Pre-Clinical Research

Animal Safety Pharmacology and Toxocology

AEOL 10150 has been evaluated for acute toxicity by the subcutaneous route in mice and rats and by the intravenous route in mice, rats and monkeys. Repeat dose toxicity studies by the subcutaneous route were conducted in mice and monkeys. Studies of genotoxic potential both in vitro and in vivo were conducted. Safety pharmacology studies were conducted evaluating cardiovascular and pulmonary parameters (in the monkey and the rat), central nervous system effects and behavior (in the mouse), and in vitro effects on the hERG ion channel and rabbit Purkinje fiber action potential duration. The toxicology studies and safety pharmacology studies were conducted by MDS, except for the monkey cardiovascular (CV)/pulmonary safety pharmacology study (MPI), the mouse 7-day IV infusion toxicology study (Inveresk), and the acute intrathecal toxicology study (Northern Biomedical). The findings of these studies are summarized in Table 10 at the end of this subsection. With the exception of exploratory and dose range-finding studies, all toxicology and safety pharmacology studies were conducted in compliance with U.S. FDA Good Laboratory Practice Regulations and/or OECD Principles of Good Laboratory Practice.

Slight decreases in RBC parameters were observed in both mouse and monkey 4-week studies, but bone marrow smears were normal. In the ongoing monkey recovery study, these RBC changes were reversible. Measures of fecal occult blood levels from treated animals were similar to controls. In short-term in vitro experiments, spiking of blood samples with concentrations of up to 80 µg/mL AEOL 10150 was without effect on hematologic (and also on clinical chemistry) measurements.

AEOL 10150 was genotoxic in both the in vitro Ames and mouse lymphoma tests but not in the in vivo mouse micronucleus test. Under conditions of high oxygen tension (atmospheric oxygen tension is 160 mm Hg), as in the in vitro assays, AEOL 10150 is able to catalyze the conversion of molecular oxygen to hydrogen peroxide, a known mutagen. This reaction is less likely to occur under the low oxygen tensions that prevail in the body (most tissue oxygen tensions are ca. 40-45 mm Hg). Abundant peroxidases also make in vivo mutagenicity by hydrogen peroxide less likely.

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AEOL 10150 has not to date been tested for efficacy in animal models of Parkinson’s disease (PD). The rationale for not directly pursuing AEOL 10150 for the possible treatment of PD is based upon recently developed data with other compounds from the Aeolus library. One of Aeolus’ pipeline compounds, AEOL 11207, has been examined preliminarily in an MPTP mouse model of PD and was found to decrease the loss of dopamine producing cells in the substantial nigra by 25% (Panel 1). AEOL 11207 has significant ability to scavenge hydrogen peroxide in vitro and can blunt hydrogen peroxide production elicited from rat brain mitochondria from MPP+ treatment (Panel 2). AEOL 11207, one of 30 compounds currently under investigation by the Company under its Pipeline Initiative, has blood brain permeability and oral bioavailability.

Downlaod Table: Summary of No-Observable-Adverse-Effect-Levels in Repeat-Dose Studies

The Comparison of Doses by Subcutaneous Route Table (below) illustrates the NOAEL and exposure in the mouse 4-week study and the exposure at the lowest level tested in the monkey 4-week study, 5 mg/kg/day, a dose that produced CV effects and lowered body temperature, but no other findings

Downlaod Table: Comparison of Doses by Subcutaneous Route

Absorption, Distribution, Metabolism and Excretion

The nonclinical absorption, distribution, metabolism and excretion (ADME) of AEOL 10150 was evaluated in the two species used in the pivotal toxicity studies, the mouse and cynomolgus monkey. In addition, an in vitro study of the metabolic stability and potential metabolic profile of AEOL 10150 in mouse, rat, dog, monkey and human hepatocytes was conducted at MDS. This section includes an overall summary of the ADME and pharmacokinetic properties of AEOL 10150, as well as specific details of the objectives, methods, results, and conclusions from each study.

The intended route of administration of AEOL 10150 in patients is by subcutaneous (SC) injection. Thus, the 7-day dose ranging studies in the monkey and the 28-day toxicity studies in mouse and monkey were performed using daily SC doses (in-life conducted at MDS, bioanalytical at BASi). In addition, several pharmacokinetic (PK) and tissue distribution studies were performed in mice using various routes of administration (in-life at Research Triangle Institute or academic laboratories, bioanalysis at LC Resources). Studies are summarized in Table 12.

Metabolic Stability of AEOL 10150

AEOL 10150 was shown to be metabolically stable in the presence of mouse, rat, dog, monkey, and human hepatocytes indicating a low potential for hepatic metabolism by cytochrome P450s or by conjugation reactions.

Species Comparison of the Metabolic Stability and Metabolite Profile of AEOL 10150 Using Mouse, Rat, Dog, Monkey and Human Primary Hepatocytes.

Cryopreserved (dog, monkey, and human) or freshly isolated (mouse and rat) hepatocytes were incubated with 1 or 10 µM AEOL 10150 for 4 hours and parent compound and formation of potential metabolites were monitored using an LC/MS/MS assay.

No measurable loss of unchanged AEOL 10150 or appearance of metabolites was observed in any of the hepatocyte incubations. In contrast, parallel incubation with positive control substrates for Phase I and Phase II metabolism showed the expected biotransformation patterns, thus confirming the viability of the test systems.

This study concluded that AEOL 10150 is metabolically stable in vitro in the presence of mouse, rat, dog, monkey, and human hepatocytes and, therefore, the typical metabolic patterns for drugs that are catalyzed by cytochrome P450s, or by conjugation reactions, may not be important determinants of the disposition of AEOL 10150.

ADME and Pharmacokinetics of AEOL 10150 in the Mouse (AA-TX-010, Table 13)

AEOL 10150 was administered by the intravenous, intraperitoneal, oral, intratracheal, and subcutaneous (SC) routes to characterize the absorption, distribution, and elimination profile of the drug in the mouse. The following conclusions were reached from the results of several studies:

• AEOL 10150 displays a biphasic pharmacokinetic profile in the mouse following intravenous administration with a terminal phase plasma half-life of 6.6 hours.
• AEOL 10150 is rapidly absorbed after intraperitoneal administration with peak concentrations in plasma, spinal cord and brain at 20 minutes, but concentrations in brain and spinal cord were 10-fold to 30-fold lower, respectively than those in plasma. AEOL 10150 accumulated in the liver with concentrations continuing to rise through two hours after dosing.
• The oral bioavailability of AEOL 10150 is low in the mouse based upon plasma and tissue concentrations obtained following gavage administration.
• Intratracheal administration in mice provided high lung and plasma concentrations of AEOL 10150 as well as high concentrations in liver tissue. The half-life in plasma was 0.78 hours and in the lungs was 33 hours.
• Twenty-four hour intravenous administration of AEOL 10150 by osmotic minipump provided high concentrations in plasma and in highly perfused organs including liver, kidneys, and lungs. Concentrations in plasma reached steady-state, but continued to rise in liver and kidneys. Concentrations in heart were lower than in the other tissues. Measurable, but low, concentrations were observed in brain.
• Conclusions from the 4-week study of daily subcutaneous doses in mice were that plasma concentrations and systemic exposure were approximately dose proportional and that systemic exposure was slightly higher in males than in females. Calculated half-lives were longer after four weeks of dosing than after a single dose but there was no indication of excessive accumulation based on AUC values.

Pharmacokinetics of AEOL 10150 in the Cynomolgus Monkey

The pharmacokinetic profile of AEOL 10150 in the cynomolgus monkey was evaluated in two multiple dose studies. In a 7-day dose ranging study, monkeys received daily subcutaneous doses of 15, 40, 50, 65 and 90 mg/kg/day and in a 28-day study daily doses of 5, 15, and 40 mg/kg were administered subcutaneously.

For the 7-day dose ranging study (AA-TX-009):

• Tmax values ranging from 0.5 to 1.0 hr indicated that AEOL 10150 was rapidly absorbed in the monkey following SC administration.
• The mean Cmax values generally increased proportionally to the increase in dose on both Day 0 and Day 6.
• The mean t½ values were quite consistent across the dosing groups on both Day 0 (range 3.4 to 4.4 hrs) and Day 6 (mean 4.2 to 5.1 hrs).
• For Day 0, AUC0 inf values increased nearly proportionately when the dose was increased from 15 to 90 mg/kg/day and on Day 6, the increase in AUC0 t was also approximately proportional to dose.
• Both the Cmax and AUC data indicate that the accumulation of AEOL 10150 in the monkey during multiple dosing is predictable and not excessive based upon the half-life and dosing interval of the drug.

For the 4-week subcutaneous toxicity study (AA-TX-011, Table 15):

• Across the dose range studied (5 to 40 mg/kg), plasma concentrations and systemic exposure were linearly dose proportional.
• Systemic exposure was slightly higher in males than in females, possibly due to differences in clearance rather than absorption.
• Calculated half-lives were longer after four weeks of dosing than after a single dose but there was no indication of accumulation based on AUC values.
• The appearance of the plasma concentration-time profiles indicates that AEOL 10150 displays multicompartmental pharmacokinetics in monkeys.

Downlaod Table: Summary of ADME Studies