-specific tolerances, depending on concentrations, volumes, dosing regimen, duration of each administration, and study duration. Current practice to approach these differences is based on individual experience and scattered literature with no comprehensive data source (the most notable exception being our 2006 publication on this same subject). Lack of formulation tolerance data results in excessive animal use, unplanned delays in the evaluation and development of drugs, and vehicle-dependent results. A consulting firm, a chemical company, and 4 contract research organizations conducted a rigorous data mining operation of vehicle data from studies dating from 1991 to 2015, enhancing the data from this author's 2006 publication (3 of the six 2015 contributors were also 2006 contributors). Additional data were found in the published literature. The results identified 108 single-component vehicles (and 305 combination formulations) used in more than 1,040 studies across multiple species (dog, primate, rat, mouse, rabbit, guinea pig, minipig, pig, chick embryo, and cat) by multiple routes for a wide range of study durations. The tabulated data include maximum tolerated use levels by species, route, duration of study, dose-limiting toxicity where reported, review of the available literature on each vehicle, guidance on syringe selection, volume and pH limits by route with basic guidance on nonclinical formulation development, and guidance on factors to be considered in nonclinical route selection.

A nonclinical safety assessment of MnTE-2-PyP, a manganese porphyrin.

Manganese (III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP or BMX-010; CASRN 219818-60-7) is a manganese porphyrin compound developed as a potential drug substance for use as a radioprotective and for the ex vivo treatment of cells, tissues, and organs intended for transplantation. In preparation for an investigational new drug filing, a full good laboratory practice nonclinical safety assessment was conducted in order to evaluate the safety of MnTE-2-PyP and included the performance of in vitro genotoxicity studies, local tissue tolerance evaluation, safety pharmacology core battery studies, and single- and repeat-dose intravenous (iv) toxicity studies in mice and monkeys. The MnTE-2-PyP was determined not to be genotoxic or hemolytic, did not demonstrate flocculation or elicit adverse pharmacologic effects on respiration, the central nervous system (CNS), and had limited transitory effects on the cardiovascular system only at levels well above the therapeutic target dose. The intended iv clinical solution did not cause venous irritation in rabbits. The no observed adverse effect level (NOAEL) in mice was determined to be 10 mg/kg/day after 18 consecutive days of bolus iv dosing once daily in the morning. The NOAEL in monkeys after 14 days of bolus iv dosing in the morning was determined to be 5 mg/kg/day. At doses relevant to clinical use in humans, neither study revealed any indication of any specific target organ toxicity, including the classic heme porphyrin kidney, liver, CNS, or cardiac toxicities, or manganese toxicity. Mortality seen shortly after dosing in individual animals at higher doses was not accompanied by any organ or clinical pathology indications, suggesting a functional pharmacological-mediated effect. Based on the results of these studies, a conservative safe initial starting clinical dose of 5.0 mg (0.083 mg/kg in a 60 kg adult) was proposed for the initiation of human trials. Because of patent life issues, use of MnTE-2-PyP as a transplantation aid or radioprotective agent is not currently being pursued past the preclinical stages. It serves as a model for the clinical development of this class of drugs.

Chronic pharmacological and safety evaluation of Hematide, a PEGylated peptidic erythropoiesis-stimulating agent, in rodents.

Hematide is a synthetic peptide-based, PEGylated erythropoiesis-stimulating agent, which is being developed for the chronic treatment of anaemia associated with chronic renal failure. To support the safety of long-term dosing of chronic renal failure patients, a comprehensive toxicology programme was implemented including rat subchronic and chronic studies. Rats were administered 0, 0.1, 1 and 10 mg/kg of Hematide every 3 weeks for 3 months via subcutaneous injection or for 6 months via intravenous injection. The dosing period was followed by a 6-week follow-up period. The primary pharmacology of Hematide resulted in erythroid polycythemia as measured by elevated haemoglobin levels that were time- and dose-dependent. The pharmacology profiles were similar regardless of administration route. For example, for male rats at Day 90, subcutaneous dosing resulted in haemoglobin increases of 2.7, 4.5 and 6.9 g/dl for 0.1, 1 and 10 mg Hematide/kg respectively, compared to 2.8, 5.7 and 7.4 g/dl increases for intravenous dosing. Histopathological changes were related to the prolonged severe polycythemia induced in normocythemic animals administered an erythropoiesis-stimulating agent. The findings included extramedullary haematopoiesis in the spleen and liver, bone marrow hypercellularity and organ congestion. Microscopic findings were reversible, demonstrating a return towards control findings within 6 weeks following cessation of dosing. Systemic exposures, based on both area under the curve (AUC) and maximum concentration (C(max)), were substantially greater for intravenous than subcutaneous administration. No Hematide-specific antibodies were detected. In conclusion, Hematide is a potent erythropoiesis-stimulating agent, and the studies provide support for the safety of clinical development, including chronic dosing, for the treatment of anaemia associated with chronic renal failure.

Chronic preclinical safety evaluation of Hematide, a pegylated peptidic erythropoiesis stimulating agent in monkeys.

Hematide is a synthetic peptide-based, pegylated erythropoiesis stimulating agent in clinical development for treatment of anemia. To support chronic clinical dosing requirements, a 9-month repeat dose IV monkey safety study was undertaken. Animals received 0, 0.2, 2 or 20 mg/kg hematide IV every three weeks for nine months followed by a 14-week recovery. Hematide administration was associated with time and dose-dependent polycythemia. Histological findings were related to exaggerated pharmacology that was secondary to the administration of an erythropoiesis stimulating agent to a normocythemic animal. In conclusion, these results support the use of repeated administration of hematide for the correction of anemia.

Preclinical safety and pharmacology of Hematide, a peptidic erythropoiesis stimulating agent (ESA), in rats and monkeys.

The pharmacology, toxicokinetics, and safety of Hematide, a synthetic peptidic erythropoiesis-stimulating agent (ESA), were characterized. Hematide was given intravenously (0, 0.5, 5, and 50 mg/kg) weekly for five weeks with a 6- (rat) and 12-week (monkey) recovery period. The pharmacological action of Hematide resulted in polycythemia. Histopathology consistent with drug-induced exaggerated pharmacology was observed primarily in rats. Secondary sequelae resulting from pronounced polycythemia was considered the cause of deaths in rats and a single high-dose monkey. Toxicokinetic analysis indicated prolonged exposure. In conclusion, Hematide is a potent ESA and the safety and efficacy profile support clinical development.

Assessment of retinal degeneration in outbred albino mice.

Evaluation of a pharmaceutical's safety includes assessment of the potential for ophthalmologic toxicity. These nonclinical studies commonly use various outbred stocks of mice. Pretest indirect ophthalmoscopic examinations in the commonly used outbred stock Hsd:ICR(CD-1) indicated that retinal degeneration was a problem in this particular outbred stock of mice. This prompted the authors to examine other stocks of outbred mice routinely used in the performance of nonclinical safety studies. Groups of mice were observed over a 13-week period to determine the progression and changing incidence of retinal degeneration. Light intensity in the room and caging was measured during the study, and it was determined that light did not play a direct role in the progression of the retinal degeneration observed during the study. Histomorphologic examination of the mouse eyes was performed at the end of the study to confirm the presence of retinal degeneration observed after ophthalmoscopic examination. The incidence of retinal atrophy in the various outbred stocks of mice was: Crl:CFW(SW)BR (98.3%), Tac(SW)fBR (80%), Tac:Icr:Ha(ICR)fBR (75%), Hsd:ICR(CD-1) (43.3%), and Crl:CF-1BR (3.0%). Retinal atrophy was not observed in the following outbred mice stocks: Crl:CD-1(ICR)BR, HsdWin:CFW1, and Hsd:NSA(CF-1). On the basis of these findings, it is highly recommended that pretest ophthalmologic screening be performed on mice to obviate pre-existing conditions from confounding or invalidating nonclinical study results.