Richard M. Fitch

Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats

OBJECTIVE The present study was to examine whether endogenous nitric oxide (NO) plays a role in the regulation of vascular stiffness. METHODS Pulse wave velocity (PWV) was determined as the time delay between the foot of pressure waves recorded simultaneously at the aortic arch and abdominal aorta (just above the bifurcation) in anesthetized Sprague-Dawley rats. A decrease in vascular compliance results in an increase in PWV. RESULTS A bolus injection of a NO synthase inhibitor, L-NAME (30 mg/kg), significantly increased PWV, accompanied by an increase in blood pressure. Since changes in blood pressure are known to affect PWV, phenylephrine (PE) was administered to mimic the blood pressure changes induced by L-NAME, thus compensating for the pressure-dependent component of the PWV changes. At each given level of mean arterial pressure (MAP), PWV was significantly higher with L-NAME than with PE treatment, suggesting that acute withdrawal of endogenous NO reduces aortic compliance independent of changes in MAP. In rats chronically treated with L-NAME (0.5 g/l in drinking water) for 3 weeks, PWV was even higher than those acutely treated with L-NAME (at MAP=150 mmHg). This additional increase in vascular stiffness may be due to the remodeling of the vascular wall as a result of chronic NOS inhibition and hypertension. CONCLUSION These data demonstrate that NO modulates vascular compliance independent of blood pressure changes and that an intact endogenous NO system is required to maintain normal vascular compliance.

Hydrophilic pyrazine dyes as exogenous fluorescent tracer agents for real-time point-of-care measurement of glomerular filtration rate.

Various hydrophilic pyrazine-bis(carboxamides) derived from 3,5-diamino-pyrazine-2,5-dicarboxylic acid bearing neutral and anionic groups were prepared and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, the dianionic d-serine pyrazine derivatives 2d and 2j, and the neutral dihydroxypropyl 2h, exhibited favorable physicochemical and clearance properties. In vitro studies show that 2d, 2h, and 2j have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that these three compounds exhibit a plasma clearance equivalent to iothalamate (a commonly considered gold standard GFR agent). In addition, these compounds have a higher urine recovery compared to iothalamate. Finally, the plasma clearance of 2d, 2h, and 2j remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration only. Hence, 2d, 2h, and 2j are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

A novel P2Y12 adenosine diphosphate receptor antagonist that inhibits platelet aggregation and thrombus formation in rat and dog models

Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y(12) receptor antagonist, BX 667. The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y(12) receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats, we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis. BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl(2), both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model. Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination. These data demonstrate that the reversible P2Y(12) receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.

Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

Increased nitric oxide accounts for decreased basal vascular tone and responsiveness in the resistance vessels of high-cholesterol-fed rabbits.

The objective of this study was to determine the effects of hypercholesterolemia on basal vascular tone and vascular responses to pharmacologic agents in hindquarter resistance vessels. Blood pressure and hindquarter blood flow were measured in conscious rabbits fed a high cholesterol diet (1%) for 17 weeks (HC) compared to age-matched rabbits fed a normal diet (control). Basal hindquarter blood flow and vascular conductance were significantly higher in HC than in control rabbits. Administration of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 mg/kg) decreased basal hindquarter blood flow and vascular conductance in a greater magnitude in HC than in control rabbits, thus, abolished the differences in both the flow and conductance between 2 groups, indicating that increased NO was responsible for reduced basal vascular tone in the HC rabbits. L- (30 mg/kg), a selective inducible NOS (iNOS) inhibitor had no effects on either flow or conductance. This result does not support the involvement of iNOS. In separate experiments, animals were anesthetized and instrumented with an extracorporeal circuit to measure perfusion pressure under constant blood flow to the hindquarter vascular bed. In the HC group, vascular responses to acetylcholine, S-nitroso-N-acetyl-penicillamine and phenylephrine were all attenuated when compared to the responses in the control rabbits. These results indicate that local overproduction of NO due to hypercholesteremia could desensitize smooth muscle reactivity, thus causing general vascular hyporesponsiveness to vasoactive agents.

Abstract 4400: Anti-tumor activity of liposomal docetaxel prodrug MNK-010 on PC3 human prostate xenografts in mice

The anti-mitotic taxanes are one of the most important families of anticancer drugs that have broad anti-tumor activity and are widely used for a variety of human cancer types. However, the taxanes are limited by a number of serious pharmacological and toxicological effects, including dose limiting myelosuppression, neurotoxicity and serious hypersensitivity reactions. We are developing a unique delivery system for docetaxel, comprised of a PEGylated liposomal nanoparticle containing a prodrug of docetaxel to improve solubility, tolerability and increase efficacy through improved pharmacokinetics and biodistribution. In this study we evaluated the antitumor activity of MNK-010 on the growth of established human PC3 xenografts in male immunodeficient mice. This study tested the hypothesis that MNK-010 can provide greater efficacy than Taxotere (docetaxel) at equivalent maximum tolerated doses (MTD) in mice implanted with human PC3 tumor cells. Male nude mice bearing PC3 human prostate xenografts were given two or four intravenous (IV) doses of MNK-010, Taxotere or saline. Dosing intervals were twenty-one days for two cycles or every four days for four cycles. The doses of Taxotere and MNK-010 were based on maximum tolerated dose (MTD) or highest dose tested for a given dose interval. Tumor volume was measured twice per week using the Biopticon tumor imaging system and tumor volume data was analyzed to determine tumor growth delay and partial tumor regression. Survival analysis was conducted and median survival time determined. MNK-010 significantly (p Citation Format: Richard M. Fitch, Jolette K. Wojdyla, James A. Blackledge, William D. McGhee. Anti-tumor activity of liposomal docetaxel prodrug MNK-010 on PC3 human prostate xenografts in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2015-4400

Real-time point-of-care measurement of impaired renal function in a rat acute injury model employing exogenous fluorescent tracer agents

Renal function assessment is needed for the detection of acute kidney injury and chronic kidney disease. Glomerular filtration rate (GFR) is now widely accepted as the best indicator of renal function, and current clinical guidelines advocate its use in the staging of kidney disease. The optimum measure of GFR is by the use of exogenous tracer agents. However current clinically employed agents lack sensitivity or are cumbersome to use. An exogenous GFR fluorescent tracer agent, whose elimination rate could be monitored noninvasively through skin would provide a substantial improvement over currently available methods. We developed a series of novel aminopyrazine analogs for use as exogenous fluorescent GFR tracer agents that emit light in the visible region for monitoring GFR noninvasively over skin. In rats, these compounds are eliminated by the kidney with urine recovery greater than 90% of injected dose, are not broken down or metabolized in vivo, are not secreted by the renal tubules, and have clearance values similar to a GFR reference compound, iothalamate. In addition, biological half-life of these compounds measured in rats by noninvasive optical methods correlated with plasma derived methods. In this study, we show that this noninvasive methodology with our novel fluorescent tracer agents can detect impaired renal function. A 5/6th nephrectomy rat model is employed.

N-alkylated aminopyrazines for use as hydrophilic optical agents

Rapid assessment of glomerular filtration rate (GFR), which measures the amount of plasma filtered through the kidney within a given time, would greatly facilitate monitoring of renal function for patients at the bedside in the clinic. In our pursuit to develop exogenous fluorescent tracers for real-time monitoring of renal function by optical methods, N-alkylated aminopyrazine dyes and their hydrophilic conjugates based on poly (ethylene glycol) (PEG) were synthesized via reductive amination as the key step. Photophysical properties indicated a bathochromic shift on the order of 50 nm in both absorption and emission compared to naked aminopyrazines which could be very useful in enhancing both tissue penetration as well as easier detection methods. Structure-activity relationship (SAR) and pharmacokinetic (PK) studies, and the correlation of in vivo optical data with plasma PK for measurement of clearance (and hence GFR) are focus of the current investigation.

Abstract 5516: Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts

The anti-mitotic taxanes belong to one of the most important families of anticancer drugs that have broad anti-tumor activity and are widely used for a variety of human cancer types. However, the taxanes are limited by a number of serious pharmacological and toxicological effects. We are developing a unique delivery system for docetaxel, comprised of a PEGylated liposomal nanoparticle containing a prodrug of docetaxel to improve solubility, tolerability and increase efficacy through improved pharmacokinetics and biodistribution. In this study we evaluated the biodistribution of MNK-010, a liposomal formulation of a docetaxel prodrug in immunodeficient mice bearing A549 Human Non-Small Cell Lung Carcinoma (NSCLC) xenografts. This study tested the hypothesis that MNK-010 can produce greater plasma and tumor accumulation of docetaxel than with delivery of standard of care free docetaxel in tumor-bearing mice. Female nude mice bearing A549 human NSCLC xenografts were given a single intravenous (IV) dose of MNK-010 (40 mg/kg docetaxel molar equivalent) or free docetaxel (50 mg/kg). For each treatment group, three animals were sacrificed at various time points between 1 hr and 504 hr post injection. After euthanasia, blood, tumor, liver, kidney, spleen, lung and skeletal muscle specimens were collected. Tissue and blood samples were assayed to determine docetaxel concentrations. Administration of MNK-010 IV provided a reservoir for the continual slow release of docetaxel in the circulation and in tumors. MNK-010 increased systemic docetaxel exposure (AUC) ten-fold greater than free docetaxel injected at similar doses. MNK-010 produced sustained docetaxel levels in tumor through the 21 day observation period, resulting in four-fold greater docetaxel tumor exposure (AUC) compared to free docetaxel. The increase in MNK-010 plasma exposure as well as the enhanced permeability effect observed with liposomes likely contributed to the increased tumor exposure. In addition to improved accumulating in tumor tissue, docetaxel derived from MNK-010 also accumulated in liver, spleen and kidney. These tissues contain phagocytic cells (macrophages or mesangial) and serve as organs of elimination for chemotherapeutics. Docetaxel derived from MNK-010 was not detectable in skeletal muscle and was only found in lung over the first 24 hours post administration. In contrast, IV injection of free docetaxel produced high initial concentrations of docetaxel in all tissues evaluated, before falling quickly over time. Tumor levels decreased below levels of quantitation after nine days. This study demonstrated that Mallinckrodt Pharmaceuticals’ PEGylated liposomal docetaxel prodrug MNK-010 provided a reservoir for the continual release of docetaxel into the systemic circulation and produced an enhanced accumulation of docetaxel in A549 human NSCLC xenografts in mice compared to the standard of care docetaxel. Citation Format: Richard M. Fitch, James H. Wible, James A. Blackledge, William D. McGhee. Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2015-5516

Development of fluorescent tracers for the real-time monitoring of renal function

Accurate measurement of glomerular filtration rate (GFR) at the bedside is highly desirable in order to assess renal function in real-time, which is currently an unmet clinical need. In our pursuit to develop exogenous fluorescent tracers as GFR markers, various hydrophilic derivatives of 3,6-diaminopyrazine-2,5-dicarboxylic acid with varying molecular weights and absorption/emission characteristics were synthesized. These include polyhydroxyalkyl based small molecules and poly(ethylene glycol) (PEG) substituted moderate molecular weight compounds, which were further sub-grouped into analogs having blue excitation with green emission, and relatively longer wavelength analogs having green excitation with orange emission. Lead compounds were identified in each of the four classes on the basis of structure- activity relationship studies, which included in vitro plasma protein binding, in vivo urine recovery of administered dose, and in vivo optical monitoring. The in vivo optical monitoring experiments with lead candidates have been correlated with plasma pharmacokinetic (PK) data for measurement of clearance and hence GFR. Renal clearance of these compounds, occurring exclusively via glomerular filtration, was established by probenecid blocking experiments. The renal clearance property of all these advanced candidates was superior to that of the iothalamate, which is currently an accepted standard for the measurement of GFR.