Development and Preclinical Evaluation of New Inhaled Lipoglycopeptides for the Treatment of Persistent Pulmonary Methicillin-Resistant Staphylococcus aureus Infections.

Chronic pulmonary methicillin-resistant Staphylococcus aureus (MRSA) disease in cystic fibrosis (CF) has a high probability of recurrence following treatment with standard-of-care antibiotics and represents an area of unmet need associated with reduced life expectancy. We developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells, the latter of which have been linked to clinical antibiotic failure. A library of next-generation potent lipoglycopeptides was synthesized with an emphasis on attaining superior pharmacokinetics (PK) and pharmacodynamics to similar compounds of their class. Our strategy focused on hydrophobic modification of vancomycin, where ester and amide functionality were included with carbonyl configuration and alkyl length as key variables. Candidates representative of each carbonyl attachment chemistry demonstrated potent activity in vitro, with several compounds being 30 to 60 times more potent than vancomycin. Selected compounds were advanced into in vivo nose-only inhalation PK evaluations in rats, where RV94, a potent lipoglycopeptide that utilizes an inverted amide linker to attach a 10-carbon chain to vancomycin, demonstrated the most favorable lung residence time after inhalation. Further in vitro evaluation of RV94 showed superior activity to vancomycin against an expanded panel of Gram-positive organisms, cellular accumulation and efficacy against intracellular MRSA, and MRSA biofilm killing. Moreover, in vivo efficacy of inhaled nebulized RV94 in a 48 h acute model of pulmonary MRSA (USA300) infection in neutropenic rats demonstrated statistically significant antibacterial activity that was superior to inhaled vancomycin.

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors.

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11beta-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC(50) values of 1.6+/-0.1 and 9.9+/-0.9 nM (mean+/-SEM, n=3-6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K(i) values of 0.8+/-0.04 and 2.2+/-0.2 nM (n=3-4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.