Bruce R. Ito

Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index.

Despite efforts spanning four decades, the therapeutic potential of thyroid hormone receptor (TR) agonists as lipid-lowering and anti-obesity agents remains largely unexplored in humans because of dose-limiting cardiac effects and effects on the thyroid hormone axis (THA), muscle metabolism, and bone turnover. TR agonists selective for the TRβ isoform exhibit modest cardiac sparing in rodents and primates but are unable to lower lipids without inducing TRβ-mediated suppression of the THA. Herein, we describe a cytochrome P450-activated prodrug of a phosphonate-containing TR agonist that exhibits increased TR activation in the liver relative to extrahepatic tissues and an improved therapeutic index. Pharmacokinetic studies in rats demonstrated that the prodrug (2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811) undergoes first-pass hepatic extraction and that cleavage of the prodrug generates the negatively charged TR agonist (3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methylphosphonic acid (MB07344), which distributes poorly into most tissues and is rapidly eliminated in the bile. Enhanced liver targeting was further demonstrated by comparing the effects of MB07811 with 3,5,3′-triiodo-l-thyronine (T3) and a non-liver-targeted TR agonist, 3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid (KB-141) on the expression of TR agonist-responsive genes in the liver and six extrahepatic tissues. The pharmacologic effects of liver targeting were evident in the normal rat, where MB07811 exhibited increased cardiac sparing, and in the diet-induced obese mouse, where, unlike KB-141, MB07811 reduced cholesterol and both serum and hepatic triglycerides at doses devoid of effects on body weight, glycemia, and the THA. These results indicate that targeting TR agonists to the liver has the potential to lower both cholesterol and triglyceride levels with an acceptable safety profile.

Reduction of hepatic steatosis in rats and mice after treatment with a liver‐targeted thyroid hormone receptor agonist

Non‐alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver disease, with a prevalence ranging from 10% to 30%. The use of thyroid hormone receptor (TR) agonists for the treatment of NAFLD has not been considered viable because thyroid hormones increase free fatty acid (FFA) flux from the periphery to the liver, induce hepatic lipogenesis, and therefore could potentially cause steatosis. MB07811 is an orally active HepDirect prodrug of MB07344, a liver‐targeted TR‐β agonist. The purpose of these studies was to assess the effects of MB07811 on whole body and liver lipid metabolism of normal rodents and rodent models of hepatic steatosis. In the current studies, MB07811 markedly reduced hepatic steatosis as well as reduced plasma FFA and triglycerides. In contrast to MB07811, T3 induced adipocyte lipolysis in vitro and in vivo and had a diminished ability to decrease hepatic steatosis. This suggests the influx of FFA from the periphery to the liver may partially counteract the antisteatotic activity of T3. Clearance of liver lipids by MB07811 results from accelerated hepatic fatty acid oxidation, a known consequence of hepatic TR activation, as reflected by increased hepatic mitochondrial respiration rates, changes in hepatic gene expression, and increased plasma acyl‐carnitine levels. Transaminase levels remained unchanged, or were reduced, and no evidence for liver fibrosis or other histological liver damage was observed after treatment with MB07811 for up to 10 weeks. Additionally, MB07811, unlike T3, did not increase heart weight or decrease pituitary thyroid‐stimulating hormone beta (TSHβ) expression. Conclusion: MB07811 represents a novel class of liver‐targeted TR agonists with beneficial low‐density lipoprotein cholesterol‐lowering properties that may provide additional therapeutic benefit to hyperlipidemic patients with concomitant NAFLD. (HEPATOLOGY 2009.)

Gradual Onset of Myocardial Ischemia Results in Reduced Myocardial Infarction Association With Reduced Contractile Function and Metabolic Downregulation

BACKGROUNDnWith moderate reductions in coronary blood flow, adjustments in myocardial metabolism can occur with a normalization of the imbalance between the decreased O2 delivery and tissue O2 demand. This state of downregulated metabolism is associated with reduced function and minimal irreversible injury and has been linked to myocardial hibernation. We hypothesized that (1) this process would occur when perfusion was reduced to severely ischemic levels, but only when flow declined at a slow rate rather than abruptly, and (2) this would result in blunted ischemia and reduced myocardial injury for a given period of low blood flow.nnnMETHODS AND RESULTSnThe left anterior descending coronary artery in anesthetized open-chest pigs was cannulated and perfused with arterial blood by an extracorporeal perfusion pump. Regional function (percent segment shortening, %SS) was measured with sonomicrometry and a regional coronary vein cannulated for blood gas analysis and lactate measurements. Coronary blood flow (CBF) was reduced to 10% of control either in a step fashion (Fast Ischemia group) or gradually in a linear manner over 70 minutes (Ramp Ischemia group). In all animals, CBF was held for 60 minutes at this 10% level and then followed by 2 hours of reperfusion. In the Ramp Ischemia group, the linear fall in CBF resulted in an initial maintenance of both %SS and myocardial oxygen consumption (MVO2) followed by linear decreases in both variables (r = .98 to .99) as flow fell to the 10% level. The relation of MVO2 to function was linear (r = .99) over the entire flow range. Although %SS, MVO2, CBF, coronary pressure, and hemodynamics during the 10% flow period were not different between groups, the increases in coronary venous lactate and PCO2 and fall in pH were blunted in the Ramp Ischemia group compared with the Fast Ischemia group. With reperfusion, a significant decrease in end-diastolic length was present only in the Fast Ischemia group. Additionally, although the region at risk was not different, infarction was markedly reduced in the Ramp Ischemia group (6.6 +/- 1.9%) compared with the Fast Ischemia group (31.4 +/- 6.9%).nnnCONCLUSIONSnThese data are consistent with the hypothesis that the downregulation of myocardial metabolism with gradually decreased flow to severe levels results in reduced myocardial injury for a given period of low flow. We propose that the rate at which blood flow decreases with myocardial underperfusion is a novel determinant of infarct injury. This may have clinical implication in situations in which there is a time-dependent component to the decrease in coronary blood flow in acute ischemic events, ie, thrombus formation at a site or coronary stenosis.

Thyroid hormone β receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys

Background and purpose:u2002 Thyroid hormone receptor (TR) agonists are in clinical trials for the treatment of hypercholesterolaemia. As statins are the standard of clinical care, any new therapies must have adjunctive activity, when given in combination with statins. As already known for the statins, the cholesterol lowering effect of TR activation involves increased expression of the low‐density lipoprotein receptor. Using animal models, we tested whether TR activation would have additive cholesterol lowering activity in the presence of effective doses of a statin.

Modulation of cardiac remodeling by adenosine: In vitro and in vivo effects

The increasing incidence of congestive heart failure has stimulated efforts to develop pharmacologic strategies to prevent or reverse the associated process of adverse cardiac remodeling. The possibility of utilizing endogenously generated factors that are capable of inhibiting this process is beginning to be explored. Adenosine, has been described as a retaliatory autacoid with homeostatic activities in the regulation of myocardial blood flow, catecholamine release, and reduction of injury resulting from periods of ischemia. Adenosine exerts a variety of actions that are consistent with the concept that it can reduce or inhibit the process of cardiac remodeling. In this manuscript, the basics of adenosine metabolism, its cell surface receptors and beneficial actions on the cardiovascular system are reviewed. In addition new, in vitro and in vivo data will be presented supporting the concept that adenosine exerts actions that may ameliorate adverse cardiac remodeling.

Effect of transient coronary occlusion on coronary blood flow autoregulation, vasodilator reserve and response to adenosine in the dog

Myocardial ischemia of short duration (15 to 20 min) produces myocardial stunning during reperfusion. The vasoregulatory and contractile status of reperfused myocardium during normal and reduced perfusion pressures is of interest in the treatment of patients with unstable angina. In the present study the effects of 15 min of reversible ischemic injury on several aspects of coronary vasoregulation were assessed with use of pressure-flow curves in anesthetized open chest dogs. The left anterior descending coronary artery was cannulated and perfused with arterial blood with use of a servo-controlled roller pump. The autoregulatory gain and an adenosine dose-response curve for coronary flow before and after ischemia and reperfusion were obtained. The maximal autoregulatory gain values in the pressure range of 140 to 60 mm Hg were not significantly different before and after ischemia and reperfusion (0.41 +/- 0.08 vs. 0.5 +/- 0.06, p greater than 0.1). The adenosine dose-response curve was significantly shifted to the right after reperfusion; however, coronary blood flows during maximal adenosine vasodilation over a large range of perfusion pressures (140 to 60 mm Hg) were significantly greater after ischemia and reperfusion. The pressure-dependent decrease in segment shortening (sonomicrometry) over the coronary pressure range of 160 to 30 mm Hg was similar in myocardium before and after stunning. Contractile function in the stunned myocardium at normal (100 mm Hg) and low (40 mm Hg) coronary perfusion pressures was similarly and significantly enhanced by the administration of adenosine. It is concluded that 1) coronary autoregulation is unchanged after brief ischemia and reperfusion; 2) although maximal coronary vascular conductance assessed with adenosine is greater after ischemia, the coronary circulation shows a decreased coronary sensitivity to exogenous adenosine; 3) the relation of contractile function to coronary pressure before and after stunning is unchanged; and 4) enhancement of function in stunned myocardium by vasodilation with adenosine occurs at low and normal perfusion pressures.

Preclinical pharmacokinetics of a HepDirect prodrug of a novel phosphonate-containing thyroid hormone receptor agonist.

The prodrug [(2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811)] of a novel phosphonate-containing thyroid hormone receptor agonist [3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxylmethylphosphonic acid (MB07344)] is the first application of the HepDirect liver-targeting approach to a non-nucleotide agent. The disposition of MB07811 was characterized in rat, dog, and monkey to assess its liver specificity, which is essential in limiting the extrahepatic side effects associated with this class of lipid-lowering agents. MB07811 was converted to MB07344 in liver microsomes from all species tested (CLint 1.23-145.4 μl/min/mg). The plasma clearance and volume of distribution of MB07811 matched or exceeded 1 l/h/kg and 3 l/kg, respectively. Although absorption of prodrug was good, its absolute oral bioavailability as measured systemically was low (3-10%), an indication of an extensive hepatic first-pass effect. This effect was confirmed by comparison of systemic exposure levels of MB07811 after portal and jugular vein administration to rats, which demonstrated a hepatic extraction ratio of >0.6 with liver CYP3A-mediated conversion to MB07344 being a major component. The main route of elimination of MB07811 and MB07344 was biliary, with no evidence for enterohepatic recirculation of MB07344. Similar metabolic profiles of MB07811 were obtained in liver microsomes across the species tested. Tissue distribution and whole body autoradiography confirmed that the liver is the major target organ of MB07811 and that conversion to MB07344 was high in the liver relative to that in other tissues. Hepatic first-pass extraction and metabolism of MB07811, coupled with possible selective distribution of MB07811-derived MB07344, led to a high degree of liver targeting of MB07344.

Intracoronary versus intravenous effects of cocaine on coronary flow and ventricular function.

BACKGROUNDnCocaine use has been associated with cardiomyopathy and ischemic coronary syndromes. However, the pathophysiological mechanisms responsible for these syndromes are not clear and have been suggested to involve direct effects of cocaine on myocyte contractility and coronary resistance as well as indirect effects via altered autonomic tone, secondary mediators, and myocardial metabolism. We sought to distinguish direct from indirect effects of cocaine on ventricular function and coronary resistance by comparison of the administration of intracoronary cocaine (0.12 to 0.36 mg/min constant infusion) with intravenous cocaine (5 mg/kg bolus infusion) in an in vivo anesthetized pig preparation.nnnMETHODS AND RESULTSnTo control for changes in coronary resistance secondary to autoregulation and myocardial metabolism, the left anterior descending coronary artery was perfused at constant coronary pressure and the interventricular vein was cannulated for coronary venous oxygen saturation measurement. Coronary blood flow, regional percent segment shortening, myocardial oxygen consumption, and serum cocaine concentrations were measured. Intracoronary cocaine produced a dose-dependent decrease in percent segment shortening in the absence of significant changes in coronary flow or systemic hemodynamics. In contrast, intravenous cocaine had mild biphasic effects on coronary resistance with an initial brief vasodilation (30.0 +/- 5% increase in flow from control) followed by more prolonged vasoconstriction (17.0 +/- 3.3% decrease in flow from control), which were independent of autoregulation or myocardial metabolism. In addition, intravenous cocaine caused an early 48% decrease in percent segment shortening, at which time the measured cocaine concentration was 20.1 micrograms/mL blood. This was comparable to the intracoronary cocaine concentration of 17.1 micrograms/mL blood, which produced a similar 48% decrease in percent segment shortening.nnnCONCLUSIONSnWe conclude that the effects of acute cocaine exposure on ventricular function are predominantly direct but of brief duration and therefore probably not clinically relevant. The effects of cocaine on coronary tone are predominantly indirect and biphasic, with early vasodilation followed by mild and more prolonged vasoconstriction. In the absence of coronary stenosis or ventricular hypertrophy, this small amount of vasoconstriction is unlikely to cause ischemia.

Thromboxane is produced in response to intracoronary infusions of complement C5a in pigs. Cyclooxygenase blockade does not reduce the myocardial ischemia and leukocyte accumulation.

Activated polymorphonuclear leukocytes (PMNs) contribute to myocardial injury during ischemia and reperfusion. There is evidence that activation of the complement pathway may be one of the mechanisms of PMN activation during ischemia. Intracoronary infusion of complement C5a during normal perfusion pressure is associated with decreased coronary Bow, contractile dysfunction, and PMN accumulation. The mechanisms responsible for these changes have not been identified. Thromboxane A2 (TXA2) is a potential mediator of this myocardial ischemic response. Activated PMNs produce TXA2, a known coronary vasoconstrictor, and TXA] was shown to be a mediator of the pulmonary hypertensive response to activated complement. The goal of the present study was to determine if an enhanced TXA2 production is associated with the myocardial response to C5a and whether cyclooxygenase blockade would reduce the myocardial ischemia. In open-chest pigs, intracoronary C5a (500 ng) caused reversible reductions in blood flow (50.0% of control), regional contractile function (25.8% of control), leukocyte trapping (1.0 × 106 cells/g myocardium or a peak artery-coronary venous difference of 5.3 × l03 cells/μl blood), and increased coronary venous TXB2 (the TXA2 breakdown product) from 1.6 pmol/ml to a peak of 6.9 pmol/ml. Cyclooxygenase blockade with aspirin or indomethacin, which prevented TXB2 production, did not alter the response in flow, function, or PMN trapping. Ibuprofen, a known direct inhibitor of PMNs in addition to Its cyclooxygenase blockade effect, reduced the response slightly. The pig coronary vascular bed was responsive to the TXA2 agonist U46619, which reduced flow and function without PMN trapping. Mechanical reductions in coronary flow to levels equivalent to those during the C5a infusions did not increase coronary venous TXB2 nor cause PMN trapping but did cause equivalent contractile dysfunction. Incubation of whole blood with C5a at concentrations equivalent to those achieved in vivo did not cause TXB3 production. We conclude that 1) TXA2 is produced in response to intracoronary C5a and 2) cyclooxygenase blockade does not prevent the C5a-induced myocardial ischemia, contractile dysfunction, and PMN trapping. The TXA2 production likely involves a vascular site or a blood cell-vascular interaction. This model system indicates the potential for persistently activated PMNs to cause continued ischemia during myocardial reperfusion.

Complement-Induced Myocardial Ischemia: Neutrophil and Vascular Mechanisms

Medical therapy for acute myocardial infarction is currently directed at preventing acute arrhythmia, limiting the extent of necrosis, and preventing ventricular dilation. Experimental studies dealing with constraining the extent of necrosis in acute coronary occlusion have had limited success in the absence of reperfusion. Restoration of blood flow was found to be by far the most effective mechanism of reducing the degree of necrosis during acute myocardial infarction. Studies in experimental animals indicate that reperfusion within 2–3 h of coronary occlusion in canine hearts results in significant salvage of tissue, defined as the restoration of contractile function to tissue which would have otherwise developed into necrosis. Thus, in the 1980s, attempts to limit injury during acute myocardial infarction are primarily directed at reperfusion. It should be noted, however, that other measures, such as the administration of beta-adrenergic blocking agents, are effective in reducing mortality and perhaps morbidity and should not be neglected.