Probenecid for the treatment of cardiomyopathy, systolic cardiac dysfunction and congestive heart failure symptoms

Abstract

To provide a composition which increases contractility of cardiac muscle without increasing the cell death in contrast to a commonly used β-agonist, isoproterenol.SOLUTION: A composition containing probenecid and administered by at least one of injection, oral administration and transdermal administration can increase the calcium concentration in a heart cell without increasing the cell death.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to previously filed, co-pending U.S. Application No. 13 / 584,713 (filed August 13, 2012), which is expressly incorporated herein by reference in its entirety.

[0002] The present invention relates generally to the treatment of cardiomyopathy, systolic cardiac dysfunction and congestive heart failure conditions with probenecid, its metabolites and related chemicals. [Background technology]

[0003] Nearly six million people live with congestive heart failure and other heart dysfunction conditions in the United States. On average, patients suffering from congestive heart failure are treated with six different drugs.

[0004] Current management of acute congestive heart failure is based on reducing afterload and increasing myocardial contractility. Therapies for the former are well established and include direct and indirect vasodilators. Unfortunately, available therapeutic options for the latter are limited to digoxin and sympathomimetics (i.e., dopamine, dobutamine, milrinone). Sympathomimetics share a common endpoint of increasing cAMP production in cardiomyocytes (directly via β-ADR stimulation or indirectly via phosphodiesterase inhibition PDI) and have been repeatedly shown to be associated with poor outcomes. Several mechanisms have been implicated in the adverse outcomes associated with these medications, including arrhythmias, stimulation of apoptotic signaling, and increased myocardial energy demands. New and safer therapies for congestive heart failure are needed, but despite billions of dollars invested in the search to date, most candidates still focus on the same pathways and have similar adverse effect profiles.

[0005] Probenecid is a highly lipid-soluble benzoic acid derivative with an excellent safety profile that was developed in the 1950s to reduce the renal tubular excretion of penicillin, and has since been used to increase the serum concentrations of several antibiotics and antivirals. Probenecid has also been found to be a competitive inhibitor of active transport processes in the brain, liver, and eye, and has been investigated in these areas, but no clinical uses other than its effects on the kidney have been established.

[0006] Unlike other drugs developed to increase serum levels of antibiotics, probenecid was initially administered by slow intravenous infusion. It was subsequently found to be rapidly absorbed after oral administration, with peak serum concentrations occurring in 1-5 hours. Water-soluble preparations of the drug are readily available in the United States, but only one orally administered lipid-soluble form is currently available and approved by the FDA for the treatment of gout.

[0007] In early studies with probenecid (called Benemid), it was observed to have a potent uricosuric effect and quickly became the standard of care for gout treatment. Probenecid was found to lower serum uric acid levels through inhibition of reabsorption of organic acids such as uric acid by the renal proximal tubules by acting as a competitive inhibitor of organic anion transporters (OATs) and thus preventing OAT-mediated reuptake of uric acid from urine into serum. Despite its minimal adverse effects profile, probenecid has been used significantly less clinically as other gout treatments have shown improved efficacy.

[0008] Previously, the diuretic effect of probenecid has been investigated in patients with "uncomplicated congestive heart failure" [Bronsky et al., (1955) Diuretic Action of [Probenecid]: Its Effects Upon The Urinary Excretion of Sodium, Chloride, Potassium, and Water In Edematous Subjects, Am. J. Med. 18, 259-266, and Kushner et al., (1954) Effect of [Probenecid] On Excretion of Water, Sodium, and Chloride In Congestive Heart Failure, Federation Proceedings, 13, 435]. In these studies, large doses of probenecid, 2 g / day or 4 g / day, were administered orally for several days to subjects with uncomplicated congestive heart failure, with the explicit goal of evaluating the potential effects of probenecid on the kidney. These studies technically found that probenecid had a strong diuretic effect, and that this effect was due to the renal profile of probenecid rather than potential cardiac effects.

[0009] More recently, research interest in probenecid has increased with the discovery that it is a selective agonist of transient receptor potential vanilloid 2 (TRPV 2). The TRP family of ion channels has been the subject of research for many years in the nephrology and neurology literature. Several TRPs have also been found to be important mediators of vascular tone (TRPC1, TRPVc6, and TRPM4), cerebral blood flow (TRPM4), neointimal hyperplasia (TRPC1), and pulmonary hypertension (TRPC6). However, until recently, only some of the channels in this family (e.g., TRPC3 / 6 / 7 in the development of cardiac hypertrophy in response to pressure overload) have been found to have direct cardiac effects. There are very interesting studies that have found direct cardiac effects regarding the TRPV family. First, it was observed that cardiac-specific overexpression of TRPV2 resulted in ventricular dilation of all cavities in the mouse heart. It was then observed that TRPV1 knockout mice had increased infarct size and reduced survival after ligation of the left anterior descending artery compared to their wild-type littermates. Interestingly, others have observed that TRPV1 activation by specific agonists confers protection from ischemia / reperfusion (I / R) injury. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Bronsky et al. (1955) Diuretic Action of [Probenecid]: Its Effects Upon The Urinary Excretion of Sodium, Chloride, Potassium, and Water In Edematous Subjects, Am. J. Med. 18, pp. 259-266. [Non-Patent Document 2] Kushner et al. (1954) Effect of [Probenecid] On Excretion of Water, Sodium, and Chloride In Congestive Heart Failure, Federation Proceedings, 13, 435 [Non-Patent Document 3] Remington: The Science and Practice of Pharmacy (19th ed.), edited by A. R. Gennaro, Mack Publishing Company, Easton, Pa., 1995. Summary of the Invention [Means for solving the problem]

[0011] While the invention will be described in connection with particular embodiments, it will be understood that the invention is not limited to these embodiments, but rather the invention includes all alternatives, modifications, and equivalents which may be included within the spirit and scope of the invention.

[0012] One aspect of the present invention is directed to a method of treating symptoms of cardiomyopathy, systolic cardiac dysfunction and congestive heart failure, collectively referred to herein as "cardiac dysfunction", in a subject, comprising administering probenecid by intravenous administration in an amount effective to treat cardiac dysfunction and symptoms of cardiac dysfunction. In one embodiment of the present invention, probenecid is administered in the range of about 1 mg / kg / day to about 100 mg / kg / day. Probenecid may be administered by at least one of a bolus injection or continuous intravenous infusion, or a bolus (loading dose) followed by intravenous infusion. In one embodiment, probenecid is administered over a period of about 8 hours to about 24 hours. Probenecid can be used for short-term treatment, i.e., treatment for less than one week, or can be administered chronically, i.e., for a period of weeks, months or even years. Probenecid can be administered in an amount sufficient to clinically improve cardiac function, resulting in improvement of cardiac dysfunction. Improvement in cardiac dysfunction may be determined by quantifiable clinical observations based, for example, on standardization of the 6-minute walk test, improvement in New York Heart Association (NYHA) classification, reduction in diuretic dose requirement, reduction in serum BNP level, normalization of serum sodium concentration, and combinations thereof.

[0013] Another aspect of the present invention relates to a method for treating cardiac dysfunction and symptoms of cardiac dysfunction in a subject, comprising administering by oral administration an amount of probenecid effective to treat symptoms of cardiac dysfunction. In one embodiment, probenecid is administered in the range of about 1 mg / kg / day to about 100 mg / kg / day. Probenecid can be administered in a ready-to-use formulation or in an extended release formulation. Extended release formulations are formulated to maintain therapeutic plasma concentrations of probenecid for a duration ranging between about 18 hours and about 24 hours. Probenecid can be used for short-term treatment, i.e., treatment for less than one week, or can be administered chronically, i.e., for a period of weeks, months, or even years. Probenecid can be administered in an amount sufficient to clinically improve cardiac function, resulting in improvement of cardiac dysfunction. Improvement in cardiac dysfunction may be determined by quantifiable clinical observations based, for example, on standardization of the 6-minute walk test, improvement in New York Heart Association (NYHA) classification, reduction in diuretic dose requirement, reduction in serum BNP level, normalization of serum sodium concentration, and combinations thereof.

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and, together with the general description of the invention given above and the detailed description given below, serve to explain the principles of the invention. [Brief description of the drawings]

[0015] [Figure 1A] Representative B-mode and M-mode echocardiograms from the long axis view at baseline and after administration of probenecid 200 mg / kg IV. [Figure 1B] 1 is a bar graph showing that probenecid produced no echocardiographic changes. [Figure 1C] 1 is a graph showing the time course of change in EF following IV administration of probenecid 200 mg / kg according to an embodiment of the present invention. [Figure 1D]1 is a graph showing dose-dependent changes in EF after injection of various concentrations of probenecid in accordance with an embodiment of the present invention. [Figure 1E] 1 is a bar graph showing the mean change in EF following IP administration of 100 mg / kg probenecid for wild type (WT), TRPV2+ / - (HET) and TRPV2- / - (KO). [Figure 2A] 1 is a bar graph showing RR intervals measured after administration of various doses of probenecid in accordance with an embodiment of the present invention. [Figure 2B] 1 is a bar graph showing PR intervals measured after administration of various doses of probenecid in accordance with an embodiment of the present invention. [Figure 2C] 1 is a bar graph showing QRS intervals measured after administration of various doses of probenecid in accordance with an embodiment of the present invention. [Figure 2D] 1 is a bar graph showing heart rate measurements following administration of various doses of probenecid in accordance with an embodiment of the present invention. [Figure 3A] 1 is a graph showing in vivo measurements of +dP / dt following administration of probenecid 30 mg / kg IV followed 5 minutes later by probenecid 100 mg / kg IV in accordance with an embodiment of the present invention. [Figure 3B] 1 is a graph showing that Langendorff perfusion measurements of the change in +dP / dt from 90 to 210 seconds during perfusion with 10 −6 probenecid are all significantly different from baseline, in accordance with an embodiment of the invention. [Figure 4] FIG. 1 shows quantitative RT-PCR products from mRNA isolated from wild-type (WT), TRPV2+ / − (HET) and TRPV2− / − (KO) mouse hearts. [Figure 5A] FIG. 1 shows a representative Western blot analysis of phosphorylated phospholamban (p-PLN) comparing saline and probenecid. [Figure 5B] 1 is a bar graph quantifying Western blot analysis of phosphorylated phospholamban (p-PLN) comparing saline with probenecid. [Figure 5C]FIG. 1 shows a representative Western blot analysis of phosphorylated ryanodine receptor 2 (p-RyR2) comparing saline and probenecid. [Figure 5D] 1 is a bar graph quantifying Western blot analysis of phosphorylated ryanodine receptor 2 (p-RyR2) comparing saline to probenecid. [Figure 6A] FIG. 1 shows representative contraction recordings of ventricular myocytes upon exposure to 10 −7 M probenecid (PROB). [Figure 6B] 1 is a graph showing the dose-response curve of probenecid on myocyte contractile force. Data points are averages from four mice and fitted with a sigmoidal nonlinear regression curve. [Figure 6C] FIG. 1 shows representative contraction recordings of ventricular myocytes pretreated with ruthenium red upon exposure to 10 −7 M probenecid (PROB). [Figure 6D] 1 is a bar graph showing average data on fractional shortening (FS) of myocytes in control and under exposure to 10 −7 M probenecid. [Figure 6E] FIG. 1 shows representative Ca 2+ transient recordings from ventricular myocytes upon exposure to 10 −7 M probenecid (PROB). [Figure 6F] 13 is a bar graph showing average data for Ca2+ transient amplitude F / F0 (left) and time constant tau (right) under control and 10-7 M probenecid exposure. [Figure 7A] FIG. 1 shows confocal microscopy line scan images of cytosolic Ca2+ from myocytes in control and upon exposure to 10-7 M probenecid for 5 min, along with a heat map showing F / F0 intensity (scaled from 0 to 2). [Figure 7B] 1 is a bar graph showing average data on Ca2+ spark frequency in control and after 5 min exposure to 10-7 M probenecid. [Figure 7C]FIG. 13 is a graph showing the time course of cytosolic Ca 2+ upon exposure to 10 −7 M probenecid in myocytes with and without pretreatment with 10 −6 M ruthenium red (RR). [Figure 7D] 1 is a bar graph showing average data for myocyte cytosolic Ca 2+ under control and 10 −7 M probenecid exposure (5 min). [Figure 7E] 1 is a bar graph showing average data on cytosolic Ca2+ in myocytes pretreated with 10-6M ruthenium red, under control and 10-7M probenecid exposure (5 min). [Figure 7F] FIG. 1 shows patch clamp data from the same myocyte showing the absence of inward Ca2+ currents upon treatment with 10-7M probenecid (left) and that an L-type Ca2+ current was evoked by a depolarizing voltage step to +10 mV. [Figure 7G] FIG. 1 shows confocal microscopy line scan images of cytoplasmic Ca 2+ from myocytes pretreated with 10 −6 M thapsigargin (TG) for 15 min before and after treatment with 10 −7 M probenecid for 5 min. [Figure 7H] 1 is a bar graph showing average data for myocyte cytosolic Ca 2+ in control and after treatment with 10 −7 M probenecid in myocytes pretreated with 10 −6 M thapsigargin. [Figure 8A] 1 is a bar graph showing peak cystolic fluorescence before and after various doses of probenecid. [Figure 8B] 1 is a graph showing linear regression showing normalized fluorescence dose-response curves for various doses of probenecid. [Figure 8C] 1 is a bar graph showing that various doses of probenecid did not result in significant cell death, whereas treatment with 1 mM isoproterenol resulted in significant cell death as early as 4 hours after treatment and was indistinguishable from the H2O2 positive control nearly 24 hours after treatment. [Figure 9]1 is a bar graph showing that there is no significant difference in cell death as indicated by TUNEL stained nuclei in mice with and without ischemic injury with or without probenecid pretreatment. [Figure 10A] FIG. 13 is a scatter plot showing the correlation between initial EF and change in EF after administration of probenecid and echocardiographic follow-up of cardiac function in mice subjected to I / R injury and grouped by initial EF of 50-60%. [Figure 10B] Graph showing correlation between initial EF and change in EF after probenecid administration, showing the most minimal damage (40-50%). [Figure 10C] FIG. 11 is a graph showing the correlation between initial EF and change in EF after probenecid administration, indicating more significant myocardial injury. [Figure 11A] FIG. 13 is a graph showing that water-treated mice have increased left ventricular diastolic volume (LV Vol;d) compared to probenecid-treated mice following I / R injury. [Figure 11B] FIG. 13 is an echocardiogram showing that probenecid mice had improved myocardial contractility as measured by greater displacement compared to water-treated mice. [Figure 11C] FIG. 11C is a graph showing the echocardiogram data of FIG. [Figure 11D] FIG. 13 Echocardiogram showing a small (but not statistically significant) increase in EF change with probenecid. [Figure 11E] FIG. 11D is a graph showing the echocardiogram data of FIG. [Figure 12A] 1 is a bar graph showing that infarct size was greater after 45 min of ischemia compared to 30 min of ischemia, but was not different between control and 200 mg / kg probenecid pretreated mice at any time point. [Figure 12B] 1 is a bar graph showing that there was no difference between control and pretreated mice at any time point with respect to EF. [Figure 12C]FIG. 1 is a bar graph showing that there was no difference in circumferential strain between control and pretreated mice at any time point. [Figure 12D] FIG. 13 is an echocardiogram showing IR pretreated with probenecid. [Figure 12E] 1 is a bar graph showing that there was no difference in radial displacement between control and pretreated mice at any time point. [Figure 13] FIG. 1 is a graph showing improvement in cardiac function as measured by echocardiography following various doses of probenecid IV in a healthy porcine model. [Figure 14] FIG. 13 shows no change in cardiac function after saline administration and a rapid increase in EF after probenecid 5 mg / kg IV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] One aspect of the present invention is directed to a novel method for treating cardiomyopathy, systolic cardiac dysfunction, and congestive heart failure symptoms, collectively referred to herein as "cardiac dysfunction," in a subject. Recently, probenecid has been identified as an agonist of the transient receptor potential vanilloid 2 (TRPV2) ion channel. TRPV2 is a weakly calcium-selective cation channel that is activated by certain agonists, such as probenecid, as well as by cell swelling and heat.

[0017] Without being bound by any particular theory, probenecid increases the contractile force of cardiomyocytes by dose-dependent changes in myocyte calcium concentration and improved calcium handling by cells. As shown by the data provided in Example 1, these processes are not mediated through phosphorylation of beta-adrenergic receptors or ryanodine receptors or phospholamban. The effects mediated by probenecid do not induce cell death and, unlike clinically used sympathomimetics, are not associated with increased infarct size during simulated myocardial infarction. Understanding the mechanism of action for the effects of probenecid, as demonstrated in Examples 2 and 3, allows the surprising discovery that cardiac dysfunction and symptoms of cardiac dysfunction can be treated with probenecid, more specifically, at lower doses of probenecid than are required for other uses, and in fact at lower doses of probenecid than those used in previous studies of congestive heart failure. These observations have led to novel treatments of subjects in acute crisis and long-term treatment of patients with therapeutically effective doses of probenecid.

[0018] As shown in the examples, probenecid acts as a positive inotrope in mouse models of heart failure.Importantly, probenecid is more active in mice with heart failure and low ejection fraction than in mice without heart failure.In mice with heart failure, probenecid normalizes ejection fraction.Similar to mouse cardiac tissue, human cardiac tissue also contains TRPV2 ion channels that can be targeted to treat cardiac dysfunction or symptoms of cardiac dysfunction with probenecid.

[0019] When treating cardiac dysfunction or symptoms of cardiac dysfunction, the therapeutically effective dose of probenecid is the dose that achieves the plasma level of probenecid and its active metabolites that increases the contractile force of the heart so that cardiac output is sufficient to alleviate at least some symptoms of cardiac dysfunction.For example, probenecid can be administered in an amount sufficient to clinically improve cardiac function, resulting in the improvement of cardiac dysfunction.Improvement of cardiac dysfunction can be determined by quantifiable clinical observations, for example, based on standardization of 6-minute walk test, improvement of New York Heart Association (NYHA) classification, reduction in diuretic dosage requirements, reduction of serum BNP level, normalization of serum sodium concentration, and combinations thereof.

[0020] One aspect of the invention is directed to a method of treating cardiac dysfunction or symptoms of cardiac dysfunction in a subject, comprising administering a therapeutically effective amount of probenecid by injection. In one embodiment, the injection is intravenous. The dose required to adequately improve cardiac function in a given patient may vary widely depending on the dosage required by the efficacy of the treatment and the rate of clearance. Thus, in one embodiment, probenecid is administered in the range of about 1 mg / kg / day to about 100 mg / kg / day. The term "day" is understood to be a 24-hour period. In another embodiment, the extended release formulation comprises a total dose of probenecid in the range of about 1 mg / kg / day to about 50 mg / kg / day. In an alternative embodiment, the extended release formulation comprises a total dose of probenecid in the range of about 1 mg / kg / day to about 25 mg / kg / day. In one embodiment, the therapeutically effective amount of probenecid is in the range of about 1 mg / kg / day to about 20 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid ranges from about 5 mg / kg / day to about 50 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid includes a dosage range from about 10 mg / kg / day to about 100 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid includes a dosage range from about 50 mg / kg / day to about 100 mg / kg / day.

[0021] The therapeutically effective amount of probenecid can be administered by bolus injection, continuous infusion, or a combination of bolus injection and continuous infusion. The term "bolus injection" is understood to be an injection in which the dose is delivered over a relatively short period of time. The term "continuous infusion" is understood to be an injection delivered, such as by intravenous infusion, in which the dose is delivered metered over the period of time desired for probenecid therapy. In one embodiment, probenecid is administered by continuous infusion for a period ranging between about 30 minutes / day to about 24 hours / day. In another embodiment, the therapeutically effective amount of probenecid is administered for about 8 hours / day to about 24 hours / day. In some circumstances, probenecid is administered by continuous infusion for at least one day, but not more than seven days. In other circumstances, probenecid can be administered for an even longer period of time, such as on multiple days over the weeks, months, or even years required to treat the subject's condition. Thus, embodiments of the present invention are directed to the chronic administration of probenecid to treat cardiac dysfunction and symptoms of cardiac dysfunction.

[0022] In some cases, a combination of bolus injection and continuous infusion may be desirable for treating a subject. For example, bolus injection can be used to deliver a loading dose, i.e., a dose of probenecid that rapidly achieves a desired therapeutic level of probenecid in the subject, and continuous infusion can be used to maintain or even titrate the desired therapeutic level over a desired duration of treatment. For example, a subject in acute distress, such as from decompensated congestive heart failure, may require immediate treatment by a bolus intravenous injection of probenecid. After an initial bolus injection, the subject may then require a subsequent maintenance dose or titration of probenecid over a period of time thereafter, such as by continuous infusion. Alternatively, the maintenance dose of probenecid can be achieved by subsequent bolus injections. Continuous infusion of probenecid is also useful for treating subjects with compensated congestive heart failure or other forms of cardiac dysfunction.

[0023] Another aspect of the invention is directed to a method of treating cardiac dysfunction and symptoms of cardiac dysfunction in a subject, comprising administering a therapeutically effective dose of probenecid by oral administration. In one embodiment, the therapeutically effective amount of probenecid ranges from about 1 mg / kg / day to about 25 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid includes a dosage range of about 5 mg / kg / day to about 25 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid includes a dosage range of about 5 mg / kg / day to about 20 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid includes a dosage range of about 5 mg / kg / day to about 15 mg / kg / day. This oral dose of probenecid can be administered in a single dose or multiple doses over a 24 hour period, typically over a period of days, weeks, months, or even years. In one embodiment, probenecid is orally administered to a subject for at least two weeks, while in alternative embodiments, probenecid is administered for multiple months or a year or more. Thus, embodiments of the present invention are directed to the long-term administration of probenecid to treat cardiac dysfunction and symptoms of cardiac dysfunction.

[0024] Another aspect of the present invention is directed to a method for treating cardiac dysfunction or symptoms of cardiac dysfunction in a subject, comprising administering a long-release formulation of probenecid that maintains therapeutic plasma concentrations of probenecid and its active metabolites for a duration ranging from about 18 hours / day to about 24 hours / day. The long-release formulation can be an oral formulation, an injected formulation, or even a transdermal formulation. In one embodiment, the long-release formulation can include a total dose of probenecid that clinically improves cardiac function, resulting in improvement of cardiac dysfunction. Improvement of cardiac dysfunction can be determined by quantifiable clinical observations, for example, based on standardization of the 6-minute walk test, improvement of New York Heart Association (NYHA) classification, reduction in diuretic dosage requirements, reduction of serum BNP levels, normalization of serum sodium concentration, and combinations thereof. Dosing of probenecid will require titration to achieve and maintain the desired effect. Thus, the dose required to adequately improve cardiac function can vary widely for a given subject as treatment progresses and between subjects. Thus, in one embodiment, probenecid is administered in the range of about 1 mg / kg / day to about 100 mg / kg / day. The term "day" is understood to be a 24-hour period. In another embodiment, the extended release formulation comprises a total dose of probenecid in the range of about 1 mg / kg / day to about 50 mg / kg / day. In an alternative embodiment, the extended release formulation comprises a total dose of probenecid in the range of about 1 mg / kg / day to about 25 mg / kg / day. In one embodiment, the therapeutically effective amount of probenecid is in the range of about 1 mg / kg / day to about 20 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid is in the range of about 5 mg / kg / day to about 50 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid comprises a dosage in the range of about 10 mg / kg / day to about 100 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid comprises a dosage ranging from about 50 mg / kg / day to about 100 mg / kg / day, hi one embodiment, the probenecid is orally administered to the subject for at least two weeks, and in an alternative embodiment, the probenecid is administered for multiple months or more than a year.Thus, embodiments of the present invention are directed to the chronic administration of probenecid to treat cardiac dysfunction and symptoms of cardiac dysfunction.

[0025] Another aspect of the present invention is directed to a method for treating cardiac dysfunction or symptoms of cardiac dysfunction in a subject, comprising administering a therapeutically effective amount of probenecid by transdermal administration, such as by gel or patch. In one embodiment, the transdermal formulation maintains therapeutic plasma concentrations of probenecid and its active metabolites for a duration ranging between about 18 hours / day and about 24 hours / day. In one embodiment, the transdermal formulation can include a total dose of probenecid that clinically improves cardiac function, resulting in improvement of cardiac dysfunction. Improvement of cardiac dysfunction can be determined by quantifiable clinical observations, such as, for example, standardization of the 6-minute walk test, improvement of New York Heart Association (NYHA) classification, reduction in diuretic dosage requirements, reduction in serum BNP levels, normalization of serum sodium concentration, and combinations thereof. Dosing of probenecid will require titration to achieve and maintain the desired effect. Thus, the dose required to adequately improve cardiac function can vary widely for a given subject as treatment progresses and between subjects. Thus, in one embodiment, probenecid is administered in the range of about 1 mg / kg / day to about 100 mg / kg / day. The term "day" is understood to be a 24-hour period. In another embodiment, the extended release formulation comprises a total dose of probenecid in the range of about 1 mg / kg / day to about 50 mg / kg / day. In an alternative embodiment, the extended release formulation comprises a total dose of probenecid in the range of about 1 mg / kg / day to about 25 mg / kg / day. In one embodiment, the therapeutically effective amount of probenecid is in the range of about 1 mg / kg / day to about 20 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid is in the range of about 5 mg / kg / day to about 50 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid comprises a dosage in the range of about 10 mg / kg / day to about 100 mg / kg / day. In another embodiment, the therapeutically effective amount of probenecid comprises a dosage ranging from about 50 mg / kg / day to about 100 mg / kg / day, hi one embodiment, the probenecid is administered transdermally to the subject for at least one week, and in an alternative embodiment, the probenecid is administered for multiple months or more than one year.Thus, embodiments of the present invention are directed to the chronic administration of probenecid to treat cardiac dysfunction and symptoms of cardiac dysfunction.

[0026] Achieving therapeutic plasma concentration can be assessed by quantifying the clinical improvement of the subject's new functions, such as by standardizing the 6-minute walk test, improving the New York Heart Association (NYHA) classification, reducing diuretic dosage requirements, lowering serum BNP levels, normalizing serum sodium concentration, and combinations thereof. Alternatively, the levels of probenecid and its metabolites can be measured in the subject's blood. For example, lowering the NYHA classification by one, for example from 4 to 3, is a quantifiable improvement. Extended release and transdermal formulations can be used to rescue subjects suffering from decompensated congestive heart failure, while these formulations are particularly useful for maintaining and titrating probenecid to subjects suffering from cardiac dysfunction and cardiac dysfunction symptoms.

[0027] Dosage and route of administration of probenecid may be combined to provide optimal treatment for the subject. For example, probenecid can be administered at a dose of 1 mg / kg to 50 mg / kg as a bolus therapy in acutely ill subjects with cardiac dysfunction, such as decompensated heart failure. If this treatment is sufficient to improve symptoms, the physician may choose to initiate a continuous infusion at a rate of 1 mg / kg / hour to a maximum of 100 mg / kg / hour, titrating as needed based on individual scenarios. Furthermore, some patients may need to transition to parenteral probenecid, which can be administered in the range of 200 mg per day to a maximum of 4 gr per day in gel form or split capsules.

[0028] Probenecid, as used herein, includes pharma- ceutically acceptable salts of probenecid, as well as prodrugs, isomers and polymorphs, and derivatives structurally related to or derived from probenecid. A composition of probenecid in a pharma- ceutically acceptable carrier can be administered in vivo. By "pharma-ceutically acceptable" is meant a material that is not biologically or otherwise undesirable. Thus, the composition can be administered to a subject without causing undesirable biological effects or adversely interacting with any other components of the pharmaceutical composition contained therein. Of course, the carrier is selected to minimize any degradation of probenecid and to minimize adverse side effects in the subject, as will be appreciated by those skilled in the art.

[0029] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th Edition) AR Gennaro, ed., Mack Publishing Company, Easton, Pa. 1995. For intravenous administration, an appropriate amount of a pharma- ceutical acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharma- ceutical acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is in the pharma- ceutical acceptable range, preferably about 5 to about 8.5, more preferably about 7.8 to about 8.2. Further carriers include sustained release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the pharmaceutical composition, the matrices being in the form of shaped articles, such as films, liposomes, or microparticles. It will be apparent to one skilled in the art that some carriers may be more preferable, for example, depending on the route of administration and the concentration of the composition to be administered. For example, one skilled in the art can select a particular carrier suitable for introduction into the body by injection as described above or by oral ingestion.

[0030] In one embodiment, the probenecid injection formulation is prepared by dissolving probenecid powder (acid form) in 0.1M sodium hydroxide. This solution is then diluted with 0.2M phosphate buffer (pH=7.4). Probenecid is then diluted to a stock solution concentration, for example, about 4.2mg / ml, with normal saline or other carrier suitable for intravenous injection to form a stock solution. The stock solution can then be diluted in saline or other injectable solution to the desired dose for administration.

[0031] For oral ingestion, the probenecid can be in tablet form, encapsulated, or dissolved or suspended in a liquid or gel, as known to those skilled in the art for oral administration of drugs. In some embodiments, the probenecid is formulated for sustained release, e.g., with one or more excipients that control the release of probenecid over a designated period of time for absorption by the subject.

[0032] Pharmaceutical compositions of probenecid can also include binders, thickeners, diluents, buffers, preservatives, surfactants, and the like in addition to probenecid and carriers.

[0033] The disclosed compositions may be suitable for systemic administration. For example, the compositions may be administered by other means known in the art, such as orally, parenterally (e.g., intravenous, intramuscular, intraperitoneal or subcutaneous injection), by suppository, or even transdermally, such as by gel or patch formulation. Such formulations may be prepared as described above or as known to those skilled in the art. EXAMPLES

[0034] Surveys of mouse and human myocardial tissues for expression of TRPV channels established that TRPV2 was the most highly expressed in whole heart samples and specifically in the left ventricle. This study examined whole animals, isolated whole hearts, and isolated ventricular myocytes to evaluate the ability of probenecid to modulate myocardial function.

[0035] method Animals - Wild-type (WT) mice (B6129SF2 / J F2 and C57BL6J, Jackson Laboratories) and TRPV2 - / - Mice (breeding pairs provided by Dr. M. Caterina (John's Hopkins, Baltimore, MD)) were male and aged 12-16 weeks.

[0036] In vivo studies - Contractile force studies using intravenous administration of probenecid. Echocardiographic assessment. To obtain dose-response curves, 12-16 week old male C57 WT (n=39) mice were anesthetized with isoflurane while intravenous access (IV) was obtained in the jugular vein under a microscope as previously described. Subsequently, echocardiographic studies in both M-mode and B-mode were obtained in the parasternal long axis (PSLAX) orientation as described below. Saline or different doses of probenecid (increasing from 2 mg / kg to 200 mg / kg) were injected (bolus IV) for early contractile force studies in WT mice.

[0037] Invasive assessment. - Once the dose range was determined, different groups of WT mice were anesthetized by intraperitoneal injection of ketamine (50 μg / g) and Inactin (thiobutabarbital, 100 μg / g, Sigma, MA). A tracheotomy was performed (PE-90) and body temperature was monitored and maintained with a feedback-controlled heating table. The right femoral artery was cannulated with liquid-filled polyethylene tubing for blood pressure measurement and connected to a low-compliance pressure transducer (COBE Cardiovascular, Arvada, CO). The right femoral vein was cannulated for drug delivery. A high-fidelity 1.2-French SciSence pressure catheter (SciSence, London, ON, Canada) was inserted into the right carotid artery and advanced to the left ventricle to monitor cardiac performance. ECG leads were placed on the left and right arms and left leg and connected to a BIOAmp (AD Instruments, Colorado Springs, CO). To measure carotid blood flow, the left carotid artery was isolated and fitted with a 0.5-PSB perivascular flow probe connected to a TS420 flowmeter (Transonic Systems, Ithaca, NY). Experimental solutions of 100 μg / μl probenecid were delivered as boluses of 30 and 100 mg / kg via the femoral vein catheter, with 5 min between doses. Hemodynamic variables were collected and analyzed using a MacLab 4 / S system (AD Instruments, Colorado Springs, CO) and Chart software.

[0038] WT, TRPV2 + / - and TRPV2 - / - Contractile force study in mice - Based on the results of the previous experiment, it was determined that a dose of 100 mg / kg of probenecid would produce the maximum contractile force response. Mice were then injected intraperitoneally (IP) with probenecid to reduce the potential stress effects of surgery. WT, TRPV2 + / - and TRPV2 - / - Mice were monitored by ultrasound for 30 minutes after injection as described below.

[0039] Echocardiography - All echocardiographic examinations were performed with a Vevo 2100 Ultrasound system [Visualsonics, Toronto, Canada] equipped with an MS400 probe (centerline frequency 30 MHz) and post-processed with Vevostrain software [Vevo 2100, v1.1.1 B1455, Visualsonics, Toronto, Canada] on a separate workstation. Images were obtained from PSLAX and short axis (SAX) sections between 2 and 10 mm depth in both M-mode and B-mode. All examinations of mice exposed to I / R injury included strain imaging in M-mode in PSLAX, B-mode and SAX. Additionally, for contractile force studies, only M-mode measurements were obtained from PSLAX. Strain imaging was performed from B-mode images, and regional radial strain and circumferential displacement were measured by regional wall and summed average values ​​from SAX images. Left ventricular lumen size and wall thickness were measured from M-mode images. Ejection fraction (EF) and fractional shortening (FS) calculations were obtained using Vevo software.

[0040] Changes in parameters derived from EF, FS, and strain were obtained by subtracting baseline values ​​from individual subjects for subsequent time points. In addition, the mean change from baseline for each time point between 5 and 30 minutes was determined for each mouse by taking measurements every 5 minutes. These means were compared between different groups.

[0041] In vivo electrophysiology - Electrocardiogram data were obtained during all echocardiographic examinations. These examinations were subsequently analyzed by an independent blinded reader at all time points to assess electrocardiogram changes and drug-induced arrhythmias. PR interval, RR interval, and QRS width parameters were measured and reported as peak changes, while the presence of supraventricular or ventricular arrhythmias was determined as the sum observed across all images acquired in a 30-minute period.

[0042] Ex vivo (Langendorff) studies - Isolated heart experiments were performed as previously described for WT mice. After the hearts reached a steady state of 400 bpm by pacing, probenecid (10 -6 Hearts were perfused with probenecid (M) by continuous perfusion using a syringe pump for up to 5 min. Measurements were taken every 1 sec. After 5 min of probenecid perfusion, hearts were removed from the cannula and flash frozen in N2 for Western blot analysis.

[0043] molecular research Quantitative RT-PCR - WT, TRPV2 + / - and TRPV2 - / - Hearts (LV) obtained from mice for RNA isolation and qRT-PCR were flash frozen and stored at -80°C. To estimate TRPV2 transcript levels, total RNA was isolated (RNeasy kit; Qiagen, Valencia, CA), and cDNA was synthesized (high Capacity RNA-to-cDNA kit; Applied Biosystems, Carlsbad, CA) using C-terminally located primers 5'-CTACTGCTCAACATGCTC-3' (sense) (SEQ ID NO: 1) and 5'-CTCATCAGGTATACCATCC-3' (antisense) (SEQ ID NO: 2), which generate a 198 base pair product, according to the manufacturer's instructions. A minimum of three independent experimental replicates were performed in triplicate for all samples, and expression differences were calculated using the δ-δCt estimation method with 18S mRNA as a loading control. Corrections for primer efficiency, when necessary, were performed using the Pfaffl method.

[0044] Western blot analysis - WT hearts used for protein expression were excised from control mice (C57, 12-16 weeks old) from the Langendorff experiment. Total protein for calcium handling proteins was isolated by homogenizing whole hearts in ice-cold buffer containing 80 mM imidazole, 300 nM sucrose, 1 mM DTT, 10 mM sodium pyrosulfite, protease inhibitor cocktail P834 [Sigma, St. Louis, MO] and phosphatase inhibitor cocktail set II [EMD, Merck, Darmstadt, Germany]. Protein concentration was measured using the BCA Protein Assay kit [Pierce, Thermo Scientific, Rockford, IL].

[0045] Aliquots of proteins were separated on SDS-PAGE gels (Novex gels, Invitrogen, Eugene, OR) and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) and blocked with 5% BSA. Membranes were analyzed with primary antibodies. Where appropriate, membranes were cut in half to allow the use of two different primary antibodies. The top half was stained with Ca 2+ One of the handling proteins, the lower half (less than 60 kD) was used for the normalization protein, actin. In situations where cutting the membrane was not feasible because the two proteins were too close in size, the membrane was stripped after the first protein. The membrane was also stripped between the phosphorylated protein and the corresponding total protein primary antibody. Table A shows the amount of each protein loaded, the size of the protein, the dilution, and the manufacturer.

[0046] 3. Isolated Myocytes, Calcium Uptake and Handling Isolation of ventricular myocytes - Hearts from WT mice (12 weeks old) were perfused with modified Krebs-Henseleit buffer (KHB) (pH=7.4) consisting of NaCl 118 mM, KCl 5.4 mM, HEPES 10 mM, NaH2PO4 0.33 mM, MgCl2 2 mM, glucose 10 mM, taurine 30 mM, butanedione monoxime 10 mM in a Langendorff system. The hearts were then digested for 10 min with an enzyme solution (KHB containing 0.7 mg / ml type II collagenase, 0.1% BSA and 25 μM CaCl2). Finally, the ventricles of the hearts were excised, minced and filtered to obtain isolated cells. After sufficient sedimentation, the supernatant was removed and the cells were resuspended.

[0047] Measurement of myocyte contractile force - Myocytes were placed on a Plexiglas chamber containing Tyrode's solution (pH=7.4) consisting of NaCl 140 mM, KCl 5.4 mM, MgCl2 1 mM, CaCl2 1.8 mM, HEPES 5 mM, glucose 10 mM. Myocytes were excited under 0.5 Hz electric field stimulation. After reaching a steady state, myocyte shortening and shortening rate (+dL / dt) and relengthening rate (-dL / dt) were imaged with a CCD camera and monitored with a video edge detector. Drugs were dissolved in Tyrode's solution and perfused into the chamber. Data were collected continuously by PCLAMP 9 software.

[0048] muscle cell Ca 2+ Transient and cytoplasmic Ca 2+ Measurement of Ca - Myocytes were loaded with Fluo-4 acetoxymethyl ester (7 μM) for 20 min at room temperature, followed by washing with Tyrode's solution for 10 min, and then placed on a plexiglass chamber for recording. 2+ For transient measurements, myocytes were excited under 0.5 and 3 Hz electric field stimulation and fluorescent signals were acquired using a Nikon TE 2000 microscope and collected by an InCyt Standard photometry system. 2+For analysis, fluorescent images from resting myocytes were recorded using a Zeiss LSM 510 confocal microscope. Line scan mode was applied with 512 pixels spaced at 0.056 μm intervals at 3.07 ms intervals. Drugs were dissolved in Tyrode's solution and perfused into the chamber. Images of myocytes were recorded every 30 s after drug application. Data analysis was performed using Clampfit 9.2 and Image J 1.44 software.

[0049] Water-soluble probenecid was used in all myocyte experiments.

[0050] Electrophysiological recordings - Isolated myocytes were incubated in a 10-mL buffer containing 137 mM tetraethylammonium chloride (TEA-Cl), 5.4 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 10 mM glucose, and 3 mM 4-aminopyridine. + and K + The cells were perfused with Ca-free solution (pH = 7.4). Whole-cell patch clamp recordings were performed using an Axopatch-1B amplifier. 2+ For current recordings, glass pipettes were filled with a solution containing 115 mM aspartate, 115 mM CsOH, 20 mM CsCl, 11 mM EGTA, 10 mM HEPES, 2.5 mM MgCl2, 0.1 mM Na-GTP, and 2 mM Mg-ATP (pH adjusted to 7.2 with CsOH). Probenecid was used to perfuse myocytes, and Na + and K + The samples were dissolved in a pH-free solution. Data were collected by an Axon Digidata 1322A data acquisition system using pCLAMP9 software. All experiments were performed at room temperature (24°C).

[0051] Statistical Analysis - All data are expressed as mean ± standard error of the mean (SEM). Results were analyzed by paired and unpaired Student's t-test and one-way ANOVA, as appropriate. P values ​​≤ 0.05 were considered significant. Power analysis was used to determine the group size required to determine whether significant differences existed between endpoint measurements in control and experimental groups as previously described. SigmaPlot was used to generate best-fit lines and EC values ​​for dose-response curves. 50 was calculated.

[0052] result Probenecid increases contractile force in vivo in WT but not in TRPV2 - / - - Administration of probenecid to WT mice increased contractile force, as measured by EF, compared to EF in saline-fed control mice (Fig. 1A). Increases in contractile force were observed within 5 min after bolus injection for all doses ≥ 75 mg / kg (peak changes 5.26 ± 3.35, 8.40 ± 2.80, and 7.32 ± 2.52 for 75 mg / kg, 100 mg / kg, and 200 mg / kg, respectively) (Fig. 1B and 1C, data not shown for 75 and 100 mg / kg). Changes in contractile force measured at 5 min intervals (for a total of 30 min) revealed a dose-dependent increase in contractile force, with the estimated EC 50 The EF was 49.33 mg / kg (Figure 1D). EF remained elevated for at least 1 hour in subjects evaluated over a longer period (n=5, dose 200 mg / kg IV) (mean increase in EF over baseline 8.9±2.57, data not shown).

[0053] Probenecid inhibits TRPV2 + / - When probenecid was administered to mice, it increased contractile force, but the peak change in contractile force was only 49% of that observed at the same dose in WT mice (Fig. 1E). - / - When administered at 100 mg / kg, no discernible cardiac response was observed during a 30-minute echocardiogram.

[0054] Probenecid does not cause measurable changes in electrical conduction in mice in vivo - commonly used inotropes are known to produce a variety of arrhythmias. No supraventricular or ventricular arrhythmias or premature atrial or ventricular beats were observed in WT mice after administration of any dose of probenecid. Furthermore, no changes were measured in any of the conduction intervals at all doses (Figures 2A, 2B, and 2C), indicating that WT and TRPV2 - / - No significant differences were observed between mice treated with probenecid (100 mg / kg) IP (Figure 2D).

[0055] Probenecid increases contractile force in in vivo hearts and in Langendorff-perfused hearts. - Invasive measurements of +dP / dt revealed no significant effect of probenecid (10 -6 A similar effect was demonstrated in Langendorff-perfused hearts used to measure the cardiac-specific effects of probenecid (M). We found that in vivo administration of probenecid rapidly increased peak +dP / dt at both low (30 mg / kg) and high (100 mg / kg) doses (Fig. 3A). The time course of mean +dP / dt in Langendorff-perfused hearts is shown in Fig. 3B. The change was found to be statistically significant from 90 to 210 s and to reach a steady state between 120 and 180 s (Fig. 3B). Relaxation (-dP / dt) was also affected by the same concentration of probenecid, increasing from 1812 ± 158 to 2309 ± 133 (P < 0.01). After treatment with probenecid, hearts were flash frozen for use in other experiments.

[0056] TRPV2 mRNA is found in cardiac tissue. - WT, TRPV2 + / - and TRPV2 - / - Quantitative real-time PCR from mice revealed that TRPV2 + / - A decrease in expression was observed in mice, and TRPV2 - / -No counts were detected for mice. The PCR products of qRT-PCR were run on a 2% agarose gel to verify specificity, and expression of approximately 200 bp was observed, but the expression was not related to TRPV2. - / - TRPV2 + / - In contrast, it decreased (Figure 4).

[0057] Expression levels of calcium handling proteins are not altered after administration of probenecid. - Using hearts isolated from Langendorff animals and age-matched control mouse hearts perfused with Tyrode's solution, we estimated the expression of phospholamban (PLN) and ryanodine receptor (RyR) (phosphorylated and total protein) in the whole heart. Despite the fact that these hearts showed an increase in contractile force, we observed no significant changes in the amount of phosphorylated or total phospholamban or phosphorylated or total ryanodine receptor in these hearts (Figure 5A and 5B), nor in the ratio of p-PLN to t-PLN (Figure 5C). Furthermore, we found that another Ca receptor that may be responsible for the increase in contractile force was not altered by probenecid. 2+ To confirm whether there were handling proteins, we examined the sodium / calcium exchanger (NCX) and SERCA2a, but found no change in their expression levels (data not shown).

[0058] There is a dose-dependent increase in contractile force by probenecid in isolated myocytes. - Probenecid was found to increase the contractile force of isolated ventricular myocytes in a dose-dependent manner (Figure 6). The fractional shortening (FS) of myocytes increased by 30.8 ± 1.4% (from 6.9% to 9.0%; n = 6, P < 0.01) when exposed to 0.1 μM probenecid at 0.5 Hz and room temperature, and from 10.1% to 13.9% (n = 4; P < 0.01) when exposed to 3 Hz and 32°C; the dose-response curve for the effect of probenecid was EC 50The maximal +dL / dt was 1.6 nM (Fig. 6A and 6B). Pretreatment of myocytes with ruthenium red, a nonselective blocker of TRPV2 channels, completely abolished the effect of probenecid on myocyte contractile force (Fig. 6C and 6D). In isolated myocytes, we measured +dL / dt and -dL / dt. At room temperature, probenecid treatment led to a change in maximum +dL / dt from 80.6 ± 12.3 to 112.5 ± 16.6 (n = 10, P < 0.01), and -dL / dt increased from 57.7 ± 7.4 μm / s to 77.6 ± 8.7 μm / s (P < 0.01).

[0059] muscle cell Ca 2+ The effect of probenecid on the Ca transient was also examined. 2+ Probenecid at a concentration of 0.1 μM significantly increased the amplitude of the transients (ΔF / F0) (Fig. 6E and 6F), consistent with the increase in the shortening rate seen in myocytes. 2+ The amplitude of the transient (ΔF / F0) increased from 3.35±0.20 to 4.25±0.25 (P<0.001), but not Ca 2+ The decay rate of the transient (tau) was unaffected by probenecid treatment ( Fig. 6<em>F ).

[0060] Probenecid-induced cytoplasmic Ca in isolated muscle cells 2+ - Increase in cytoplasmic Ca 2+ and SR Ca 2+ The effect of probenecid on SR release was further examined using confocal imaging. Probenecid (0.1 μM) induced a significant increase in SR release (Ca 2+ (measured by spark frequency) and cytoplasmic Ca 2+ This caused a stepwise increase in the concentration of Ca (Figures 7A and 7B). On average, the spark frequency increased by a factor of two, from 1.45 ± 0.09 to 2.99 ± 0.24. 2+ began to increase 1 min after probenecid treatment and typically peaked after about 5 min (Fig. 7C). Once a steady state was reached, cytoplasmic Ca 2+The probenecid-treated myocytes increased cytosolic Ca by 2.5 ± 0.2-fold compared to the control (Fig. 7D). When ruthenium red (1 μM) was applied to myocytes, the probenecid-induced cytosolic Ca 2+ The increase in Ca was completely blocked (Fig. 7E). Patch clamp experiments showed that probenecid did not induce significant transmembrane Ca 2+ Holding myocytes at -70 mV and exposing them to 0.1 μM probenecid did not trigger measurable inward Ca influx. 2+ No currents were detected (Fig. 7F, left). However, depolarization of the cell membrane from -70 mV to +10 mV elicited robust L-type Ca currents. 2+ SR Ca currents were evoked (Fig. 7F, right). 2+ The increase in release of cytoplasmic Ca 2+ Emptying the SR contents with the SERCA blocker thapsigargin reduced the cytosolic Ca levels induced by probenecid. 2+ The changes in the IL-1 expression level disappeared (Fig. 7G and Fig. 7H).

[0061] Probenecid has previously unrecognized positive inotropic properties, the effects of which are mediated by TRPV2 channels.

[0062] These data show that probenecid increased contractile force in mouse hearts as measured by echocardiography and in a dose-dependent manner by invasive measurements. However, these studies required the introduction of a jugular catheter, which is traumatic for the animals and may stimulate a sympathetic response. IP injection of a lower dose (100 mg / kg) was observed to have a similar effect with a simpler method of administration (hence this method and dose was used in the remaining studies).

[0063] Probenecid has been documented to cause a transient increase in certain monoamines in the brain, CSF and plasma. These studies suggested that probenecid therapy could increase the concentrations of 5-HIAA and HVA (which are acid metabolites of serotonin and dopamine, respectively) in dogs and the levels of norepinephrine or DOPAC (also acid metabolites of dopamine) in humans. A series of experiments was performed to determine whether the β-adrenergic (β-ADR) pathway was stimulated by the administration of probenecid. It was concluded that the increase in contractile force observed in the in vivo experiments occurs through a calcium-dependent mechanism and not through β-ADR-mediated phosphorylation of RyR or PLN. This is important because it has been widely documented that β-ADR stimulation has cardiotoxic effects, resulting in increased infarct size due to increased oxygen demand, induction of cardiomyocyte apoptosis and hypertrophy, and because β-antagonism is known to be cardioprotective and safe in the treatment of heart failure. Thus, cardiac β-ADR stimulation (direct or indirect) increases contractile force, but is accompanied by increased ischemia and cell death and is considered to be harmful in patients with ischemic heart disease. The data presented here demonstrate, by ex vivo and in vivo experiments, that the mechanism of action of probenecid (i.e., causing increased contractile force) is independent of these pathways and may be potentially useful as a positive inotropic agent in patients with ischemic heart disease. Furthermore, WT and TRPV2 - / - A very thorough analysis of all ECG recordings before, during and after IP or IV administration of probenecid to mice demonstrated the absence of arrhythmias and significant changes in any of the ECG variables measured, which is in stark contrast to commonly used inotropes that produce significant arrhythmias including atrial tachycardia and AV conduction defects (digoxin) as well as ventricular and supraventricular arrhythmias (dopamine, dobutamine, isoprotenelol).

[0064] Although the initial in vivo experiments described only an increase in contractile force, without describing the mechanism, we subsequently performed a series of Langendorff experiments to dissociate the systemic from the cardiac-specific effects of probenecid. These experiments showed an increase in contractile force similar to that observed in vivo. -6 The concentration of M indicated that this dose produced the most reproducible responses. Furthermore, the fact that these experiments produced corresponding changes in contractile force within a similar time frame (5 min in each case) clearly argues against a significant systemic increase in adrenergic drive as the primary cause of the positive inotropic effect. We also observed that the increase in contractile force was associated with an increase in the rate of relaxation in isolated myocytes when we measured the fractional shortening (+dL / dt) and fractional relengthening (-dL / dt), which are comparable, if not identical, to the fractional shortening (+dP / dt) and fractional relaxation (-dP / dt) seen in the ex vivo studies.

[0065] Probenecid was identified as a TRPV2 agonist in 2007 using TRPV2-expressing HEK293 cells. Fluro-3 AM calcium imaging experiments showed that probenecid suppressed cytoplasmic Ca in TRPV2-expressing cells. 2+ It has been demonstrated that TRPV2 induces a significant increase in Ca in cells expressing other thermo-TRP channels, including TRPV1, TRPV3, TRPV4, TRPM8, and TRPA1, but not in cells expressing other thermo-TRP channels, including TRPV1, TRPV3, TRPV4, TRPM8, and TRPA1. As a member of the thermo-TRP channel family, TRPV2 is a Ca channel that is activated by noxious heat (>52°C). 2+ Activation of TRPV2 is a selective channel that upregulates Ca in neuronal cells localized to the dorsal root ganglion. 2+ It has been shown that cytoplasmic Ca influx 2+ It was hypothesized that the stimulatory effect of probenecid on myocyte contractile force involves activation of the TRPV2 channel. This hypothesis was supported by the finding that ruthenium red (RR), a blocker of the TRPV2 channel, reduces contractile force and cytoplasmic Ca 2+This is supported by the fact that RR can completely abolish the effects of probenecid on both calcium levels and calcium release from ryanodine receptors. RR is established as a general antagonist of TRPV channels, acting by blocking their aqueous pore, but it has also been shown to affect mitochondrial calcium uptake and calcium release from ryanodine receptors.

[0066] TRPV2 - / - Mice were obtained and in vivo functional experiments were performed, showing that after exposure to probenecid (100 mg / kg), TRPV2 + / - In mice, contractile force was reduced by about 50%, but TRPV2 - / - There was no change in function in the mice. Although these findings argue for a requirement of the TRPV2 receptor for increased contractile force (as it is a whole-body, not a cardiac-specific, knockout), they do not establish that the effect is directly dependent on these receptors in cardiomyocytes.

[0067] At the cellular level, data indicate that probenecid increased contractile force in mouse ventricular myocytes, although cardiac fibroblasts were not examined separately. TRPV2 is known to be present and active in various types of fibroblasts, which could potentially be involved in the observed effects. Also, probenecid increased Ca 2+ It was also found to increase the amplitude of the cytoplasmic Ca transient in resting muscle cells. 2+ A stepwise increase in Ca concentration was observed. Interestingly, no measurable inward Ca 2+ No current was detected upon exposure to probenecid, indicating that Ca 2+ Probenecid-triggered cytoplasmic Ca influx 2+ These results suggest that SR Ca was not the direct cause of the increase in Ca levels. 2+ Increased release of cytoplasmic Ca 2+ Probenecid appeared to play a major role in increasing Ca 2+Significantly increasing spark frequency and emptying the SR with a SERCA blocker increased cytoplasmic Ca 2+ It was found that the effect of probenecid on EXAMPLES

[0068] The data in Example 1 demonstrate that probenecid has a previously unrecognized positive inotropic property. The data in Example 1 also demonstrate that probenecid has no significant adverse electrophysiological properties (i.e., no arrhythmias were observed) and therefore may be a useful drug for the treatment of heart failure. Since other positive inotropes are known to be cytotoxic, the data in Example 2 demonstrate the potential cytotoxic / apoptotic properties of probenecid and its potential use as a positive inotrope in a mouse model of ischemic heart disease. These data suggest that probenecid functions by a different mechanism than traditionally used inotropes and increases contractile force and improves post-ischemic cardiac function without shortening cell survival or increasing apoptosis. In addition to an established, clinically relevant mouse ischemia / reperfusion (I / R) model, data were generated in vitro using the HL-1 cardiac cell line and cardiac function was monitored by echocardiography.

[0069] method Cell viability assay - Cell viability was estimated using the ApoTox-Glo Triplex Assay (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, HL-1 cells were cultured at 1 × 10 4Cells were seeded at a density of 1000 cells / well and grown overnight in Claycomb medium supplemented with 10% FBS, 100 U / ml penicillin / streptomycin, and 2 mM L-glutamine. Cells were treated with the indicated doses of probenecid or isoproterenol in a total volume of 100 μl of supplemented growth medium and incubated overnight. GF-AFC (GF-AFC viability) was then added to ApoTox-Glo Assay Buffer at a ratio of 10 μl of GF-AFC to 2.0 ml of Assay Buffer, 20 μl of this mixture was added to each well, and cells were incubated at 37° C. for 30 min. Fluorescence was measured at 400 nm (ex) / 505 nm (em) and data were normalized to untreated control cells.

[0070] Water-soluble probenecid was used in all of the experiments.

[0071] Ca 2+ Imaging and measurements of fluorescence. -HL-1 cells were obtained as described previously. Dissociated cells were plated on glass coverslips (GG-25-polylysine #1, Neuvitro, CA) placed inside each well of a 6-well plate (BD Falcon). Image acquisition was performed at 40-65 Hz using an inverted Axiovert 200 M BP [Carl Zeiss Microscopy, LLC, Thornwood, NY] and an LSM 510 Meta system equipped with LSM5 software. Image acquisition was performed with a Plan-Apochromat 40x / water at frame size 512 (X) and 256 (Y). To optimize acquisition speed, the acquisition area was limited to a few cells. Ca2+ diluted in Tyrode's solution [Sigma-Aldrich, St. Louis, MO] to a final concentration of 1 μM was used. 2+Cells were loaded with the indicator dye FLUO4-AM [Molecular Probes, Life Technologies, Carlsbad, CA] for 20 min at 37°C and then washed twice for 5 min in Tyrode's solution. Coverslips with dye-preloaded adherent HL1 cells were placed in a recording chamber mounted on an inverted microscope and subsequently submerged in Tyrode's solution before being perfused with probenecid. After focusing and taking baseline images, probenecid (10 in various experiments) was added to the wells and washed twice for 5 min in Tyrode's solution. -8 ~10 -4 Cells were perfused with 0.5 ml / 15 s of 100 mM NaCl. For these experiments, image exposure time was 5 ms, camera on-chip multiplication gain was 500-600, and 700 cycles were performed for each run of various concentrations. A 488 nm line of a multiline argon laser excited FLUO-4, and an electronic shutter controlled cell exposure to the laser. Offline image processing was performed by LSM5 software, and statistical analysis of the data was performed using MS Excel (Microsoft) and SAS 9.2 [SAS Institute, Cary, NC].

[0072] After image acquisition from the stored LSM files, the peak fluorescence before perfusion (F0) and peak fluorescence after perfusion (F) were determined manually. A circular area within the cytoplasm of each cell in the stored images for the entire duration of each experiment was used to determine the change in fluorescence before and after perfusion.

[0073] Normalized fluorescence, determined as the quotient of F / F0, was used as the variable to compare the relative changes in fluorescence at various increasing concentrations in statistical analysis. Maximal likelihood estimates of the dependent variable (log[F / F0]) were determined as a function of probenecid (log[probenecid], M) dose concentration using proc mixed modeling for repeated measures in SAS. Dose-response relationships were modeled using linear regression and linear regression plots of the model.

[0074] Detection of cell death. - Hearts were harvested from mice exposed to probenecid or sugar water (control) under various conditions. Mice in these experiments were divided into 4 groups, including control (n=4), probenecid-treated water (n=4), control after I / R injury (n=4), and probenecid-treated water after I / R injury (n=4). Hearts were removed, rinsed in PBS, fixed in 3.7% buffered formaldehyde (buffer was changed after 30 min), and dehydrated (hearts were placed in 70% ethanol after 18-24 h). Samples were embedded in paraffin and sectioned 6 μm thick with 10 μm intervals between each section, and two heart sections were mounted on each slide. Slides were prepared using the in situ apoptosis detection kit CardioTACS [Trevigen, Gaithersburg, MD] according to the manufacturer's instructions. After staining, slides were placed on a microscope (Olympus 1X71) and the number of apoptotic cells was counted in 5 fields for each heart section by two independent blinded readers. Total cell numbers were also determined in the same fields and apoptotic cells were calculated as a percentage of total cells. These values ​​were then averaged for each group of probenecid and control mice.

[0075] Animals - All mice [C57BL6J, Jackson Laboratories, Bar Harbor, ME] were male and aged 12-16 weeks.

[0076] Ischemia / reperfusion - Mice were anesthetized with sodium pentobarbital and the heart was exposed through a left thoracotomy at the level of the fourth intercostal space. Mice were intubated and placed on a mini-ventilator (Harvard apparatus, Holliston, MA). A loop occluder was placed around the left anterior descending artery (LAD) and tightened to occlude the LAD for 30 or 45 minutes. After surgery, the thorocotomy was sutured over and the animals were allowed to recover.

[0077] All mice underwent echocardiographic measurements as described 24 hours after IR to determine the severity of injury in vivo.

[0078] Echocardiography - All echocardiographic examinations were performed as previously described for Example 1.

[0079] Ischemia / reperfusion followed by probenecid bolus - I / R studies were performed with 45 min of ischemia as previously described. Echocardiograms were performed 24 h after I / R and weekly thereafter. Mice were grouped into four groups: saline and probenecid (n=5 for saline and n=6 for probenecid) for subgroups of mice with post-I / R EF of 50-60% or saline and probenecid (n=6 for saline and n=6 for probenecid) with subgroups of mice with post-I / R EF of 40-50%. Probenecid mice were treated with a 100 μl (100 mg / kg) IP bolus, whereas the saline group received only 100 μl of saline IP. Mice were imaged at 5 min intervals for a total of 30 min.

[0080] Ischemia / reperfusion followed by oral probenecid - I / R studies were performed as described above with 45 min of ischemia, and echocardiograms were performed 24 h after I / R and weekly thereafter for 4 weeks. After 24 h, mice were randomly assigned to cages with probenecid-treated or non-treated water. Probenecid was dissolved in water containing 5% sucrose at a concentration of 0.5 μg / ml. Water was changed twice weekly and the volume was measured before and after consumption to determine the approximate dose. Mice were housed two per cage to more accurately estimate the total dose administered.

[0081] Probenecid bolus followed by ischemia / reperfusion - saline or probenecid (100 mg / kg) was administered IV 15 min before 45 min of coronary artery occlusion (i.e., at the time of maximal increase in contractile force). 24 h after I / R, hearts were removed for echocardiography followed by measurement of infarct size and normalization to area at risk.

[0082] Statistical Analysis - All data are expressed as mean ± standard error of the mean (SEM). Results were analyzed with paired and unpaired Student's t-tests and one-way ANOVA, as appropriate. P values ​​≦0.05 were considered significant. For all in vivo studies, power analysis was used to determine the group size required to determine whether significant differences existed between endpoint measurements in control versus experimental groups as previously described.

[0083] result Probenecid does not induce cell death in vitro. - In these experiments, HL-1 cells were used to assay the effect of probenecid. -8 No significant differences were observed in the measured median peak cytoplasmic fluorescence before and after perfusion of probenecid. Additionally, the median peak fluorescence after perfusion of higher concentrations of probenecid was significantly higher than the preperfusion value (Figure 8A).

[0084] Increasing concentration (10 -8 ~10 -4 A log dose-response curve for the log of normalized fluorescence (F / F) at 100 nm showed a linear response as the dose of probenecid increased. The R of the best-fit regression line of the dose-response 2 The value was 0.67 (Figure 8B). The lowest probenecid concentration was 10 -8 Statistically significant differences were observed in the responses at higher concentrations versus the other (p values ​​ranged between 0.001 and 0.01 for these pairwise comparisons).

[0085] In addition, treatment of HL-1 cells with increasing doses of probenecid did not result in detectable cytotoxicity at 4, 8, or 24 hours after treatment (Figure 8B). However, similar treatment with 1.0 mM isoproterenol induced a significant amount of cell death at 4 and 8 hours after treatment ( * p<0.001), and caused significant cell death after 24 hours of treatment ( ** p < 0.001), viable cells were less than 20% of the cells and were indistinguishable from the H2O2 positive control (Figure 8C).

[0086] Probenecid does not cause significant cell death of muscle cells in vivo. Oral probenecid therapy in healthy mice did not induce significant apoptosis after 2 weeks of treatment where the average intake of probenecid was estimated to be 190.56±6.33 mg / kg / day based on the water consumption of paired mice per cage. Furthermore, mice subjected to I / R and then randomly assigned to probenecid-treated or non-treated water showed no significant difference in the amount of cell death between probenecid-treated and non-treated water (Figure 2A). As expected, mice subjected to I / R injury showed higher levels of apoptosis compared to non-I / R mice (Figure 9).

[0087] Bolus probenecid acutely improves function in ischemically damaged hearts. After I / R injury, IP administered probenecid caused a statistically significant increase in EF in all treated mice. Interestingly, treatment with probenecid was able to increase function more strongly in mice exhibiting lower initial EF, as shown by the strong correlation between the probenecid-induced increase in EF and initial EF (Figure 10A). This effect appeared in a similar time frame as previously reported, with the first increase observed at 1 and 5 min in both groups. However, the peak effect was seen at 10 min in mice with higher initial EF (50-60%) (change in EF was 19.72 ± 4.10%), whereas probenecid continued to increase contractile force in mice with lower EF (40-50%) for the entire 30 min experimental period (Figures 10B and 10C).

[0088] Oral probenecid improves function after ischemia and reperfusion injury. Twenty-four hours after I / R injury, there were no significant differences in EF or LV Vol;d between mice randomized to water control and subsequently treated with probenecid (EF: 43.44±2.05 and 41.81±3.40, respectively, p=0.65; LV Vol;d: 66.51±2.65 and 71.99±5.44, respectively, p=0.39).

[0089] In the probenecid group, LV cavity size (LV Vol;d) showed minimal dilation 1 week after the experiment but did not deteriorate during the experiment. However, in sham-treated mice, LV cavity size (LV Vol;d) was further increased (water 22.55±4.47 and probenecid 7.59±6.08, p=0.076, FIG. 11A). In addition, radial displacement was reduced in water-treated mice compared to probenecid-treated mice (representative echocardiograms shown in FIG. 11B and FIG. 11C). This indicates that myocardial function was preserved (water 0.018±0.018 and probenecid 0.071±0.013, p<0.05, FIG. 11D and FIG. 11E). Thus, despite equal initial ischemia / reperfusion injury, probenecid-treated mice had preserved function compared to controls.

[0090] Probenecid does not exacerbate ischemia and reperfusion injury. Adrenergic stimulation increases myocyte contractile force, but is clearly associated with cell damage and death by several mechanisms, including increased metabolic demand and induction of apoptotic mechanisms. This study was performed to evaluate the effect of increased contractile force observed with probenecid administration during an ischemic event. Infarct size, as measured by histological examination, was not significantly different in mice pretreated with probenecid compared to mice pretreated with saline for both the 30- and 45-min ischemia groups after 24 h of reperfusion (Figure 12A). Furthermore, contractile function (measured by EF and circumferential global strain) in these mice was not significantly different between saline and probenecid-treated groups (Figures 12B and 12C). Interestingly, when regional wall motion was determined by strain imaging measuring radial displacement, there was a significant difference in anterior wall function after 30 min of ischemia compared to 45 min of ischemia, but no difference between treatment groups (30 min, saline 0.43 ± 0.02 mm vs. probenecid 0.46 ± 0.06 mm, and 45 min, saline 0.36 ± 0.03 mm vs. 0.35 ± 0.06 mm, p > 0.05 between both saline and probenecid treatment groups) (Figures 12D and 12E).

[0091] Consideration The data from Example 1 demonstrate that probenecid, an FDA-approved drug, has inotropic properties that have gone unrecognized for decades. These experiments demonstrate that probenecid increases cytoplasmic Ca 2+ We demonstrated that adrenergic stimulation increases myocardial metabolic demand, stimulates apoptotic pathways, and results in increased contractile force independent of β-adrenergic signaling. The lack of stimulation of the β-AR pathway initiated the studies presented herein, since adrenergic stimulation has already been shown to increase myocardial metabolic demand, stimulate apoptotic pathways, and cause myocyte hypertrophy. This is reflected in many clinical trials involving traditional β1-agonists (dobutamine, dopamine) and PDIs (i.e., milrinone), which found increased short- and long-term mortality in patients exposed to these drugs despite improvements in symptoms and hemodynamic parameters.

[0092] Two major findings of this study address these issues: 1) the lack of cell death associated with improved inotropic response, and 2) the positive inotropic effect of probenecid in ischemically injured hearts.

[0093] Regarding the latter, we used a clinically relevant model of ischemic heart disease in which vessels are opened after a short period of ischemia. This pathophysiology is not different from the typical clinical course of acute myocardial infarction, in which revascularization occurs for a short period and the subsequent ischemic damage is minor. The model uses a radiofrequency probe, which allows the detection of small decreases in systolic function, allowing us to dichotomously divide mice into mildly damaged (EF 50-60%) and moderately damaged (EF 40-50%). The present finding that probenecid not only increases contractile force as previously described, but also inversely correlates with postischemic EF (the moderately damaged group with EF 40-50% showed a larger increase in EF), importantly lends credence to the translational potential of this drug for use in patients with moderate systolic dysfunction.

[0094] Although the mechanism of action of probenecid in uninjured / non-ischemic muscle cells has already been reported by our group, we are now exploring the effect of TRPV2 channels as modulators of contractile force in response to stressors (e.g., ischemia). There is some evidence outside the cardiovascular system that describes TRPV2 channels found to be located in intracellular compartments, mainly in urothelial cells, pancreatic cells and neuroendocrine cells, and activated and / or translocated to the cell membrane after stretch stimuli. Although this study did not aim to resolve the role of TRPV2 in this process, the results of this study demonstrate that the effect of probenecid on the heart is enhanced in ischemically damaged myocardium. This is consistent with previous studies demonstrating that translocation and activation of TRPV2 is mediated by stress (i.e., by stretch, increased cAMP) and may be a useful compensatory mechanism after injury.

[0095] As further evidence for the use of probenecid as a potential therapeutic agent in I / R, it was also demonstrated that treatment with oral probenecid reduced the deterioration of cardiac function (as evidenced by less ventricular dilation and improved systolic function) compared to sham-treated mice.

[0096] The second important finding of this study was that probenecid did not cause cell damage both in vivo and in vitro. This is a dramatic finding, since most clinically available inotropes have been shown to increase mortality and stimulate cardiotoxic pathways leading to increased infarct size and apoptosis. However, even high-dose therapy did not result in significant cell death (compared to untreated water) after 2 weeks of therapy in vivo or at several different time points in vitro and at concentrations substantially higher than those achieved when administered clinically. This finding could be potentially important, since only digoxin can be used orally as a positive inotrope for long periods of time, but digoxin has a very narrow therapeutic window and requires strict monitoring. This is in stark contrast to probenecid, which has had a positive safety profile for decades and is safe even when administered at very high doses in mouse models.

[0097] In summary, the present data represent the next step in the translational process that began with the intriguing discovery that probenecid is a positive inotropic agent. These data demonstrate that probenecid improves function after I / R injury and does not induce an apoptotic response in vivo or in vitro. These findings, together with previous studies shown in Example 1 that demonstrate a β-AR-independent mechanism of action for probenecid, indicate that probenecid can be safely used to treat heart failure in humans. EXAMPLES

[0098] Probenecid induces an increase in intracellular calcium concentration in HL-1 cells in vitro without increasing cell death. We confirmed that probenecid increases intracellular calcium but does not damage cardiac cells. This is different from isoproterenol (a commonly used β-agonist), which also increases contractile force at clinically relevant concentrations but clearly increases cell death. Even at supraphysiological concentrations, probenecid does not cause cell death and in fact tends to extend cell survival. We attribute this to the fact that probenecid nudges the heart with a small increase in intracellular calcium without activating pro-cell death pathways. Isoproterenol, on the other hand, triggers signaling that leads to both a more dramatic increase in calcium levels and activation of pro-cell death and hypertrophy pathways known to be damaging. Estimation of the activity of some of these pathways demonstrates that probenecid does not activate β-AR-dependent pathways (target of isoproterenol and dobutamine) and does not alter the phosphorylation state of calcium handling proteins that are regulated by isoproterenol to increase calcium levels.

[0099] There is no difference in cardiac cell death between untreated and probenecid-treated mice with or without ischemic injury (corresponding to a heart attack). This confirms that probenecid is safe for oral use (administered in water) after a heart attack in mice and confirms that probenecid does not increase cell death in high-stress injury models. Similar studies with β-AR agonists show increased infarct size, suggesting further damage.

[0100] Furthermore, mice treated with probenecid had less ventricular dilation (increased heart size) and functioned better than untreated mice.

[0101] Following ischemic insult (heart attack), a more potent effect of probenecid (administered intraperitoneally) is seen in mice with the most damage (ie, larger infarct areas).

[0102] We measured cardiac function (EF) in mice after injury. The lower the EF after myocardial infarction (MI), the stronger the inotropic effect. This lends credence to its use in ischemically damaged hearts and suggests that even low doses may be effective in damaged or failing hearts.

[0103] Probenecid improves function as measured by echocardiography when administered by continuous infusion at 5 mg / kg / h (first 60 min) and 10 mg / kg / h (second 30 min) in healthy porcine models. This effect was also seen when probenecid was administered as a bolus of 5 mg / kg IV.

[0104] While the present invention has been illustrated by one or more embodiments thereof and the embodiments have been described in considerable detail, these embodiments are not intended to limit the scope of the appended claims or in any way restrict its scope to such details. The various features shown and described herein can be used alone or in any combination. Further advantages and modifications will readily occur to those skilled in the art. Thus, the invention in its broader aspects is not limited to the specific details, representative apparatus and methods, and examples shown and described. Accordingly, departures may be made from such details without departing from the general inventive concept.

Claims

1. A composition for treating cardiac dysfunction in a subject, comprising an amount of probenecid effective to treat the cardiac dysfunction, the composition being administered by at least one of injection or transdermal administration, wherein the therapeutically effective amount of probenecid is administered for a period of about 8 hours / day to about 24 hours / day.

2. The composition of claim 1, wherein the injection is an intravenous infusion.

3. 2. The composition of claim 1, wherein the injection is a continuous intravenous infusion.

4. 10. The composition of claim 1, which maintains a therapeutic plasma concentration of probenecid for a duration ranging between about 18 hours / day to about 24 hours / day.

5. 10. The method of claim 1, wherein probenecid is administered at a dosage ranging from about 1 mg / kg / day to about 100 mg / kg / day.

6. 10. The composition of claim 1, wherein the subject is treated with a therapeutically effective amount of probenecid for at least one week.

7. 10. The composition of claim 1, wherein the subject is treated with a therapeutically effective amount of probenecid for at least one month.

8. 2. The composition of claim 1, wherein the cardiac dysfunction is congestive heart failure.

9. The composition of claim 8, wherein the congestive heart failure is decompensated heart failure.

10. A composition for treating cardiac dysfunction in a subject, comprising probenecid in an amount effective to treat cardiac dysfunction, the composition being an extended release formulation administered by oral administration, the extended release formulation maintaining a therapeutic plasma concentration of the probenecid for a duration ranging from between about 18 hours / day to about 24 hours / day.

11. The composition of claim 10, wherein probenecid is administered at a dosage ranging from about 1 mg / kg / day to about 100 mg / kg / day.

12. The composition of claim 10, wherein the subject is treated with a therapeutically effective amount of probenecid for at least one week.

13. The composition of claim 10, wherein the subject is treated with a therapeutically effective amount of probenecid for at least one month.

14. The composition of claim 10, wherein the cardiac dysfunction is congestive heart failure.

15. The composition described in claim 14, wherein the congestive heart failure is decompensated heart failure.

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