WO2015130568A1

WO2015130568A1 - Combination therapy for resistant hypertension

Info

Publication number: WO2015130568A1
Application number: PCT/US2015/016818
Authority: WO
Inventors: Mark Charles Kowala
Original assignee: Eli Lilly And Company
Priority date: 2014-02-28
Filing date: 2015-02-20
Publication date: 2015-09-03
Also published as: AR099416A1

Abstract

The present invention provides a method of treating resistant hypertension, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil.

Description

COMBINATION THERAPY FOR RESISTANT HYPERTENSION

The present invention relates to a combination of the mineralocorticoid receptor (MR) antagonist [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-l l-ylidene)methyl]-l- [(lR)-l-methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, with tadalafil, including fixed-dose combinations thereof, and to methods of using the combination to treat resistant hypertension.

The present invention is in the field of treatment of resistant hypertension.

Resistant hypertension is a serious cardiovascular disorder that affects millions of patients worldwide. Resistant hypertension has been defined as uncontrolled blood pressure (BP) despite use of > 3 antihypertensive agents from different classes, or controlled BP with the use of > 4 agents, and has an estimated prevalence of 10-15% among all participants treated for hypertension (See Calhoun et al., Hypertension, 2014; 63:451-458, Calhoun et al., Hypertension, 2008; 51: 1403-1419). Alternatively, resistant hypertension refers to blood pressure in patients that does not reach the recommended goals of systolic blood pressure (SBP) <140 mmHg (for patients 30 to 59 years old) or <150 mmHg (for patients >60 years old) or diastolic blood pressure (DBP) <90 mmHg, despite treatment with 3 or more recommended first-line therapies (See 2014 Evidence-Based Guideline for the Management of High Blood Pressure in Adults, James et al., JAMA, 2014; 311(5): 507- 520). Thus, by clinical definition, the resistant hypertension patient represents one whose hypertension persists despite treatment with multiple antihypertensive agents representing different mechanisms of action. These agents may include thiazide-type diuretics, angiotensin converting enzyme-inhibitors (ACEi); angiotensin receptor blocker (ARB); a adrenergic receptor antagonists; β adrenergic receptor antagonists, central acting agents; calcium-channel blockers (CCB); and/or mineralocorticoid receptor antagonists (MRA). Patients with resistant hypertension are more likely to have cardiovascular disease, manifest as stroke, myocardial infarction, or congestive heart failure when compared with patients with more easily controlled hypertension (See Calhoun et al., Hypertension, 2014; 63:451-458). Thus it is believed that that even partial reduction of elevated blood pressure in resistant hypertension patients might result in a significant long-term cardiovascular benefit, providing desirable clinical improvements for resistant hypertension. In view of current treatments, notably including multiple and various drug combinations, which afford only partial, incomplete, or no benefits to the resistant hypertension patients, there is a significant unmet need in the treatment of resistant hypertension.

When used separately, MR antagonists and PDE 5 inhibitors have each previously been reported to have blood pressure lowering effects in patients with hypertension, which is generally also referred to as essential or primary hypertension. Studies have shown that hyperaldosteronism may be common in patients with resistant hypertension (See Pimenta and Calhoun, Circulation, 2012; 125: 1594-1596). Aldosterone, the primary endogenous mineralocorticoid, regulates hemodynamic homeostasis by promoting sodium and water reabsorption and potassium excretion following interaction with the mineralocorticoid receptor (MR). Because of aldosterone's role in maintaining electrolyte and water balance, MR antagonists have been used for the treatment of numerous physiological disorders including hypertension. Some existing MR antagonists produce effects which limit their safety and /or effectiveness. For example,

spironolactone is nonselective and cross reacts with other nuclear hormone receptors (e.g. the androgen receptor (AR), the progesterone receptor (PR), or the glucocorticoid receptor (GR)) which mediate other physiological processes. Spironolactone therapy has been associated with hyperkalemia as well as gynecomastia, erectile dysfunction, reduced libido, irregular menses, as well as gastric distress. Eplerenone, though selective for MR relative to the other nuclear hormone receptors, has also been associated with

hyperkalemia. Thus, there remains a need in the art for alternatives to existing MR antagonist therapies.

The MR antagonist compound [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-l l- ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, alternatively represented as (E)-N-(5-((E)-3-fluoro-6H-dibenzo[b,e]oxepin-l l- ylidenemethyl)-l-((R)-l-methyl-2-morpholin-4-yl-ethyl)-l,3-dihydro-benzoimidazol-2- ylidene)-urea, alternatively represented as (E)-[6-[(E)-(3-fluoro-6H- benzo[c] [ 1 Jbenzoxepin- 11 -ylidene)methyl] -3 - [( 1 R)- 1 -methyl-2-morpholino-ethyl] - 1H- benzimidazol-2-ylidene]urea, can also be represented by the structural formula I, shown below:

Formula I.

An MR antagonist of Formula I, and methods of making and using said compounds as useful therapeutic agents for therapeutic indications such as hypertension, are recited in US 20090163472, published June 25, 2009, incorporated herein by reference, and see WO2009/085584. An MR antagonist of Formula I is a potent and selective antagonist of MR.

Phosphodiesterase 5 (PDE5) inhibitors are selective blockers of the enzyme PDE5, which degrades the potent vasodilator cyclic guanosine monophosphate (cGMP). The vascular tone, systemic vasodilation, and circulation are regulated through the endothelial production of NO from the systemic arteries and veins. After its production, NO diffuses into the adjacent smooth muscle cells and enhances the production of cGMP and leads to both vascular smooth muscle relaxation and an increase in systemic vasodilation. Inhibition of PDE5 results in an increase in the intracellular levels of cGMP and prolonged duration of cGMP action. Since PDE5 is widely distributed in the body, artisans have believed that inhibition of PDE5 could lead to significant vasodilation, which could benefit patients with cardiovascular disease such as coronary artery disease. This led to the development of PDE5 inhibitors with the first being sildenafil citrate. Studies of sildenafil in patients with coronary artery disease demonstrated a modest cardiovascular effect but a potent action on penile erection in men, resulting in sildenafil becoming the first approved PDE5 inhibitor for treatment of erectile dysfunction (ED). Yet, despite the success of PDE5 inhibitors in the treatment of ED, in some patients ED is particularly difficult to treat and cannot be adequately treated with PDE5-monotherapy, and these patients can be considered to have PDE5 -monotherapy resistant ED. Because PDE5 is found in smooth muscle cells of the systemic arteries and veins throughout the body, PDE5 inhibitors have mild vasodilator effects and thus have the potential to impact the cardiovascular system. Sildenafil, vardenafil, and tadalafil are mild vasodilators and may cause small drops in blood pressure, however the degree of the decrease in blood pressure is usually small (Kloner, Circulation, 2004; 110:3149-3155). Use of PDE5 inhibitors for essential hypertension has been a subject of clinical research (Oliver JJ, Melville VP, Webb DJ. Hypertension, 2006; 48:622-627), but none have progressed to FDA approval for essential hypertension. In contrast, tadalafil and sildenafil have each been approved for treatment of pulmonary arterial hypertension (PAH).

Tadalafil, also represented as (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6- (3,4-methylenedioxyphenyl)-pyrazino[2',l':6,l]pyrido[3,4-b]indole-l,4-dione, can be represented by the structural fo

Formula II.

Tadalafil, and methods of making and using tadalafil as a therapeutic agent for indications including hypertension, are recited in U.S. Patent No. 5,859,006, incorporated herein by reference, and WO 1995/19978 published July 27, 1995. The inhibition of PDE5 by tadalafil results in an increase of the concentration of cGMP. Tadalafil tablets are available, where approved, in 2.5 mg, 5 mg, 10 mg, and 20 mg dosages, and contain the inactive ingredients lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, magnesium stearate, hydroxypropyl cellulose and sodium lauryl sulfate. Tadalafil is a potent inhibitor of PDE5 in vitro with an inhibitory concentration of 50% (IC50) for human recombinant PDE5 of 1 nM. In vitro, tadalafil is 700-fold more selective for PDE5 than for the retinal rod enzyme PDE6, and at least 9000-fold more selective for PDE5 than for PDEs 1 through 4 and 7 through 10. Thus, it is believed that targeting MR with an MR antagonist of Formula I, in combination with targeting PDE5 with tadalafil, will facilitate the lowering of blood pressure in resistant hypertension patients. At the same time, it is believed that this therapeutic combination will alleviate erectile dysfunction in resistant hypertension patients having comorbid resistant hypertension and erectile dysfunction. It is further believed that this therapeutic combination will alleviate erectile dysfunction in patients whose ED has been poorly or unsuccessfully treated by PDE5 inhibitors alone, that is, the combination of an MR antagonist of Formula I with tadalafil will treat PDE5- monotherapy resistant erectile dysfunction. It is believed that an MR antagonist of Formula I has specific advantages over other MR antagonists such as spironolactone or elplerenone, such as preferable specificity and lack of side effects, for use in a therapeutic combination for resistant hypertension, and it is believed that tadalafil has specific advantages over other PDE5 inhibitors, such as sildenafil and vardenafil, such as advantageous PDE selectivity and a relatively long half-life. Thus the particular combination of the present invention is unexpectedly advantageous for use in treatment of resistant hypertension. The present invention provides a combination of an MR antagonist of Formula I with tadalafil, administered separately or in a fixed-dose combination, for treatment of resistant hypertension.

PDE5 inhibitors increase intracellular cGMP, activate Protein Kinase G (PKG) and decrease intracellular calcium, leading to smooth muscle relaxation and reducing BP. Treatment with the PDE5 inhibitor sildenafil has been reported to increase the expression of the Renin, Angiotensin and Aldosterone System (RAAS) as a counter regulatory response to the decrease in BP (Thiesson et al., Am J Physiol Renal Physiol 2005; 288: F1044-F1052). Resistant hypertension patients would typically be treated with either an ACE inhibitor, or an angiotensin receptor blocker (ARB), and therefore angiotensin II signaling would typically be blocked. Thus, it is believed that treatment of resistant hypertension patients with the combination of an MR Antagonist of formula I and tadalafil will have the benefit of lowering blood pressure, while blocking potential activation of the RAAS pathway, particularly aldosterone, by PDE 5 inhibition, and in so doing be particularly and unexpectedly useful in therapy for resistant hypertension. Accordingly, the present invention provides a method of treating resistant hypertension, comprising administering to a patient in need of such treatment an effective amount an MR antagonist of Formula I in combination with an effective amount of tadalafil. A combination of an MR antagonist of Formula I with tadalafil is desired to provide treatment for resistant hypertension, wherein the combination may be more effective than either drug alone. For example, treatment with said combination may allow for use of lower doses of either or both drugs, as compared to each drug used alone, potentially leading to lower side effects while maintaining efficacy. Alternatively, treatment with said combination may allow for use of either or both drugs at higher doses, with the combination potentially leading to a mitigation of side effects, as compared with one drug of the combination used alone, while at the same time maintaining efficacy. Further, treatment with the combination of the MR antagonist of Formula I and tadalafil may provide desirable cardiovascular and or metabolic benefits, such as an unexpectedly beneficial reduction in vascular dysfunction and/or insulin resistance as compared to each drug used alone.

More specifically, the present invention provides a method of treating resistant hypertension, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, represented structurally as:

or a pharmaceutically acceptable salt thereof; in combination with an effective amount of tadalafil, represented structurally as:

The present invention also provides a method of treating a disease that is characterized by resistant hypertension, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil.

The present invention further provides a method of reducing blood pressure in a patient having resistant hypertension to levels wherein systolic blood pressure (SBP) is less than 140 mmHg (for patients 30 to 59 years old) or less than 150 mmHg (for patients >60 years old), or diastolic blood pressure (DBP) is less than 90 mmHg, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil.

The present invention further provides a method of treating PDE5-monotherapy resistant erectile dysfunction, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil.

The present invention also provides a method of treating refractory hypertension, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil.

Furthermore, the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil, for use in therapy, in particular for the treatment of resistant hypertension.

The invention further provides a pharmaceutical composition, comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients, in combination with a pharmaceutical composition of tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

The invention further provides a pharmaceutical composition, comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the combination is a fixed-dose combination. The invention further provides a pharmaceutical composition, comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the combination is a fixed-dose combination for oral administration.

In addition, the invention provides a kit, comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and tadalafil. The invention further provides a kit, comprising a pharmaceutical composition, comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients, and a pharmaceutical composition, comprising tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients. As used herein, a "kit" includes separate containers of each component, wherein one component is a compound of Formula I, or a pharmaceutically acceptable salt thereof, and another component is tadalafil, in a single package. A "kit" may also include separate containers of each component, wherein one component is a compound of Formula I, or a pharmaceutically acceptable salt thereof, and another component is tadalafil, in separate packages with instructions to administer each component as a combination.

The invention further provides the use of a combination of a compound of the Formula I, or a pharmaceutically acceptable salt thereof, and an effective amount of tadalafil, for the manufacture of a medicament for the treatment of resistant hypertension.

As used herein, the phrase "resistant hypertension" in the broadest embodiment refers resistant hypertension as known to one of skill in the art. In another embodiment "resistant hypertension" refers to uncontrolled blood pressure (BP) despite use of > 3 antihypertensive agents from different classes, or controlled BP with the use of > 4 agents. In a preferred embodiment resistant hypertension as used herein refers to blood pressure in patients that does not reach the recommended goal of SBP <140 mmHg (for patients 30 to 59 years old) or <150 mmHg (for patients >60 years old) or DBP <90 mmHg, despite treatment with 3 or more recommended first-line therapies (See 2014 Evidence-Based Guideline for the Management of High Blood Pressure in Adults, James et al., JAMA, 2014; 311(5): 507-520). In another preferred embodiment resistant hypertension as used herein refers to blood pressure in patients that does not reach the recommended goals from the 2014 Evidence-Based Guideline for the Management of High Blood Pressure in Adults of SBP <140 mmHg (patients 30 to 59 years old) or <150 mmHg (patients >60 years old) or DBP <90 mmHg despite treatment with 3 or more recommended first-line therapies, or black (African American) hypertensive patients with 3 or more recommended first-line therapies excluding angiotensin-converting enzyme [ACE] inhibitors (2014 Evidence-Based Guideline for the Management of High Blood Pressure in Adults, James et al., JAMA, 2014; 311(5): 507-520). In another embodiment resistant hypertension refers to patients having refractory hypertension (Calhoun et al., Hypertension, 2014; 63:451-458). Methods for identifying resistant hypertension patients, and studying drug candidates as therapies for these patients, and for measuring blood pressure in these patients, are known to one of skill in the art and are taught for instance in WO 2007/098390, published August 30, 2007, which is incorporated herein by reference.

As used herein, the term "patient" refers to a human. As used herein, the terms

"treating", "to treat", or "treatment", include restraining, slowing, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. As used herein, the terms lower blood pressure or lowering blood pressure refers to a decrease in SBP or DBP.

As used herein, the term "effective amount" refers to the amount or dose of compound of Formula I, or a pharmaceutically acceptable salt thereof, and/or to the amount or dose of tadalafil which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment. It is understood that the combination therapy of the present invention is carried out by administering a compound of Formula I, or a pharmaceutically acceptable salt thereof, together with the tadalafil, in any manner which provides effective levels of the compound of Formula I and tadalafil in the body. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques, and by observing results obtained under analogous circumstances. In determining the effective amount for a patient, a number of factors are considered by the attending diagnostician, including, but not limited to the patients size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

The compound of Formula I and its pharmaceutically acceptable salts are generally effective over a broad dosage range in the combination of the present invention. For example, dosages per day of individual agents normally fall within the range of about 1 mg/day to about 200 mg/day, preferably about 5 mg/day to about 100 mg/day, and most preferably about 5 mg/day to about 50 mg/day. Most preferably the compound of

Formula I is used at a doses per day selected from 5 mg, 6 mg, 10 mg, 13 mg, 15 mg, 20 mg, 24.5 mg, 25 mg, 26 mg, or 30 mg per day. In addition, tadalafil is generally effective over a wide dosage range in the combination of the present invention dosages per day normally fall within the range of about lmg/day to about 100 mg/day. Preferably, dosages per day normally fall within the range of about 2.5 mg/day to about 40 mg/day. Preferably tadalafil is used at doses per day selected from 5 mg/day to 20 mg/day, and most preferably selected from 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg per day. A preferred combination comprises 15 mg of tadalafil and 25 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 5 mg of tadalafil and 25 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 10 mg of tadalafil and 25 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 20 mg of tadalafil and 25 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 15 mg of tadalafil and 13 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 5 mg of tadalafil and 13 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 10 mg of tadalafil and 13 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 20 mg of tadalafil and 13 mg of the compound of Formula I per day in a fixed dose combination. Another preferred combination comprises 20 mg of tadalafil and 6 mg of the compound of Formula I per day in a fixed dose combination.

Another embodiment (A) preferably comprises a pharmaceutical composition, comprising [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-ll-ylidene)methyl]-l-[(lR)-l- methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients, in combination with a pharmaceutical composition of tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients. Another embodiment (B) preferably comprises a pharmaceutical composition comprising [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, in combination with tadalafil, and one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the combination is a fixed-dose combination. Another embodiment preferably comprises the pharmaceutical composition of (A) or (B) above for simultaneous, separate or sequential daily administration of 25 mg of [5-[(E)-(3- fluoro-6H-benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, and 20 mg of tadalafil. Another embodiment preferably comprises the pharmaceutical composition of (A) or (B) above for simultaneous, separate or sequential daily administration of 25 mg of [5 - [(E)-(3 -fluoro-6H-benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2- morpholino-ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, and 15 mg of tadalafil. Another embodiment preferably comprises the pharmaceutical composition of (A) or (B) above for simultaneous, separate or sequential daily administration of 13 mg of [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-ll- ylidene)methyl]-l-[(lR)-l-methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, and 20 mg of tadalafil. An MR antagonist of Formula I and tadalafil are preferably formulated as pharmaceutical compositions administered by any route which makes the compounds bioavailable. The route of administration may be varied in any way, limited by the physical properties of the drugs and the convenience of the patient and the caregiver. Preferably, an MR antagonist of Formula I is formulated for oral or parenteral administration including intravenous or subcutaneous administration. In addition, the tadalafil is formulated for oral or parenteral administration, including intravenous or subcutaneous administration. Preferably an MR antagonist of Formula I and the tadalafil are each formulated for oral administration. Preferably an MR antagonist of Formula I and the tadalafil are formulated together in a fixed-dose combination for oral

administration. Providing the MR antagonist of Formula I and the tadalafil formulated together in a fixed-dose combination for oral administration is believed to enhance patient compliance in taking this combination, as prescribed by his or her health care provider, and this enhanced compliance for this particular combination is believed to provide unexpectedly improved lowering of blood pressure in resistant hypertension patients. Such pharmaceutical compositions and processes for preparing same are well known in the art. (See, e.g., Remington: The Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006).

As used herein, the phrase "in combination with" refers to the administration of an MR antagonist of Formula I, or a pharmaceutically acceptable salt thereof, with tadalafil, simultaneously or sequentially in any order, or any combination thereof. The two molecules may be administered either as part of the same pharmaceutical composition, or in separate pharmaceutical compositions. An MR antagonist of Formula I can be administered prior to, at the same time as, or subsequent to administration of the tadalafil, or in some combination thereof. Where an MR antagonist of Formula I is administered at repeated intervals (e.g. during a standard course of treatment), the tadalafil can be administered prior to, at the same time as, or subsequent to, each administration of an MR antagonist of Formula I, or some combination thereof, or at different intervals in relation to therapy with an MR antagonist of Formula I, or in series of dose(s) prior to, at any time during, or subsequent to the course of treatment with the an MR antagonist of Formula I. Preferably for the treatment of resistant hypertension the [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or pharmaceutically acceptable salt thereof, and tadalafil are administered simultaneously. Preferably for the treatment of PDE5 -monotherapy resistant erectile dysfunction the [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-l 1- ylidene)methyl]-l-[(lR)-l-methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, or pharmaceutically acceptable salt thereof, and tadalafil are administered simultaneously. Preferably for the treatment of resistant hypertension the [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea or pharmaceutically acceptable salt thereof, is administered prior to the administration of tadalafil. Preferably for the treatment of resistant hypertension the tadalafil is administered prior to the administration of the [5-[(E)-(3- fluoro-6H-benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea.

The following paragraphs describe preferred groups, substituents, and

configurations of the present invention. It is preferred that the compound of Formula I be in the free base form. It is preferred that the tadalafil be in the free base form.

The following methods, preparations and examples further illustrate the invention. Unless noted to the contrary, the compounds illustrated herein can be named and numbered using Accelrys® Draw version 4.0 (Accelrys, Inc., San Diego, CA.),

IUPACNAME ACDLABS, or ChemDraw® Ultra 12.0. Certain stereochemical centers may have been left unspecified and certain substituents may have been eliminated in the following schemes for the sake of clarity and are not intended to limit the teaching of the schemes in any way. Furthermore, individual isomers, enantiomers, and diastereomers may be separated or resolved by one of ordinary skill in the art at any convenient point in the synthesis of compound of Formula I by methods such as selective crystallization techniques or chiral chromatography (See for example, J. Jacques, et al., "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981, and E.L. Eliel and S.H. Wilen," Stereochemistry of Organic Compounds", Wiley-Interscience, 1994).

Designations "isomer 1" and "isomer 2" refer to the compounds that elute from chiral chromatography first and second, respectively, and if chiral chromatography is initiated early in the synthesis, the same designation is applied to subsequent intermediates and examples. Additionally, certain intermediates described in the following schemes may contain one or more nitrogen protecting groups. The variable protecting group may be the same or different in each occurrence depending on the particular reaction conditions and the particular transformations to be performed. The protection and de -protection conditions are well known to the skilled artisan and are described in the literature (See for example "Greene's Protective Groups in Organic Synthesis", Fourth Edition, by Peter G.M. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).

An MR antagonist of Formula I, or pharmaceutically acceptable salts thereof, or tadalafil, may be prepared by a variety of procedures known in the art, some of which are illustrated in the schemes, preparations and examples below. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different procedures, to prepare a compound of Formula I, or salts thereof, or tadalafil. The products of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In addition, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

One of ordinary skill in the art will appreciate that an MR antagonist of Formula I can exist in tautomeric forms, as depicted in Figure 1. When any reference in this application to one of the specific tautomers of the compound of Formula I is given, it is understood to encompass all tautomeric forms and all mixtures thereof. Structures A and B and Structure C in Figure 1, shown below, each represent different tautomeric forms of an MR antagonist of Formula I, which also can be represented as (E)-N-(5-((E)-3-fluoro- 6H-dibenzo[b,e]oxepin-l l-ylidenemethyl)-l-((R)-l-methyl-2-morpholin-4-yl-ethyl)-l,3- dihydro-benzoimidazol-2-ylidene)-urea, or alternatively as [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or alternatively as (E)-[6-[(E)-(3-fluoro-6H- benzo [c] [ 1 Jbenzoxepin- 11 -ylidene)methyl] -3 - [( 1 R)- 1 -methyl-2-morpholino-ethyl] - 1H- benzimidazol-2-ylidene]urea. Figure 1: MR antagonist of Formula I showing alternate tautomer forms.

C

For illustration, an MR antagonist of Formula I can be prepared as follows in Example 1.

Example 1

(i¾-N-(5-((ii)-3-Fluoro-6H-dibenzo[b,e]oxepin-ll-ylidenemethyl)-l-((/?)-l-methyl-2- morpholin-4-yl-ethyl)-l,3-dihydro-benzoimidazol-2-ylidene)-urea, alternatively described as

(E)- [6- [(E)-(3 -fluoro-6H-benzo [c] [ 1 Jbenzoxepin- 11 -ylidene)methyl] -3 - [( 1 R)- 1 -methyl-2- morpholino-ethyl]-lH-benzimidazol-2-ylidene]urea

alternatively described as

[5- [(E)-(3 -fluoro-6H-benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 -methyl-2- morpholino-ethyl]benzimidazol-2-yl]urea,

^ N H ₂

O

(E)-[6-[(E)-(3-fluoro-6H-benzo[c][l]benzoxepin-l l-ylidene)methyl]-3-[(lR)-l- methyl-2-mo^holino-ethyl]-lH-benzimidazol-2-ylidene]cyanamide (2.23 g, 4.3 mmol) is dissolved in 4 N HCl in dioxane (23 mL) and stirred at room temperature overnight. The reaction is then neutralized with 4 N NaOH, extracted with dichloromethane, and concentrated to dryness to give the title compound (1.42 g, 60%). LCMS m/z 528.2 [M+H]. One of skill in the art will recognize that the title compound may form tautomers which may be represented by alternative naming.

For example, the MR antagonist compound of Formula I for oral administration can be composed of the micronized compound of Formula I in a dry blend formulation in an oral capsule form. The MR antagonist compound of Formula I formulation is composed of micronized MR antagonist compound of Formula I prepared according to Example 1 above, and the following inactive ingredients: colloidal silicon dioxide, sodium stearyl fumarate, sodium starch glycolate, and lecithin and lactose monohydrate. The MR antagonist compound of Formula I containing capsules can be supplied as 0.2, 1.5, 6, 10, 13, 24.5, 25, or 26 mg of the compound as the free base, or at other desired doses.

U.S. Patent No. 5,859,006, and U.S. Patent No. 7,550,479 , each

incorporated herein by reference, disclose the synthesis of (6R,12aR)- 2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino- [2',l':6,l]pyrido[3,4-b]indole-l,4-dione, also known as tadalafil, as well as

methods for making and using tadalafil by one of ordinary skill in the art. U.S.

Patent No. 6,821,975 and U.S. Patent No. 7,182,958, each incorporated herein by reference, disclose methods of preparing, formulating, and using tadalafil by one of ordinary skill in the art. Tadalafil has two asymmetric carbon atoms wherein the nonhydrogen substituents of the asymmetric carbon atoms are in the cis configuration. Compound (I), also known as tadalafil, can be prepared by the two synthetic pathways disclosed in U.S. Patent No. 5,859,006, and U.S. Patent No. 7,550,479 also describes methods of making tadalafil, and both are incorporated herein by reference. A first synthetic pathway (A), from D-tryptophan, has few steps, but the yield of the desired diastereomer (i.e., Compound II) is poor and requires a separation step from the irans-stereoisomer (Compound Ila). Pathway (A) also utilizes the highly corrosive trifluoroacetic acid (i.e., TFA or CF₃CO₂H). The key step in pathway A is a classic Pictet-Spengler reaction using D- tryptophan methyl ester and piperonal to yield substituted tetrahydro- -carboline Compounds (II) and (Ila). A second pathway (B) provides a better yield of the desired Compound I, but requires numerous synthetic steps. In each synthetic pathway the key intermediate in the synthesis of Compound (I) is Compound (II). Compound (I) then is synthesized from Compound (II) in two straightforward synthetic steps.

Pathway A

D-Tryptophan Piperonal

methyl ester

Compound (II) Compound (Ila)

(cis -isomer) (42% yield) {trans -i somer ) (28% yield)

(desired) (undesired)

Pathway B

hydrochloride CH₂C1₂

10

Pathway from Compound (II) to tadalafil

Compound (II)

Compound I, also referred to as tadalafil.

The overall yield of tadalafil using synthetic pathway (A) or (B) can be about 25% to about 30%. Pathway (B) requires several synthetic steps. A key step in the synthesis of tadalafil is the preparation of Compound (II) in the shorter synthetic pathway (A). The preparation of Compound (II) in pathway (A) utilizes a Pictet- Spengler cyclization between D-tryptophan methyl ester and piperonal in dichloromethane (CH₂CI₂) with two equivalents of trifluoroacetic acid at 4°C which provides, after five days, a mixture of two diastereoisomers, i.e., the desired ds-isomer tetrahydro- -carboline Compound (II) ((1R,3R)) and the undesired irans-isomer tetrahydro- -carboline Compound (Ila) ((1S,3R)) in a ratio of about

60/40. From this mixture, the pure ds-isomer (i.e., Compound (II)) can be

obtained by fractional crystallization in a 42% yield (ee>99% (chiral HPLC)).

The following illustrates the modified Pictet-Spengler reaction (Step 2), and the subsequent synthesis of Compound (I) (tadalafil) from Compound (II) (Steps 3 and 4).

)

Compound (II

Compound (I)/tadalafil

In general, the synthesis of compound (I) (tadalafil) using the above method involves a four-step synthetic pathway. The first step is an esterification in methanol (MeOH) using thionyl chloride (SOCl₂) under reflux. The product is crystallized and isolated by filtration. The second step involves a simplified variation of the Pictet- Spengler reaction, wherein D-tryptophan methyl ester hydrochloride is admixed with piperonal in isopropyl alcohol (i-PrOH) and heated under reflux to form a mixture of di- astereomeric adducts. Because the desired ds-diastereomer (Compound (II)) is substantially insoluble in isopropyl alcohol at reflux temperature and below, the cis- diastereomer crystallizes from solution leaving a dynamic cis-trans equilibrium in solution. As the ds-diastereomer precipitates from the isopropyl alcohol, the equilibrium is driven towards the ds-diastereomer until the concentration of the ds-diastereomer is sufficiently low to remain in solution. The desired diastereomer is isolated in greater than 90% yield by crystallization and filtration.

The third step involves an aqueous tetrahydrofuran (THF) acylation of the amino (NH₂) moiety of Compound (II), followed by crystallization and filtration. Ring closure with methylamine (MeNH₂) completes the ring-forming sequence. After solvent exchange, the product is crystallized from aqueous isopropyl alcohol or other suitable solvent, and filtration provides Compound (I) (tadalafil) in an overall yield of about 77%.

As used herein,"DMSO" refers to dime thylsulf oxide; "ee" refers to enantiomeric excess; "EtOAc" refers to ethyl acetate; "hr refers to hour or hours; "min" refers to minute or minutes; and "SEM" refers to standard error of the mean.

In vitro Assays for an MR antagonist of Formula I and pharmaceutically acceptable salts thereof:

For in vitro enzymatic and cellular assays, test compounds can be prepared in DMSO to make up a 10 mM stock solution. The stock solution can be serially diluted in DMSO to obtain a ten-point dilution curve with final compound concentrations ranging from 10 μΜ to 1 nM in a 96-well round-bottom plate before conducting the in vitro enzymatic and whole cell assays.

As used herein, refers to the equilibrium dissociation constant for a ligand- receptor complex; "Kj" refers to the equilibrium dissociation constant for drug-receptor complex, and is an indication of concentration of drug that will bind to half the binding sites at equilibrium; refers to the equilibrium dissociation constant for an antagonist-receptor complex; "IC50" refers to the concentration of an agent which produces 50% of the maximal inhibitory response possible for that agent or, alternatively, to the concentration of an agent which produces 50% displacement of ligand binding to the receptor; "EC50" refers to the concentration of an agent which produces 50% of the maximal response possible for that agent; and "ED50" refers to the dose of an administered therapeutic agent which produces 50% of the maximal response for that agent, "qd" refers to administration once daily, and "bid" refers to administration twice a day.

Steroid Hormone Nuclear Receptor - Binding Assay:

The MR is a member of the steroid receptor subfamily of nuclear hormone receptors (NHRs) and is most closely homologous to the glucocorticoid receptor (GR), the progesterone receptor (PgR) and the androgen receptor (AR). The level of ligand selectivity an MR antagonist of Formula I for MR relative to other steroid nuclear receptors is determined using ligand binding and cell-based functional reporter assays. Binding is evaluated in HEK293 cells expressing human MR, GR, AR or PgR. Cell lysates from human embryonic kidney HEK293 cells overexpressing human MR

(mineralocorticoid receptor), GR (glucocorticoid receptor), AR (androgen receptor), or PR (progesterone receptor) are used for receptor-ligand competition binding assays to determine K; values.

Briefly, steroid receptor competition binding assays are run in a buffer containing 20 mM HEPES buffer (pH = 7.6), 0.2 mM EDTA, 75 mM NaCl, 1.5 mM MgCl2, 20% glycerol, 20 mM sodium molybdate, 0.2 mM DTT (dithiothreitol), 20 μg/mL aprotinin and 20 μg/mL leupeptin (assay buffer). Typically, steroid receptor binding assays include radio-labeled ligands, such as 0.25 nM [^HJ-aldosterone for MR binding, 0.3 nM [¾]- dexamethasone for GR binding, 0.36 nM [^HJ-methyltrienolone for AR binding, and 0.29 nM [³H]-methyltrienolone for PR binding, and either 20 μg 293-MR lysate, 20 μg 293- GR lysate, 22 μg 293-AR lysate, or 40 μg 293-PR lysate per well. Assays are typically run in 96-well format. Competing test compounds are added at various concentrations ranging from about 0.01 nM to 10 μΜ. Non-specific binding is determined in the presence of 500 nM aldosterone for MR binding, 500 nM dexamethasone for GR binding, or 500 nM methyltrienolone for AR and PR binding. The binding reactions (140 μΕ) are incubated overnight at 4 °C, then 70 μΕ of cold charcoal-dextran buffer (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) is added to each reaction. Plates are mixed for 8 minutes on an orbital shaker at 4 °C. The plates are then centrifuged at 3,000 rpm at 4 °C for 10 minutes. An aliquot of 120 μΕ of the binding reaction mixture is then transferred to another 96-well plate and 175 μΕ of Wallac Optiphase Hisafe 3™ scintillation fluid is added to each well. Plates are sealed and shaken vigorously on an orbital shaker. After an incubation of 2 hours, plates are read in a Wallac Microbeta counter. The data are used to calculate an estimated IC50 and percentage inhibition at 10 μΜ. The Kd for [^HJ-aldosterone for MR binding, [¾]- dexamethasone for GR binding, [^HJ-methyltrienolone for AR binding, or [¾]- methyltrienolone for PR binding, is determined by saturation binding. The IC50 values for compounds are converted to Ki using the Cheng-Prusoff equation.

Following a protocol essentially as described above, the MR antagonist compound of Formula I shows binding to MR with high affinity, with an inhibition constant (Ki) of 0.33 + 0.35 nM (Mean + SEM), thus demonstrating that the MR antagonist compound of Formula I is a potent ligand of human MR. Selectivity for binding to MR is 800- to 7500-fold over that for GR, AR, and PgR.

Steroid Nuclear Hormone Receptor Modulation - Functional Assays:

Aldosterone exerts it physiological effects through interaction with the mineralocorticoid receptor. Following cytoplasmic binding of aldosterone to MR, the ligand receptor complex translocates to the cell nucleus where it binds to hormone response elements on DNA to initiate expression of target genes. To demonstrate the ability of the MR antagonist compound of Formula I to modulate the activity of steroid hormone receptors (i.e. either agonize, partially agonize, partially antagonize, or antagonize), bioassays are performed which detect functional modulation of target gene expression in cells transiently transfected with a nuclear receptor protein and a hormone response element-reporter gene construct. The solvents, reagents, and ligands employed in the functional assay are readily available from commercial sources, or can be prepared by one of ordinary skill in the art.

Functional activity is evaluated in HEK293 cells expressing the individual NHR and the respective response element/TK promoter- luciferase reporter construct

(glucocorticoid response element [GRE] for MR, GR and PgR; and androgen response element [ARE] for AR). Transfected 293 cells are exposed to the MR antagonist compound of Formula I alone (agonist activity evaluation) or together with low concentrations of the specific agonist for each respective receptor (antagonist activity evaluation), and cell lysates are subsequently measured for luciferase activity. For antagonist activity, percent efficacy of the MR antagonist compound of Formula I plus specific agonist is determined versus maximum stimulation obtained using the individual specific agonist alone. Testing assays can be conducted as described below. A. Nuclear Hormone Receptor Panel Screen

Human embryonic kidney HEK293 cells are transfected with steroid hormone receptor and reporter gene plasmids using a suitable transfection reagent such as

Fugene™ . Briefly, the reporter plasmid containing two copies of probasin ARE and TK(thymidine kinase) promoter upstream of the luciferase reporter cDNA, is transfected into HEK293 cells with a plasmid constitutively expressing human androgen receptor (AR) using viral CMV (cytomegalovirus) promoter. The reporter plasmid containing two copies of GRE and TK promoter upstream of the luciferase reporter cDNA is transfected with a plasmid constitutively expressing either human glucocorticoid receptor (GR), human mineralocorticoid receptor (MR), or human progesterone receptor (PR) using viral CMV promoter. Cells are transfected in T 150 cm flasks in DMEM media with 5% charcoal-stripped Fetal Bovine Serum (FBS). After an overnight incubation, transfected cells are trypsinized, plated in 96 well dishes in DMEM media containing 5% charcoal- stripped FBS, incubated for 4 hours and then exposed to various concentrations of test compounds ranging from about 0.01 nM to 10 μΜ. In the antagonist mode for the assays, low concentrations of agonist for each respective receptor are added to the media (0.08 nM aldosterone for MR, 0.25 nM dexamethasone for GR, 0.66 nM of methyltrienolone for AR, and 0.08 nM of promegestone for PR). After 24 hours incubation with test compounds, cells are lysed and luciferase activity is determined using standard techniques.

Data are fitted to a four parameter-fit logistic curve to determine EC50 values. The percentage efficacy (compounds with saturated maximum responses) or the percent maximum stimulation (compounds with maximum responses that do not saturate) are determined relative to maximum stimulation obtained with the following reference agonists: 30 nM aldosterone for MR assay, 100 nM methyltrienolone for AR assay, 30 nM promegestone for PR assay, and with 100 nM dexamethasone for GR assay. IC50 values are determined similarly using antagonist mode assay data. In the antagonist mode, percent inhibitions are determined by comparing test compound activity in the presence of low concentration of agonist (0.08 nM aldosterone for MR, 0.25 nM dexamethasone for GR, 0.66 nM of methyltrienolone for AR, and 0.08 nM of promegestone for PR) to the response produced by the same low concentration of agonist in the absence of test compound.

B. hMR Competitive Antagonist Assay:

Human embryonic kidney HEK293 cells are transfected with human MR using the same transfection reagents, plasmids, promoters, reporter constructs, buffers, and procedures as described above for the Nuclear Hormone Receptor Panel Screen.

Transfected cells are trypsinized, plated in 96 well dishes in DMEM media containing 5% charcoal-stripped FBS, incubated for 4 hours and then exposed to various concentrations (10 dilutions) of aldosterone (ranging from about 0.001 nM to 0.03 μΜ. The ability of aldosterone to agonize the hMR is determined in the absence and presence of fixed concentrations of test compound and is monitored by measuring luciferase activity using standard techniques. The test compound Κ¾ may then be determined using a Schild analysis plotting log (dose ratio - 1) against log of antagonist concentration using the equation: Log (DR-1) = Log [Antagonist] - Log Kb where the dose ratio (DR) represents the ratio of the aldosterone EC50 in the presence of test compound to the aldosterone EC50 in the absence of test compound).

Assays essentially according to the above procedures indicate that the MR antagonist compound of Formula I shows pure MR antagonist activity, with 97.3% inhibition of aldosterone activity and an IC₅₀ of 27.9 nM, thus demonstrating that the MR antagonist compound of Formula I is a potent antagonist of human MR. When tested in a Schild plot analysis, the binding affinity constant can be determined to be 13.3 nM. The MR antagonist compound of Formula I can demonstrate a 40-fold IC₅₀ selectivity against PgR (IC₅₀ of 1220 nM), and can show no cross-reactivity in antagonist assays against GR or AR, thus showing the selectivity of the MR antagonist compound of Formula I. The above data indicate that the MR antagonist compound of Formula I to be potent and selective MR ligand with functional antagonist activity on MR, and selectivity against other steroid receptors.

In order to demonstrate that the MR antagonist compound of Formula I in combination with tadalafil has a reduced incidence or likelihood of producing hyperkalemia, the following model may be employed. In vivo Assay of Electrolyte Modulation

Male Sprague Dawley rats (240-280 g) are adrenalectomized then maintained on 5001 rodent chow and 1% NaCl drinking solution for 6 days after surgery. Animals are then fasted overnight and 1% saline drinking water is replaced with house water ad lib. The morning of the study, fasted animals are randomized to treatment on the basis of fasted body weight. Control animals (e.g . those that receive no aldosterone or MR antagonist compound of Formula I in combination with tadalafil) are given 10 mL/kg of test compound vehicle comprising 0.5% CMC / 0.25% polysorbate 80 / 2.7% NaCl by oral gavage, and 1 mL/kg of aldosterone vehicle (0.01% DMSO/water) by subcutaneous injection. Vehicle animals are given the same test compound vehicle by oral gavage and aldosterone 3 g/kg, s.c. MR antagonist compound of Formula I in combination with tadalafil at desired doses of each are suspended in the carboxy methylcellulose/NaCl vehicle. The MR antagonist compound of Formula I in combination with tadalafil test treatment groups receive test substances suspended in the carboxy methylcellulose/NaCl vehicle and aldosterone 3 g/kg s.c. Immediately after dosing, animals are placed in metabolic racks with ad lib access to house water. Urine samples are collected 5 hours after dose administration and electrolyte excretion is assayed. Data are presented as log Na/K excretion ratio. Compounds can be tested alone or together, at various doses, to determine whether the MR antagonist compound of Formula I in combination with tadalafil results in a modulation of the urinary Na/K ratio (an index of increased serum potassium concentration), as compared to either compound alone.

In vivo Inhibition of Aldosterone Mediated Hypertension

The MR antagonist compound of Formula I, alone or in combination with tadalafil, at various doses of each, can be evaluated for effects on hypertension using a rat model in which uninephrectomized rats are administered 6% NaCl in the diet concomitant with subcutaneous delivery of aldosterone (0.25 μg/hr). This treatment results in a gradual increase in mean daily BP over a 2- week treatment period. The MR antagonist compound of Formula I effects are, for example, directly compared with those of eplerenone (EPL) in this model. The MR antagonist compound of Formula I (range 1- 30 mg/kg/day), administered once daily by gavage, dose-dependently blocks the hypertensive effects of aldosterone in the presence of salt throughout the 14-day treatment period. At the maximum dose of 30 mg/kg/day, the MR antagonist compound of Formula I produces an 89% reduction in BP from the vehicle peak response. While EPL (range 3- 100 mg/kg/day) is also effective in reducing BP in this model, the maximum reduction in aldosterone-mediated hypertension after EPL administration is about 34% less than that observed with the MR antagonist compound of Formula I (55% and 89% for EPL and the MR antagonist compound of Formula I respectively). The EC5₀ and 90% CI values calculated from PK PD analysis of blood pressure data are 250 ng/mL (range 130— 500 ng/mL) and 200 ng/mL (range 130-320 ng/mL) for the MR antagonist compound of Formula I and EPL, respectively.

Further, analysis of the diurnal BP after 14 days of aldosterone infusion reveals that the MR antagonist compound of Formula I, at 3, 10, and 30 mg/kg, reduces the peak- to-nadir difference observed in the vehicle group (30 mmHg) leading to complete control of BP over the 24-hour diurnal period. Concomitant with the gradual increase in pressure, the expected bradycardic reflex is noted in vehicle treated animals. This reflex bradycardia is not suppressed upon administration of the MR antagonist compound of Formula I. Thus, the MR antagonist compound of Formula I is a potent antagonist of aldosterone-mediated hypertension in this model. In Vivo Combination Study

Male SHR rats weighing 250-300 gm with age of 7 weeks are obtained from Charles River Laboratories (Wilmington, MA) and fed with regular chow and water ad libitum. The rats are housed in temperature controlled quarters with a 12-hr light/dark cycles. Rats are anesthetized with 3% Isoflurane. Under aseptic conditions, a 2-3 cm incision is made at the inner thigh above the left femoral triangle, and the femoral artery is carefully isolated. A mid-abdominal incision is carried out and the electric transmitter (model TA11PA-C40; Data Sciences International, St. Paul, MN) is sutured to inside of abdominal muscles. The catheter of the transmitter is punched and passed through the abdomen into left femoral triangle with the leading of 14-gauge 1.5-inch syringe needle as a trocar, followed by inserting into femoral artery and going in for about 5 cm until reaching to the position of renal arteries. The transmitter is fixed within the peritoneal cavity by suturing to the abdominal muscles. The proximal suture is tied around the vessel and catheter, and the distal suture is tied around the catheter to secure. Both incisions are closed and the rats are allowed to recover from surgery for two weeks and then are placed in individual cages in a quiet telemetry facility room for measurement of baseline mean arterial pressure (MAP) and heart rate (HR). Digitized pressure signals are acquired for 20 seconds every 5 minutes using DSI Dataquest IV 4.0 software. The digitized values are stored in a computer. In order to evaluate combinational blood pressure lowering therapy of the MR antagonist compound of Formula I and tadalafil in combination a cohort of SHR rats (n=40) are prepared as described above and randomized by mean arterial pressure (MAP) into two groups (N=20). Starting mean arterial pressure is 153 mm Hg. Group A is orally dosed with tadalafil (TAD) 6 mg/kg for 5 weeks. Group B is orally dosed with tadalafil (TAD) 6 mg/kg/day for 5 weeks, and for the first week is given no MRA, then each week simultaneously dosed with the MR antagonist compound of Formula I (MRA), for 1 week each, at doses 0.6, 1.2, 4 and 10 mg/kg/day, to result in a weekly dose escalation as illustrated in the Table 1 below. Based on exposure, TAD 6 mg/kg/day is roughly equivalent to giving a human dose of 20 mg qd. Similarly the doses of the MR antagonist compound of Formula I are roughly equivalent to human doses of 0, 1, 3, 10, 25 mg qd. The maximum reduction of blood pressure, at the end of each week, is summarized in Table 1 below. Data in Table 1 are represented as 24-hr means, and statistically analyzed with JMP® 9.1 software (*p<0.05 or p<0.001, one way ANOVA comparison). Tadalafil alone at 6 mg/kg/day for 5 weeks significantly reduces MAP between 10.9 and 12.7 mm Hg compared pre-treatment MAP of 153 mm Hg. Tadalafil plus the MR antagonist compound of Formula I at 0.6 or 1.2 mg/kg/day has no effect on MAP compared to tadalafil alone. Tadalafil plus the MR antagonist compound of Formula I at 4 and 10 mg/kg/day significantly reduces MAP by 15 and 17.4 mm Hg, respectively, versus tadalafil alone at weeks 4 and 5. Tadalafil with or without MR antagonist has no effect on heart rate in this SHR model. In view of advantageous properties of the MR antagonist compound of Formula I, and the advantageous properties of tadalafil, and in particular the properties of these compounds when used in combination, and results such as those which can be observed in the hypertension assays described herein, it is believed that the particular combination of the MR antagonist compound of Formula I and tadalafil, taken individually or in a fixed-dose combination, will lower blood pressure in patients with resistant hypertension. On this basis it is further believed that the particular combination of the MR antagonist compound of Formula I and tadalafil, taken individually or in a fixed-dose combination, will treat PDE5-monothereapy resistant erectile dysfunction. Table 1:

MAP = mean arterial pressure, TAD = tadalafil at the indicated dose, MRA = the MR antagonist compound of Formula I at the indicated dose, N/A = not applicable for this data table.

Clinical Dose Assessment and/or Combination Therapy Assessment Study

A pilot pharmacokinetic and pharmacodynamic study can be performed in humans, with the MR antagonist compound of Formula I or pharmaceutically acceptable salt thereof (MRA), and optionally tadalafil, in order to define doses that provide minimal to marked blood pressure reduction in patients with primary hypertension and/or resistant hypertension according to methods known to one of skill in the art. The doses of MR antagonist in this study can be selected from 5 mg, 6 mg, 10 mg, 13 mg, 15 mg, 20mg, 24.5 mg, 25 mg, and 30 mg. The doses of tadalafil in a pilot study can be selected from 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, and 30 mg.

The pilot studies can be followed by a study or studies in patients with resistant hypertension where the combination doses of MR antagonist plus tadalafil can be determined. Methods of such studies and clinical identification of resistant hypertension patients and measurement of blood pressure in those patients are known to one of skill in the art (See for example WO 2007/098390 published August 30, 2007).

For example, such a study could be conducted with a patient population who have systolic BP >140 while on 3 or more antihypertensive drugs at full doses, wherein full dose can be defined as hydrochlorothiazide (HCTZ) >25mg, and other agents at the highest labeled dose, or the highest usual dose in the local practice, or the highest tolerated dose, or the highest appropriate dose according to the investigator's best clinical judgment. Patients can be randomized after a run-in period, in a placebo controlled, single blind fashion, to ensure BP remains stable and to continue to meet entry criteria.

Four combination doses of MR antagonist plus Tadalafil can be studied for daily administration of each agent or a fixed dose combination in resistant hypertension patients. These include 1) an MRA high dose + a tadalafil high dose, 2) an MRA high dose + tadalafil low dose, 3) an MRA low dose + a tadalafil high dose, and 4) an MRA low dose + a tadalafil high dose. In addition, up to 3 doses of the MR antagonist compound and up to 3 doses of tadalafil can be included for comparison to the effect of combinations on blood pressure lowering. The doses of MR antagonist in this study can be 6 mg, 13 mg, and 24.5 mg, or doses estimated based on PK PD modelling of the sort known to one of skill in the art. For tadalafil, doses of 5mg, lOmg, and 30mg can be used, or doses estimated based on PK PD modelling of the sort known to one of skill in the art, and these dose levels can roughly cover the dose and exposure-response curve.

Approximately 50 patients per arm can be enrolled, and the number per arm can adjusted based on the design of the study according to methods known in the art. The treatment duration can be 2 months. Blood pressure and other relevant clinical parameters can be assessed by methods known to one of skill in the art, such as those described in WO 2007/098390. The primary end point, for example, of BP change from baseline, including office BP and ambulatory BP, each assessed by methods known to one of skill in the art. These studies may show that the combination therapy of the MR antagonist compound of Formula I and tadalafil will result in enhanced blood pressure reductions in resistant hypertension patients, and alleviation of erectile dysfunction in the same patients who may have comorbid ED, relative to the individual mono-therapies alone.

Claims

WE CLAIM:

1. A method of treating resistant hypertension in a patient, comprising

administering to a patient in need of such treatment an effective amount of the compound [5-[(E)-(3-fluoro-6H-benzo[c] [2]benzoxepin- 1 l-ylidene)methyl]- 1-

[( 1R)- 1 -methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, structurally represented by the formula:

or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil, struc a:

The method of claim 1 wherein the tadalafil comprises a dose of 20 mg per day.

The method of claim 1 wherein the tadalafil comprises a dose of 15 mg per day. The method of claim 1 wherein the tadalafil comprises a dose of 10 mg per day.

The method according to claims 1 to 4 wherein the [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea comprises a dose of 6 to 30 mg per day.

6. The method according to claims 1 wherein the resistant hypertension

comprises uncontrolled blood pressure (BP) in a patient despite use of > 3 antihypertensive agents from different classes, or controlled BP with the use of > 4 agents.

7. The method according to claims 1 wherein the resistant hypertension

comprises blood pressure in a patient that does not reach the recommended goal of SBP <140 mmHg (for patients 30 to 59 years old) or <150 mmHg (for patients >60 years old) or DBP <90 mmHg, despite treatment with 3 or more recommended first-line therapies.

8. A pharmaceutical composition, comprising [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients, in combination with a pharmaceutical composition of tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients. 9. A pharmaceutical composition comprising [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, in combination with tadalafil, and one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the combination is a fixed-dose combination.

10. The pharmaceutical composition of claim 8 or 9 for simultaneous, separate or sequential daily administration of 25 mg of [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, and 20 mg of tadalafil.

11. The pharmaceutical composition of claim 8 or 9 for simultaneous, separate or sequential daily administration of 25 mg of [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, and 15 mg of tadalafil.

12. The pharmaceutical composition of claim 8 or 9 for simultaneous, separate or sequential daily administration of 13 mg of [5-[(E)-(3-fluoro-6H- benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino- ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, and 20 mg of tadalafil.

13. A kit, comprising [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-l l- ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2-morpholino-ethyl]benzimidazol-2- yljurea, or a pharmaceutically acceptable salt thereof, and tadalafil.

14. A kit, comprising a pharmaceutical composition, comprising [5-[(E)-(3-fluoro- 6H-benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1R)- 1 -methyl-2- morpholino-ethyl]benzimidazol-2-yl]urea,

or a pharmaceutically acceptable salt thereof, with one or more

pharmaceutically acceptable carriers, diluents, or excipients, and a

pharmaceutical composition, comprising tadalafil, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

15. [5 - [(E)-(3 -fluoro-6H-benzo [c] [2]benzoxepin- 11 -ylidene)methyl] - 1 - [( 1 R)- 1 - methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, or a pharmaceutically acceptable salt thereof, in combination with tadalafil, for simultaneous, separate, or sequential use in therapy.

16. A method of treating PDE5-monotherapy resistant erectile dysfunction,

comprising administering to a patient in need of such treatment an effective amount of [5-[(E)-(3-fluoro-6H-benzo[c][2]benzoxepin-l l-ylidene)methyl]-l- [( 1R)- 1 -methyl-2-morpholino-ethyl]benzimidazol-2-yl]urea, structurally represented by the formula:

or a pharmaceutically acceptable salt thereof, in combination with an effective amount of tadalafil, structurally represented by the formula: