BACKGROUND OF THE INVENTION

This invention relates to methods for employing diphenylhalonium ion to elicit various pharmacological effects treating different types of cardiovascular dysfunction. In accordance with the present invention, diphenylhalonium ion has been found to be useful in stimulating guanylate cyclase activity, increasing the contractility of cardiac muscle and inhibiting the aggregation of blood platelet cells, . each effect being desirable in ameliorating symptoms associated with particular pathological or pathogenic conditions.

Sodium nitroprusside ( SNP) , administered intravenously, is an extremely potent relaxant of vascular smooth muscle in both arterial and venous vessels. Prolonged administration of SNP can lead to symptoms of thiocyanate toxicity. Thyroid hormone insufficiency has also been reported during prolonged infusions of SNP. Nevertheless, SNP has become the drug of choice for treating

hypertensive crises, and is also widely used in the acute management of cardiac failure. The potent activity in vivo of SNP has been linked to the drug ^' s ability to stimulate guanylate cyclase in most mammalian tissues. The resulting elevation of guanosine 3 ^' ,5 '-cyclic phosphoric acid (cyclic guanosine onophosphate, hereinafter "cyclic GMP") levels can result, for example, in a relaxation of vascular smooth muscle and a decrease in the tendency for blood platelet cells to aggregate. See, e.g. , Bohme et al , "Effects of sodium nitroprusside and other smooth muscle relaxants on cyclic GMP formation in smooth muscle and platelets," ADV. CYCLIC NϋCLEOTIDE RES. 9: 131-43 (1978).

Although SNP is used to treat cardiac failure from a variety of causes, the cardiac glycosides, such as digitalis, still play a principal role in the treatment of chronic heart failure, in part because they can be taken orally, unlike SNP , which must be administered intravenously. But in addition to a comparatively narrow toxic-to-therapeutic ratio, cardiac glycosides also generally display a relatively weak inotropic effect. This deficiency in the pharmacological profile of cardiac glycosides has prompted an ongoing search for potent inotropic agents that can be employed in the managment of severe congestive heart failure. See Bairn et al, "Evaluation of a new bipyridine inotropic agent - Milrinone

- in patients with severe congestive heart failure," NEW ENGL. J. MED. 309: 748-56 (1983).

Ideally, a single hemodynamic agent would combine SNP-like activity with inotropic activity, and would be suitable for administration by more than one route, e.g. , orally as well as intravenously. Such an agent would be extremely useful in treating heart failure, since it could act to increase the force of contraction of the failing ventricle while concomitantly decreasing the total

peripheral resistance against which the weakened heart must work. See Cohn and Franciosa, "Vasodilator therapy of cardiac failure," NEW ENGL . J. MED. 297: 27-31, 254-58 (1977). To the extent that the agent's SNP-like activity included elevating cGMP levels in blood platelets, the agent could also inhibit platelet aggregation, thereby ameliorating the atherosclerotic condition which is often present in patients with heart failure and to which platelet aggregation contributes. While there is an extensive literature concerning candidates for therapeutic hemodynamic agents , there has apparently been no recognition in the art of pharmacological activity for diphenylhalonium-based compounds in this regard. One bivalent iodine compound, dipheyleneiodonium, and several of its derivatives have been identified as potent hypoglycemic agents, causing substantial , irreversible decreases of sugar levels in the blood of several animal species when administered orally in relatively low dosages. Gatley and Martin, "Some aspects of the pharmacology of diphenyleneiodonium, a bivalent iodine compound," XENOBIOTICA 9: 539-46 (1979). At the cellular level, diphenyleneiodonium catalyzes an exchange of Cl and OH ions across biological membranes, and, independently, diminishes the rate of respiration in mitochondria by inhibiting the oxidation of NADH-linked substrates. Id.

The hypoglyce ia-inducing activity of diphenyleneiodonium has been linked to the compound's ability to impair gluconeogenesis secondarily, via inhibition of mitochondrial NADH oxidation. Holland et al, "Mechanism of action of the hypoglycemic agent diphenyleneiodonium," J BIOL. CHEM. 218:

6050-59 (1973).

In addition , the oral administration of diphenyliodonium salts, such as diphenyliodonium chloride (DIC) , is a proven means for selectively inhibiting

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microbial deamination in ruminant animals and, thereby, protecting aminated components in the animals ^' diet from ruminal fermentation. Chalupa, "Manipulating rumen fermentation," J. ANIMAL SCI. 46: 585-99 (1977). Chalupa et al, U.S. Patent No. 3,862,333, specifically discloses a method for inhibiting the deamination of amino acids by rumen microbes, comprising the oral administration to a ruminant of an effective, nontoxic quantity of a diphenyliodonium salt. Broderick and Balthrop, "Chemical inhibition of amino acid deamination by ruminal microbes in vitro," J. ANIMAL SCI. 49: 1101-11 (1979) , similarly conclude that DIC effectively inhibits deamination at very low ruminal concentrations, and suggest DIC 's utility as a feed additive. Thus, the above-summarized literature on the physiological effects of bivalent iodine compounds makes no mention of hemodynamic activity. In particular, disclosures in the art concerning a sodium nitroprusside-like effect by any species of diphenylhalonium ion are unknown.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a pharmaceutical composition which can be used in animals and humans to elicit sodium nitroprusside-like effects, including stimulation of guanylate cyclase activity in. vivo.

It is another object of the present invention to provide a method for inhibiting the aggregation of blood platelets in mammals.

It is still another object of the present invention to provide a hemodynamic agent which can be used to enhance the contractility of cardiac muscle, for example, in the treatment of congestive heart failure.

, __OMPI _^ ^

It is a further object of the present invention to provide a method for relaxing vascular tone in systemic blood vessels, using an active ingredient which is capable of providing a diphenylhalonium ion i-n vivo. In accomplishing the foregoing objects, there has been provided, in accordance with the present invention, in preferred embodiments of the present invention, the sodium nitroprusside-like effect comprises stimulating guanylate cyclase activity and vasodilation , respectively. In accordance with another aspect of the present invention, a composition is provided which is capable of supplying diphenylhalonium ion i _j vivo for use in inhibiting blood platelet aggregation.

There has also been provided, in accordance with yet another aspect of the present invention, a method for increasing myocardial contractility in an animal or a human, using diphenylhalonium ion as the active agent.

Further objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while inicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit of the scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF DRAWINGS

FIGURE 1 shows the dose-related effect of diphenyliodonium hexaf luorophosphate (DIFP) and SNP, respectively on soluble guanylate cyclase activity. Each point represents group mean +_ S.E. combined from two separate experiments (n=8).

FIGURE 2 depicts the cumulative dose-response of DIFP for three cardiovascular parameters in the anesthetized dog. Increasing amounts of DIFP were injected (i.v.) at the times indicated by the arrows to produce cumulative doses of 0.1, 0.4, 1.4, 4.4 mg/kg. Each point represents mean +_ S.E. (n=3) .

FIGURE 3 shows the effect of DIFP on myocardial contractility in the anesthetized dog.

FIGURE 4 shows the comparison of the cardiovascular effects of DIFP and SNP in the dog. SNP was administered as a 60-minute infusion (10 ,ug/kg/min) and DIFP was given as a bolus (1 mg/kg i.v.) at time zero. Values are group means +

S.E. (n=3).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Compounds capable of providing substituted or unsubstituted diphenylhalonium (DPHO) ion are known to the art. Methods for synthesizing dissociable diphenyliodonium, diphenylbromonium , and dipheny lchloronium salts are disclosed, for example , by Nesmeyanov et al , "Diazylbremonium salts from trif luorbromine and symmetrical azyl compounds of mercury," IZV. AKAD. NAU SSSR, Ser. Khim. 255(5): 1136-40 (Russian) and Nesmeyanov et al, "Synthesis of diarylbromonium salts from B.-F-, and azenes," IZV. AKAD. NAUK SSSR, Ser. Khim. 254(3): 652-56 (Russian), and in U.S. Patents No. 3,151,175; No. 3238,394; and No. 3,981,897. For the purpose of this description, "diphenylhalonium" is defined broadly enough to include a biphenyl-phenyliodonium structure, as disclosed by Koser et al, "New methodology in iodonium salt synthesis," J. ORG. CHEM. 45 : 1543-44 (1980), and an oligomeric stucture represented by the formula

where Ha denotes halogen, as sclosed by Wiley a s and carcinolytic ^X nd Sal ⁾ ter, "Synthesi activity of some diary liodonium salts," J. MED. CHEM. 9: 228-31 (1966). The respective contents of each the documents cited in this paragraph are hereby incorporated by reference.

In general, the DPHO ion can be complexed, in accordance with the present invention, with an anion derived from a nontoxic, pharmaceutically acceptable inorganic or organic acid. The anion could be, for example, chloride, bromide, phosphate, hexaf 1 uorophosph te , iodide, dichloroiodate, hydroxide, sulfate, bisulfate, edisylate, nitrate, benzenesulf onate , methanesulf onate , tosylate, acetate, haloacetate, halosulf onate , propionate, benzoate, fumarate, maleate, lactate, citrate, picrate and tartrate. Because of their relative toxicity, f luoroborate , hexaf luoroarsenate , and benzesul f onate salts are not recommended for use in humans .

Oral application of a DPHO-based compound is preferred in the present invention, although the combination of hydrophilic (halonium) and lipophilic (phenyl) moieties in the compound ^' s structure renders it well-suited for topical application, for example, in combination with a lanolin-petrolatum base as a carrier. DPHO-based compounds can also be given intr venously, intramuscularly or subcutaneously since they are generally soluble, in therapeutical ly effective concentrations, in standard physiological solutions. Since DPHO-based compounds are generally light-sensitive, their preparation for parenteral administration should take into account a probable, gradual loss of efficacy in solution upon exposure to light. For

example, a particular compound could be packaged as an anhydrous powder in a light-resistant glass vial and dissolved just prior to use in sterile buffered saline.

For topical applications, a DPHO-based compound could be combined in a lanolin-petrolatum base as an ointment and packaged, for example, in a tube or in an application patch containing a premeasured dose. Such an ointment, once applied, would be covered by an occlusive cellophane or paper dressing to enhance adsorption. Applied directly or in an application patch, the ointment would be applied to body skin covered by clothing to decrease the chance of photodecomposition.

For oral application, a DPHO-based compound can be combined in light-resistant gelatin capsules with an inert bulk filling agent, such as talc or kaolin. Similarly, for sublingual application, a DPHO-based compound can be combined in tablet form with a suitable filler and a binder which permits rapid dissolution of the tablet when the tablet is placed on the buccal mucose . In addition, a nonvolatile fixing agent, polyethylene glycol 4000, can be added to stabilize the DPHO-based tablet formulation. Preferably, carbohydrate-based fillers like lactose and starch 1500 should be avoided, since the halonium moiety may react with carbohydrates. DHPO ion can stimulate guanylate cyclase activity, and thereby elevate cyclic GMP levels, in approximately the same concentation range (about 10 to 10 M) as does SNP. The effect of DPHO ion on cyclic GMP levels is reflected in its ability to relax systemic vessels, thereby reducing arterial blood pressure and total peripheral resistance.

Like SNP, DPHO ion can also inhibit blood platelet aggregation, possibly because of its SNP-like effect on guanylate cyclase activity. In addition, aside from its properties which parallel those of SNP, DPHO ion acts as an

inotropic agent, increasing the contractility of cardiac muscle and, hence, the force of myocardial contractions.

Studies show that the effective vasodilating dose of DPHO is less than 5 mg/kg/day, with dosages approximately 0.1 to less than 5 mg/kg/day falling within a reasonable therapeutic range. Relatively small doses of DPHO can cause an immediate sustained reduction in mean systemic arterial pressure (MAP) and systemic vascular resistance, accompanied by a slight fall in heart rate, i.e. , without the reflex tachycardia observed with phentolamine and other vasodilators. In addition, cardiac output (CO), stroke volume and the velocity of left ventricular contraction, normalized to peak left ventricular pressure, are all enhanced after DPHO treatment, while pulmonary arterial pressure and pulmonary vascular resistance are relatively unaffected. These pharmaco logic properties of DPHO evidence a utility for DPHO-based compounds in treating both systemic hypertension and chronic or acute left ventricular ' heart failure. In its ability to lower MAP and SVR without a reflexive increase in heart rate, DPHO resembles N,N-di-n-

• propyldopamine and N-n-propyl-N-n-butyl dopamine, both of which dopamine derivatives have been identified as therapeutical ly useful vasodilators. Fennell et al ,

"Hypotensive effects of N,N-Di-n-prop ldopamine in the anesthetized dog: Comparison with sodium nitroprusside," J.

CARDIOVAS PHARM. 2 : 247 ( 1980 ) ; Fennel et al , "Propylbutyldopa ine: Hemodynamic effects in conscious dogs, normal human volunteers and patients with heart failure," CIRCULATION 67: 829 (1983). The following examples illustrate, according to the present invention, DPHO 's different pharmacological properties in standard laboratory preparations. The DPHO- based compound used in aniodonium salt, diphenyliodonium hexaf 1 uorophos phate (DIFP) . Since the products of

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metabolism of DIFP in vivo may include a benzyl moiety, the use of unsubstituted DIFP in treating humans, in accordance with the present invention, is not -recommended. Like other unsubstituted, DPHO-based compounds, DIFP is readily substituted at the bis-4 positions. Substitution at one or both of the 4-positions would make less likely the possibility that a benzyl moiety would be among the metabolic products of a DPHO-based compound. For example, modification to the bis-4-methyl form (metabolic product comprising a toluene moiety) should be considered for human applications. By the same token, either or both 4-positions of the DHPO ion can be substituted by ethyl, butyl, t-butyl, methoxy, ethoxy, amino, -CH-,S0 ₂ , carboxyl , methylcarboxyl and/or ethylcarboxyl .

Examples 1 : Stimulation of guanylate cyclase by DIFP

. Measurement of guanylate cyclase activity . he conversion of [ 3 H]-guanosine triphosphate ([ 3 H]-GTP) to

[ H]-cGMP was assayed using a crude soluble preparation from rat lung, as adapted from the methods of Garbers and Murad,

"Guanylate cyclase methods," ADV. CYCLIC NULEOTIDE RES. 10: 57-67 (1979 ) , and Bohme et al, supra. Rats were anesthetized with sodium pentobarbital (Nembutal ) and the lungs were perfused iri situ with ice-cold 0.9% saline and quickly removed. The whitish lungs were then homogenized (Pol tron-setting #6, 60 seconds) in ice-cold 25 mM TRIS/HCl (pH 7.6) containing 0.25 M sucrose (5 ml/g tissue) and centrifuged at 105,000 x g (4°C) for 60 minutes. The cyclase reaction consisted of 25 mM TRIS-HC1 (pH 7.6) containing isobutylmethylxanthine (3 mM) , creatine kinase (33 units/ml) , creatine phosphate (20 M) , MnCl ₂ (3 mM) , dithioreitol (5 mM) , cGMP (1 M) , [ ³ H]-GTP (0.75 mM, 2.5 x

10 cpm/ml ) and enzyme preparation (approximately 750 ,ug

protein, as determined by the method of Lowry et al, "Protein measurement with the Folin phenol reagent, " J. BIOL. CHEM. 193: 265-75 (1951)). The reaction was initiated by the addition of the [ ³ H ] -GTP/MnCl ₂ /cGMP to tubes containing the remaining constituents, incubated for 15 minutes (37 C) and terminated by adding 150 ,uml 0.4 M zinc acetate followed by 150/Ul 0.2 M sodium carbonate. The tubes were frozen in dry ice/acetone, thawed and centrifuged (2500 x g, 15 minutes) to precipitate unreacted [ H]-GTP. The H-cGMP in the supernatant was isolated by ion-exchange column chromatography using polyethylenimine cellulose (Sigma Chemical Co. , St. Louis, MO) , as taught by Garbers and Murad, supra, and counted by liquid scintillation spectrometry at an efficiency of about 38%.

B. Stimulation of guanylate cyclase. As shown in Table 1 and in Figure 1, respectively, both DIFP and SNP in the above-described assay activated rat lung guanylate cyclase in an concentration-dependent manner and in the same

-5 -3 concentration range (approximately 10 to 10 M), DIFP • having about one-third the maximal activity of SNP. Both compounds produced peak effects at SOO .um, with a loss of activity at higher concentrations .

The guanylate cyclase-stimulating activity of DIFP was tested under several incubation conditions known to influence the activity of SNP. When the cyclase reaction was conducted without dithiothreitol , the activity of SNP was completely abolished, and the effects of DIFP were strongly inhibited. Similar results were obtained when the oxidant methylene blue was included, with the activity of both compounds being clearly reduced.

Table 1. Comparison of sodium nitroprusside (SNP) and diphenyliodonium hexafluorophosphate (DIFP) to activate guanylate cyclase: effects of dithiothreitol (OTT) or methylene blue.

Guan ldite Cyclase Activity*

Treatmeiit (/iM) Control* (X Charige) (-DTT) (X Change)

I. None 0.36 + 0.02 (-) 0.28 + 0.03 (--)

^• SNP ( : ιoo) 1.85 + 0.22 (+410) 0.34 + 0.03 (+21)

SNP ( :3oo) 2.59 +_0.19 (+720) 0.25 + 0.01 (-11)

DIFP (3) 0.23 + 0.02 (-36) . 0.22 + 0.02 (-21)

DIFP (10) 0.35 + 0.01 (-3) 0.30 + 0.02 (+7)

DIFP (30) 0.71 + 0.05 (+97) 0.41 + 0.03 (+46) ^•

DIFP (100) 1.31 + 0.05 (+260) 0.50 + 0.03 (+79)

DIFP (300) 1.51 + 0.02 (+320) 0.56 + 0.01 (+100)

Methylene

Control* {% Change) Blue (1 M) (5. Change)

I. None o.« + 0.02 (--) 0.47 + 0.03 (--)

SNPdo) 0.51 + 0.02 (+21) 0.46 + 0.06 (-2)

SNP (30) 0.62 + 0.04 (+48) 0.46 + 0.02 (-2)

SNP (100) 1.91 + 0.07 (+360) 1.74 + 0.04 (+270)

SNP (300) 4.27 +_ 0.21 (+920) 1.18 +_ 0.02 (+150)

DIFP (3) 0.46 + 0.01 (+10) 0.51 + 0.01 (+9)

DIFP (10) 0.63 + 0.03 (+50) 0.53 + 0.01 (+13)

DIFP (30) 0.97 + 0.02 (+130) 0.71 + 0.01 (+51)

DIFP (100) 1.46 + 0.01 (+250) - 0.91 + 0.02 (+94)

DIFP (300) 1.50 ^* + 0.01 (+260) 1.02 + 0.04 (+120)

Values represent group means + S.E. (n=3) in pmoles/mg protein/min.

Control groups contained 5 mM DTT during the 15 minute incubation.

Example 2: Cardiovascular Effects of DPHO Ion

A. Measurement of cardiovascular function in dogs. Mongrel dogs, each weighing between about 10 and 20 kg, ere anesthetized with Delvinal sodium (50 mg/kg), intubated, and ventilated with a Harvard volume-cycled ventilator.

Catheters were placed from the femoral arteries retrograde into the left ventricle and mid-abdominal aorta to measure left ventricular and systemic arterial pressures, respectively. A Swan-Ganz thermister-tipped pulmonary artery catheter (American Edwards Laboratories, Santa Anna,

CA ) was placed from the right femoral or right external jugular vein into the pulmonary artery. DIFP was administered as a bolus intraveneous (i.v.) injection, while

SNP was given by continuous infusion to obtain a sustained reduction in MAP.

Cardiac output (CO) was measured with an American Edwards 9520 A cardiac output computer using thermal dilution technique. Ten mis of ice-cold (1-5 C) 5% dextrose in water was injected as a bolus in the right ventricle on two occasions within 2 minutes, and the values averaged for the calculation of the CO. The abdominal aortic catheter was connected to a Hewlett Packard transducer, and blood pressure was recorded on a Hewlett Packard 77588 polygraph. Maximum left ventricular (IV dP/dt) and left ventricular pressure (LVP), as well as left ventricular end diastolic pressure (LVEDP), were measured via the catheter inserted into the left ventricle through the right femoral artery. Cardiac contractility was measured as max dP/dt, or as max dP/dt divided by LVP at max dP/dt . Lead aVF electrocardiogram was monitored continuously and 5000 units of heparin were administered to avoid clotting. In addition to the directly obtained parameters, total peripheral

vascular resistance (TPR dynes • sec • cm ) was calculated (as mean aortic pressure/CO x 80).

B . Reduction in MAP and enhancement of cardiac contractility. Mean systemic arterial pressure was reduced within 10 minutes after i.v. injection of DIFP. A cumulative dose of 4.4 mg/kg i.v. , given over a 30-minute period, reduced MAP about 70 mmHg (Figure 2A). There was no recovery in mean arterial pressure during the next 130 minutes. The fall in MAP was associated with a clear reduction in total peripheral resistance (Figure 2B) .

Cardiac output (measured by thermal dilution) was transiently reduced during the injection period (Figure 2C) , but subsequently rose by the end of the injection period and reached maximum value at 70 min. The rate of ventricular contraction (dP/dt) , normalized for left ventricular pressure, increased steadily after administration of DIFP (Figure 3 ) , indicating an increase in myocardial. contr ac t i 1 i t .

A 60-minute infusion with SNP ( 10 ,ug/kg/min } produced a maximal fall in MAP of comparable magnitude to 1 mg/kg (i.v.) DIFP given as a bolus (Figure 4A) . The effect of SNP persisted for as long as the infusion was maintained, but once the infusion was terminated, MAP returned to preinjection values within 20 minutes (Figure 4A) . The profile of the blood pressure response to SNP differed from that of DIFP the latter producing a more gradual and longer lasting decline (3 hours) in MAP. In general, heart rate was lowered by about 10% for the duration of the 3-hour experiment (Figure 4B). Negative dP/dt was also reduced by both SNP and DIFP, following an almost identical time course as that for MAP (compare Figures 4 A and 4C) .