Karen L. Steinmetz

The basics of preclinical drug development for neurodegenerative disease indications.

Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drugs mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimers disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.

Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes

Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded less than 0 NG. Treatment with benzo[a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole-O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: evaluation of 24 drug candidates.

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 24 drug candidates which were tested as coded compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of biological activity in a variety of preliminary drug efficacy screens. None of the compounds reported here produced an increase in UDS, and therefore, the UDS results with these materials do not suggest potential for mutagenesis or carcinogenesis.

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.

Evaluation of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) following treatment of rats and mice with p‐dichlorobenzene

p-Dichlorobenzene (PDCB) has been reported to produce tumors in the male and female mouse liver and in the male rat kidney in 2-year gavage studies (NPT, 1987). To elucidate the possible mechanisms of carcinogenicity more fully, UDS and RDS were evaluated in B6C3F1 mouse hepatocytes and F-344 rat kidney cells by autoradiography following in vivo administration of PDCB. All corn oil gavage doses of PDCB (300, 600, and 1,000 mg/kg) and the negative control resulted in < 0 net grains/nucleus (NG) in the mouse liver and rat kidney, indicating that PDCB does not induce UDS in either tissue. Compared to controls with < or = 0.29% hepatocytes in S-phase (%S), treatment of mice induced 0.46, 1.90, and 1.52 %S (males) and 2.61, 1.18, and 4.45 %S (females), which indicates that PDCB acts as an inducer of cell proliferation in the liver. In male rat kidney cells, the same doses produced 0.87, 0.67, and 1.01 %S (0.38% in controls) and in females 0.48, 0.43, and 0.32 %S (0.52% in controls), indicating that PDCB induces cell replication in the male but not the female rat kidney. Therefore, these data demonstrate that PDCB is not genotoxic in the mouse liver or rat kidney at single oral doses comparable to the daily doses given in the National Toxicology Program (NTP) bioassay (NTP, 1987). Furthermore, the increases in RDS support the hypotheses that mouse liver tumor formation occurs via stimulation of hepatocyte proliferation and male rat kidney carcinogenesis via increased renal cell proliferation.

P2-448: Preclinical evaluation of CDD-0102, a selective M1 agonist with potential utility in Alzheimer's disease

Background: Agonists that selectively activate M1 muscarinic receptors might be useful in treating memory and cognitive deficits, while preventing the formation of amyloid plaques and neurofibrillary tangles associated with Alzheimer’s disease. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102J) displays functional selectivity for M1 receptors and enhances memory function in animals with cholinergic deficits. The hydrochloride salt (CDD-0102A) also promotes alpha-secretase activity and reverses the apoptotic effects of Abeta in differentiated PC12 cells. Methods: CDD-0102A was evaluated in a series of studies to determine pharmacokinetic parameters, drug metabolism and toxicity. Results: Preclinical toxicology studies indicate that CDD-0102A is negative in bacterial mutagenicity, mammalian cell clastogenicity, and mouse micronucleus assays. Moreover, CDD-0102A does not inhibit the HEK-hERG channel current. In a 28-day repeat-dose toxicity study with CDD-0102A in male and female rats, excessive cholinergic stimulation was apparent at high doses yet no overt toxicities were observed. Dogs were more sensitive to CDD-0102A in a comparable study with dose-dependent increased frequency and severity of cholinergic symptoms, although no significant effects on cardiovascular and pulmonary safety parameters were observed. Studies in rats yielded linear pharmacokinetics with dose proportional increases in Cmax and AUC and an estimated t1/2 of 3-5 hr. Comparative drug metabolism studies indicated significant metabolism only in rabbit S9 preparations, with limited metabolism in liver microsomal and S9 preparations from mice, rats, dogs, monkeys and humans. Three potential metabolites were identified from rabbit liver microsomes. Conclusions: The preclinical data are being used in planning Phase I clinical studies of CDD-0102A in healthy adult volunteers. Taken together, the data suggest that CDD-0102A may be useful in treating the symptoms and some of the underlying causes of Alzheimer’s disease.

Neurological assessments after treatment with the antimalarial β-arteether in neonatal and adult rats

The World Health Organization currently recommends combinatorial treatment including artemisinins as first-line therapy against drug-resistant Plasmodium falciparum malaria. Although highly efficacious, artemisinin and its derivatives, including β-arteether (βAE), are associated with ototoxicity, tremors, and other autonomic and motor impairments in the clinic. Similar neurological symptoms, as well as brainstem lesions, have been observed in adult laboratory species (mice, rats, dogs, and non human primates) following acute treatment with βAE; however, few long-term, nonclinical studies have been conducted. Furthermore, the majority of deaths attributed to malarial infection occur in children under age five, yet no laboratory studies have been initiated in neonatal or juvenile animals. In the current study, neonatal 7-day-old rats were administered intramuscular doses of 1-90mg/kg βAE in sesame oil for up to eight treatment cycles (one cycle=7days treatment+7days without treatment). Neonates were tested for changes in sensorimotor function, and the same animals were tested as adults in the Functional Observational Battery, for motor activity, and in the 8-arm radial maze. Pups receiving a single cycle of 60 or 90mg/kg died within a week of treatment but had few behavioral changes and no brainstem pathology. In the long-term study, behavioral and motor changes and brainstem lesions were observed in a dose- and time-related manner. Rats given repeated cycles of 1 or 5mg/kg βAE showed subtle motor abnormalities (e.g., slight loss of righting reflex) while repeated cycles of 10mg/kg βAE treatment resulted in obvious motor and behavioral changes. Rats receiving 1mg/kg βAE had no brainstem lesions whereas some rats treated with 5mg/kg βAE and all rats treated with 10mg/kg βAE had brainstem lesions. Brainstem lesions were observed after as few as five cycles and were characterized by gliosis, satellitosis and progressive necrosis in motor neurons of the trapezoid, vestibular, and olivary nuclei. This study shows that repeated treatment with clinically relevant doses of βAE causes motor deficits associated with brainstem damage in rodents and suggests that repeated treatment with βAE in children may elicit neurological damage.

Analysis of Unscheduled Dna Synthesis and S-Phase Synthesis in F344 Rat Kidney After in Vivo Treatment with Mercuric Chloride

A variety of short-term test have been developed to predict the outcome of carcinogenicity bioassays. Unfortunately, the majority of these do not address the issue of tissue-specificity. Tests that focus on tissuespecific responses such as the in vivo — in vitro unscheduled DNA synthesis (UDS) and S-phase synthesis (SPS) assays (Mirsalis, et al., 1985; Mirsalis, 1987) have been reasonably successful in the prediction of hepatocarcinogenic potential. The most widely used system employs hepatocyte cultures derived from animals treated in vivo (Mirsalis and Butterworth, 1980); however, systems for the kidney (Tyson and Mirsalis, 1985; Loury et al., 1987), pancreas (Steinmetz and Mirsalis, 1984), trachea (Doolittle and Butterworth, 1984), stomach (Furihata et al., 1984), and spermatocytes (Working and Butterworth, 1984) have recently been developed.