Preclinical Pharmacokinetic Profile of Topical Ophthalmic and Intravenous Delivery of QLS-101, a Novel ATP-Sensitive Potassium Channel Opening Ocular Hypotensive Agent.

Purpose: To evaluate the pharmacokinetic profiles of the ocular hypotensive agent QLS-101, a novel ATP-sensitive potassium channel opening prodrug, and its active moiety levcromakalim, following topical ophthalmic and intravenous dosing of normotensive rabbits and dogs. Methods: Dutch belted rabbits (n = 85) and beagle dogs (n = 32) were dosed with QLS-101 (0.16-3.2 mg/eye/dose) or formulation buffer for 28 days. Pharmacokinetic profiles of QLS-101 and levcromakalim were evaluated in ocular tissues and blood by LC-MS/MS. Tolerability was assessed by clinical and ophthalmic examinations. Maximum systemic tolerated dose was evaluated in beagle dogs (n = 2) following intravenous bolus administrations of QLS-101 (0.05 to 5 mg/kg). Results: Plasma analysis following topical dosing of QLS-101 (0.8-3.2 mg/eye/dose) for 28 days indicated an elimination half-life (T1/2) of 5.50-8.82 h and a corresponding time (Tmax) range of 2-12 h in rabbits, and a T1/2 of 3.32-6.18 h with a Tmax range of 1-2 h in dogs. Maximum tissue concentration (Cmax) values ranged from 54.8-540 (day 1) to 50.5-777 ng/mL (day 28) in rabbits, and 36.5-166 (day 1) to 47.0-147 ng/mL (day 28) in dogs. Levcromakalim plasma T1/2 and Tmax were similar to QLS-101, while Cmax was consistently lower. Topical ophthalmic delivery of QLS-101 was well tolerated in both species, with sporadic mild ocular hyperemia noted in the group treated with the highest concentration (3.2 mg/eye/dose). Following topical ophthalmic dosing, QLS-101 and levcromakalim were found primarily in the cornea, sclera, and conjunctiva. Maximum tolerated dose was determined to be 3 mg/kg. Conclusions: QLS-101 was converted to its active moiety levcromakalim and showed characteristic absorption, distribution, and safety profiles of a well-tolerated prodrug.

Ocular Hypotensive Properties and Biochemical Profile of QLS-101, a Novel ATP-Sensitive Potassium (KATP) Channel Opening Prodrug.

Purpose: To characterize the ocular hypotensive and pharmacological properties of QLS-101, a novel ATP-sensitive potassium (KATP) channel opening prodrug. Methods: Ocular hypotensive properties of QLS-101 were evaluated by measuring IOP with a handheld rebound tonometer after daily topical ocular instillation of 0.2% (n = 5) or 0.4% QLS-101 (n = 10) in C57BL/6J mice. KATP channel specificity was characterized in HEK-293 cells stably expressing human Kir6.2/SUR2B subunits and assessed for off-target interactions using a receptor binding screen. Conversion of QLS-101 prodrug to its active moiety, levcromakalim, was evaluated in vitro using human ocular tissues and plasma samples and after incubation with human phosphatase enzymes (2.0 nM-1.0 µM). Results: C57BL/6J mice treated once daily with 0.2% QLS-101 exhibited significant (P < 0.01) IOP reductions of 2.1 ± 0.4 mmHg after five days; however, a daily attenuation of the effect was noted by 23h post-dose. By comparison, treatment with 0.4% QLS-101 lowered IOP by 4.8 ± 0.7 mm Hg (P < 0.0001) which was sustained for 24 hours. Unlike levcromakalim, QLS-101 failed to induce KATP channel activity in HEK-Kir6.2/SUR2B cells consistent with its development as a prodrug. No off-target receptor effects were detected with either compound. In vitro ocular tissue conversion of QLS-101 prodrug was identified in human iris, ciliary body, trabecular meshwork, and sclera. Alkaline phosphatase was found to convert QLS-101 (mean Km = 630 µM, kcat = 15 min-1) to levcromakalim. Conclusions: QLS-101 is a novel KATP channel opening prodrug that when converted to levcromakalim shows 24-hour IOP lowering after once-daily topical ocular administration.

CMX-2043 Mechanisms of Action In Vitro.

α-Lipoic acid has been shown to provide cytoprotection in some tissues through antioxidant and antiapoptotic mechanisms. We have enhanced these properties by synthetic modification, resulting in a new chemical entity, CMX-2043, with proven efficacy in an animal model of cardiac ischemia-reperfusion injury. The present studies compare cytoprotective cellular pathways of R-α-lipoic acid and CMX-2043. Biochemical and cellular assays were used to compare antioxidant potency, tyrosine kinase activation, and protein kinase B (Akt) phosphorylation. CMX-2043 was more effective than lipoic acid in antioxidant effect, activation of insulin receptor kinase, soluble tyrosine kinase, and Akt phosphorylation. Activation of insulin-like growth factor 1 receptor was similar for both. CMX-2043 stimulation of Akt phosphorylation was abolished by the phosphatidylinositide 3-kinase inhibitor LY294002. Consistent with Akt activation, CMX-2043 reduced carbachol-induced calcium overload. The S-stereoisomer of CMX-2043 was less active in the biochemical assays than the R-isomer. These results are consistent with cytoprotection through activation of Akt and antioxidant action. CMX-2043 may thus provide a pharmacological approach to cytoprotection consistent with established anti-apoptotic mechanisms.

CMX-2043 Efficacy in a Rat Model of Cardiac Ischemia-Reperfusion Injury.

α-Lipoic acid (LA) has been shown to offer protection against ischemia-reperfusion injury (IRI) in multiple organ systems. N-[(R)-1,2-dithiolane-3-pentanoyl]-L-glutamyl-L-alanine (CMX-2043), a novel analogue of LA, was studied as part of a preclinical development program intended to identify safe and efficacious drug candidates for prevention or reduction in myocardial IRI. This study was designed to evaluate the efficacy of CMX-2043 in an animal model of myocardial IRI and to establish effective dosing conditions. CMX-2043 or placebo was administered at different doses, routes, and times in male Sprague-Dawley rats subjected to 30-minute left coronary artery ligation. Fluorescent microsphere injection defined the area at risk (AR). Animals were euthanized 24 hours after reperfusion, and the hearts were excised, sectioned, and stained with triphenyltetrazolium. Cytoprotective effectiveness was determined by comparing the unstained myocardial infarction zone (MI) to the ischemic AR. The reduction in the MI-AR ratio was used as the primary measure of drug efficacy relative to placebo injections. Treatment with CMX-2043 reduced myocardial IRI as measured by the MI-AR ratio and the incidence of arrhythmia. The compound was effective when administered by injection, both before and during the ischemic injury and at reperfusion. The most efficacious dose was that administered 15 minutes prior to the ischemic event and resulted in a 36% (P < .001) reduction in MI-AR ratio compared to vehicle control.

Pre-clinical and Clinical Safety Studies of CMX-2043: a cytoprotective lipoic acid analogue for ischaemia-reperfusion injury.

CMX-2043 is an α-lipoic acid analogue targeted to reduction of cellular injury and organ damage due to ischaemia-reperfusion injury (IRI). It has been shown to be effective in a rat model of cardiac IRI. The studies here reported evaluate its safety and pharmacokinetic profile in preparation for human clinical studies in procedures associated with IRI. Safety and tolerability were tested in standard pre-clinical in vitro and animal models and in a Phase 1 human clinical trial. CMX-2043 did not bind to a wide range of receptors and specific targets at approximately 4 µg/mL (10 µM). It was not mutagenic by Ames assay, did not produce chromosome aberrations in Chinese hamster ovary (CHO) cells, and was negative for clastogenic potential. Toxicological studies in rats including both single and 14-day repeat intravenous doses and in dogs (single intravenous dose) with a 2-week recovery period were conducted. The NOAEL in rats and dogs was 30 and >10 mg/kg, respectively. No serious adverse events were reported in a placebo-controlled, sequential dose escalation Phase 1 clinical trial. The low toxicity in the pre-clinical studies and the absence of adverse events in the Phase 1 trial have supported investigation of CMX-2043 in a human efficacy trial.

Lipoic acid analogs with enhanced pharmacological activity.

Lipoic acid (1,2-dithiolane-3-pentanoic acid) is a pharmacophore with unique antioxidant and cytoprotective properties. We synthesized a library based upon the condensation of natural and unnatural amino acids with the carboxylic acid moiety of lipoic acid. SAR studies were conducted using a cardiac ischemia-reperfusion animal model. Cytoprotective efficacy was associated with the R-enantiomer of the dithiolane. Potency of library compounds was dictated by the acidic strength of the adduct. α-N-[(R)-1,2-dithiolane-3-pentanoyl]-L-glutamyl-L-alanine, designated CMX-2043, was chosen for further pharmacologic evaluation.

Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides.

The transactivator responsive region (TAR) present in the 5'-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA-protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNA(TAR)) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418-428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNA(TAR)-peptide conjugates where uptake of the PNA(TAR)-penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC50 values in the range of 28-37 nM while IC50 for inhibition of HIV-1 replication was lowest with PNA(TAR)-transportan-27 (0.4 microM) followed by PNA(TAR)-tat (0.72 microM) and PNA(TAR)-penetratin (0.8 microM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.

A PNA-transportan conjugate targeted to the TAR region of the HIV-1 genome exhibits both antiviral and virucidal properties.

We have earlier reported that anti-TAR PNA conjugated with the membrane-transducing peptide transportan inhibits transactivation of the HIV-1 LTR resulting in decreased production of HIV-1 virions by chronically infected H9 cells (N., Kaushik, A., Basu, P., Palumbo, R.L., Myers, V.N., Pandey, 2002. Anti-TAR polyamide nucleotide analog conjugated with a membrane permeating peptide inhibits HIV-1 production. J. Virol. 76, 3881-3891). In this study, we have found that the PNA(TAR)-transportan conjugate is efficiently internalized by cells and kinetics analysis reveals a sigmoidal curve with a cooperativity index of 6, indicating very rapid cellular uptake. Additionally, analysis of uptake at varying temperatures or in the presence of phenylarsine oxide revealed that the mechanism of uptake is neither receptor-dependent nor occurs via endocytosis. We also found that the PNA(TAR)-transportan conjugate exhibits potent virucidal activity as HIV-1 virions pretreated with the conjugate were rendered noninfectious, suggesting that the conjugate may also permeate the virus envelope. The anti-HIV-1 virucidal activity of the conjugate may be useful either in topical formulations designed to block HIV-1 infection or as a prophylactic agent for inactivation of HIV-1 in the circulating plasma prior to attachment and entry.