Pre-clinical drug development of 9-aminoacridines for malignant glioma and meropenem prodrugs for drug-resistant tuberculosis

Abstract

The pre-clinical drug development of investigational molecules with encouraging in vitro efficacy require drug metabolism and pharmacokinetic experiments that ultimately characterize "what the body can do to the drug." In chapter one of this thesis, the absorption, distribution, and metabolic properties of a series of 9-amnoacridines (Acridine 1 - 4) with potent anti-proliferative activity are described by investigating their metabolic stability, cellular accumulation in MDCK cells with or without transfected efflux transporters (PgP or BCRP), plasma protein binding, and tissue accumulation in mouse pharmacokinetic studies. Additionally, acridine 2 was chosen as the lead candidate and evaluated in a mouse orthotopic glioblastoma model. Metabolic stability experiments in pooled human liver microsomes indicated slow rates of oxidation (apparent t1/2 from 2.2 - 4.1 hours) and negligible glucuronidation. In addition, acridine 1, 2, and 4 accumulated in MDCK-WT cells with values 2.7, 2.2, and 4.3 times greater than propranolol. The accumulation of acridine 3 was equal to that of propranolol. In accumulation experiments with MDCK-MDR cells, it was discovered that acridine 1 and 3 were moderate substrates for Pgp whereas acridine 2 and 4 were not substrates. Accumulation experiments with MDCK cells overexpressing BCRP identified acridine 1, 3, and 4 as substrates for BCRP, but not 2. Interestingly, it was discovered that the 9-aminoacridines were substrates for the organic cation transporter (OCT). A mouse pharmacokinetic study following a 60 mg/kg oral dose with Acridine 1 and 2 demonstrated low penetration into the brain (Cmax = 0.25 uM and 0.6 uM), but high uptake in kidney (Cmax = 30 uM and 300 uM) and liver (Cmax = 125 uM and 225 uM) relative to total peak concentrations in plasma (Cmax = 2.25 uM and 20 uM). Subsequently, an intravenous pharmacokinetic study with Acridine 2 following a 15 mg/kg dose produced peak concentrations in the brain (1.7 uM), kidney (212 uM), and liver (78 uM) at 2.0 hours relative to a 2.0 uM peak concentration in the plasma. Acridine 2 bioavailability was 83.8%. Acridine 2 significantly increased the median survival of mice in an orthotopic glioblastoma model suggesting compounds in this series may offer new strategies for the design of chemotherapeutics for treating brain cancers with high oral bioavailability and improved efficacy. Chapter 2 of this thesis describes the synthesis and evaluation of meropenem prodrugs for extensively drug-resistant tuberculosis (XDR-TB). Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of bacterial infectious disease mortality worldwide. Due to the emergence of multi- and extensively drug-resistant strains of TB, new chemical agents are desperately needed. Although B-lactams are the most widely used group of antibiotics, they have never been systematically utilized in TB therapy due to their poor penetration of the mycobacterial cell wall as well as their inactivation by the chromosomally encoded B-lactamase (BlaC). In 2009, Hugonnet et al. shattered this long held dogma demonstrating that meropenem and clavulanate were highly effective against (XDR-TB) strains in vitro. Due to meropenem's short half-life (1 hr) and lack of oral bioavailability, the synthesis and evaluation of meropenem prodrugs as potential therapeutics for the treatment of TB are described. The initial approach to improving the oral bioavailability of meropenem was to synthesize the isopropoxycarbonyloxymethyl (proxetil) ester of meropenem. Second generation prodrugs with more lipophilic promoieties (benzosuberyl, tetralyl, and indanyloxycarbonyloxymethyl) were subsequently synthesized as well as a simple benzyl ester derivative. The aqueous stability of prodrugs at biologically relevant pH 1.2 (stomach), 6.0 (intestinal), and 7.4 (blood) is reported in terms of prodrug half-life or percent remaining after a 2-hour incubation at 37 °C. The most stable prodrug at pH 7.4 and 6.0 was the proxetil ester of meropenem (6 - 11% degradation over 2 hours); however, the prodrugs containing the 1-benzosuberyl, 1-tetralyl, and 1-indanyloxycarbonyloxymethyl promoieties were all unstable in aqueous solution at pH 7.4 (half-lives of 139, 5.8 and < 1 min, respectively) and resulted in the formation of a racemic alcohol indicative of a SN1 solvolysis mechanism. The prodrugs containing the 2-benzosuberyl, 2-tetralyl, and 2-indanyloxycarbonyloxymethyl promoieties were significantly more stable at physiological pH 7.4 and intestinal pH 6.0 (20 - 30% degradation in 2 hours) as a result of carbonate attachment at the 2-poistion and not the benzylic position. The plasma stability of the most aqueous stable prodrugs was very short (1 - 6 min) implying a rapid release of the parent compounds. Interestingly, the simple benzyl ester derivative had the longest stability (8 min) in plasma compared to all the other prodrugs. Experiments to determine the relative bioavailability of meropenem were conducted with the proxetil ester of meropenem as well as the 1-(S), 1-(R), and racemic 2- benzosuberyloxycarbonyloxymethyl prodrugs in jugular vein catheterized guinea pigs. The bioavailabilities were calculated to be 1.9%, 6.2%, 4.4%, and 5.9%, respectively.