WO1998031380A1

WO1998031380A1 - Phalloidin derivatives and analogs to treat congestive heart failure

Info

Publication number: WO1998031380A1
Application number: PCT/US1998/000952
Authority: WO
Inventors: Anna E. Boukatina; Kenneth B. Campbell; Lawrence L. Kunz; Sudhakar Kasina; Louis J. Theodore; Alan R. Fritzberg
Original assignee: Washington State University Research Foundation; Neorx Corporation
Priority date: 1997-01-16
Filing date: 1998-01-16
Publication date: 1998-07-23
Also published as: AU6030098A

Abstract

A method to treat congestive heart failure with an analog or derivative of phalloidin is provided. Also provided is a method to treat other cardiopathologies associated with reduced heart muscle contractile strength, as well as novel analogs or derivatives of phalloidin, pharmaceutical compositions comprising analogs or derivatives of phalloidin, and intermediates useful for preparing analogs or derivatives of phalloidin.

Description

PHALLOIDIN DERIVATIVES AND ANALOGS TO TREAT CONGESTIVE HEART FAILURE

Background of the Invention

Congestive heart failure (CHF) results from the inability of the heart to pump adequate amounts of blood for tissue perfusion (Cohn, New Eng. J. Med., 335:490-498, 1996). CHF is almost always accompanied by retention of fluids or the accumulation of fluids in certain tissues. There are many causes of CHF but, ultimately, CHF manifests as an inability of the heart either to fill with blood when the heart muscle is relaxed, i.e., diastolic CHF, or to adequately discharge blood contained within its chambers when heart muscle contracts, i.e., systolic CHF (Goldsmith and Dick, Am. J. Med., 95:645-665, 1993). In systolic CHF, the heart muscle has lost some of its normal contractile strength. Thus, the heart muscle in a heart from a patient with systolic CHF produces less than normal contractile force or, if allowed to shorten against a load during contraction, shortens less rapidly and to a lesser extent. Such impairment of heart muscle function results in an inability of the heart to deliver a normal stroke volume when the heart is filled to a normal end-diastolic volume. An adequate stroke volume to sustain a cardiac output can be delivered by hearts of CHF patients only when the heart has been filled to greater than normal end-diastolic volumes. At best, this is a life commensurate with reduced activity and exertional effort.

As a result, hearts in patients with systolic CHF undergo marked structural remodeling and enlargement. There are a variety of patterns to this remodeling including concentric enlargement with thickening of the walls relative to chamber dimensions, eccentric enlargement with walls and chamber retaining the same relative dimensions, and dilated enlargement with thinning of the walls relative to the chamber dimensions. Regardless of the pattern, left ventricular remodeling, when carried to an extreme, complicates the functional disturbances associated with the loss of myocardial contractility. Therefore, not only has the heart muscle lost contractility in systolic CHF, but the structural environment in which this muscle must work undergoes a change that is disadvantageous to the transformation of muscle contractile function into heart blood-pumping function. Concomitantly with remodeling of the heart, there are adaptive changes in the vascular system resulting in increased total peripheral resistance and, through the sympathetic-renin-angiotensin neuro-humoral axis, adaptive changes in the entire circulatory body fluid system. These changes result in expanded circulating and interstitial body fluid volumes. Rather than alleviate the problems of CHF, these vascular and circulatory-body-fluid adaptive changes actually exacerbate CHF symptoms to the point that they may become life threatening.

Treatment of CHF is currently directed at remediation of symptoms through lifestyle modifications, including proper diet and exercise, and through pharmacologic therapy. Pharmacologic therapy is primarily directed at relieving CHF symptoms (diuretics, vasodilators, cardiac inotropic agents) and impeding or reversing structural changes in the heart and vascular system that exacerbate these symptoms (ACE inhibitors, angiotensin blocking agents).

Currently, cardiac inotropic agents for therapeutic uses fall into one of three categories: digitalis glycosides; β-agonists; and phosphodiesterase (PDE) III inhibitors. All of these agents enhance cardiac muscle contraction by making more intracellular Ca²⁺ available to cardiac myofilaments during muscle activation. Unfortunately, increased intracellular Ca²⁺ has three undesirable effects: 1) it increases cellular electrical excitability and thus, increases the potential of arrhythmia induction or subsequent fibrillation; 2) it increases the energy costs of contraction by increasing the energy needed to handle the extra Ca²⁺ during a contraction cycle, thereby increasing cardiac load; and 3) it adds an extra Ca²⁺ load to debilitated heart muscle cells that already have difficulty maintaining intracellular Ca²⁺ homeostasis which can result in cell death.

The increase in intracellular Ca²⁺ that results from treatment with one of the currently used cardiac inotropes often exacerbates the condition of patients with CHF. This is because functionally impaired heart muscle cells in patients with CHF barely maintain appropriate excitable rhythm, a balance of their energy needs with their energy supply, or intracellular Ca²⁺ levels low enough to prevent the cascade of events that lead to cell death resulting from an increase in intracellular Ca²⁺. For example, lethal cardiac arrhythmias often develop, as well as expanded areas of necrosis in border zones surrounding ischemic regions which lead to further loss of cardiac function.

For these reasons, the strong effects of β-agonists preclude their use in chronic CHF. Furthermore, the PDE III inhibitors have been associated with increased mortality during clinical trials (Packer et al., New EngJMed., 325:1468- 75, 1991), indicating that they too cause excessive disturbance of the intracellular Ca²⁺ environment. The digitalis glycosides have a narrow margin of safety and their efficacy in CHF has been seriously questioned (Bigger et al., Am. J. Cardiol, 55:623-30, 1985). Thus, problems with the currently available cardiac inotropes either contraindicate or limit their use in the treatment of CHF.

A new class of cardiac inotropic drugs, termed Ca²⁺-sensitizing cardiac inotropes, has been described. These agents enhance force development by cardiac myofilaments in the presence of fixed amounts of Ca²⁺, i.e., without an increase in intracellular Ca²⁺ concentrations (Lee and Allen, Modulation of Calcium Sensitivity, Oxford Univ. Press, Inc, 1993). Some Ca²⁺-sensitizers, e.g., levosimedan, increase the Ca²⁺ binding affinity of troponin C (TnC; Pollesello et al., J. Biol. Chem., 269:28584-28590, 1994), while others do not, e.g., EMD 57033 and CGP 48506 (Solaro et al., Circ. Res., 73:981-990, 1993; Harold et al, J. Med. Chem., 38:2946-2954, 1995). It has been proposed that compounds which do not increase the Ca²⁺ binding affinity of TnC change the distribution of cycling crossbridges so that more crossbridges accumulate in force-bearing states (Palmer et al, Cardiovasc. Res., 32:411-421, 1996).

One problem with Ca²⁺ sensitizing compounds is their specificity of action. For example, several Ca²⁺ sensitizers, e.g., sumazol, pimobendan, levosimendan and MCI 154, are also known to be PDE III inhibitors. This is also true of EMD 53998, which was found to consist of a racemic mixture in which the (-) enantiomer was a PDE III inhibitor while the (+) enantiomer, EMD 57033, was primarily a Ca²⁺ sensitizer (Gambassi et al., Am. J. Physiol, 264:H728-738, 1993; White et al., Circ. Res., 73:61-70, 1993; Solaro et al., Circ. Res., 73:981-990, 1993).

Thus, a need exists for a Ca²⁺ sensitizing cardiac inotrope which is useful to treat systolic CHF, and which does not increase intracellular Ca²⁺ concentrations in heart muscle.

Summary of the Invention The invention provides a method to treat a mammal having, or prevent a mammal at risk of, a condition characterized by reduced heart muscle contractile strength. The method comprises the administration of an amount of at least one therapeutic agent effective to increase the contractile force of the heart in said mammal. Preferably, the agent binds to cardiac actin more strongly than to skeletal actin. Also preferably, the agent is a non-toxic phalloidin derivative or analog, e.g., a compound of formula (I)-(IN) (see below). Conditions characterized by reduced heart muscle contractile strength include, but are not limited to, CHF, ischemic myocardial infarction, hypertension, myocarditis, epicarditis, endocarditis, pulmonary edema, heart failure secondary to ischemic heart disease, valavular heart disease, hypodynamic heart such as occurs in cardiogenic shock, cardiac arhythmia, atrial or ventricle arrhythmias, other cardiomyopathies or acute heart conditions, e.g., septic shock leading to cardiogenic shock or post-myocardial infarction. As used herein, "cardiomyopathies" includes, but is not limited to, dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy, as well as cardiomyopathies of known origin, e.g., from infection by viruses, bacteria or fungi, metabolic disorders, general system diseases, hereditary muscle and neurological disorders and the like. The term "mammals" includes human patients, domestic animals and other animals.

The invention also provides a method to prevent or treat systolic congestive heart failure (CHF) in a mammal, e.g., a human. The method comprises the administration to a mammal of an amount of at least one non-toxic phalloidin analog or derivative effective to reduce or inhibit at least one of the symptoms associated with systolic CHF, e.g., a decrease in discharge of blood from the heart, a decrease in contractile force of the heart, and/or an increase in the structural remodeling and enlargement of the heart. While phalloidin derivatives have described as being useful to treat interleukin-2 mediated edema (Shepro et al., U.S. Patent No. 5,278,143), interleukin-2 mediated edema is an inflammatory edema related to increased endothelial permeability which results in an exudate having proteins, e.g., plasma proteins, and polymorphonuclear leukocytes. In contrast, the non-inflammatory edema which is associated with CHF is protein poor, and is a clinically distinct pathology. Preferably, the administration of a phalloidin analog or derivative of the invention enhances contractile vigor of the heart muscle without increasing intracellular Ca²⁺ levels, i.e., it is a Ca²⁺-sensitizing agent. Preferred phalloidin analogs or derivatives are ones that are specific in their Ca²⁺-sensitizing action, i.e., they have high affinity for cardiac actin relative to skeletal actin. Also preferably, the administration of a phalloidin analog or derivative of the invention enhances contractile vigor of the muscle fibers without increasing oxygen consumption by the heart. In addition, phalloidin analogs or derivatives of the invention that do not increase arrhythmia induction and/or cardiac load are particularly useful for prolonged or chronic use.

As used herein, "non-toxic" with respect to a phalloidin analog or derivative means that the administration of the phalloidin analog or derivative does not result in detrimental consequences to an organism, e.g., liver toxicity, that outweigh the therapeutic benefit provided by the administration. The toxicity of a phalloidin analog or derivative can be measured by methods well known to the art, e.g., by determining the LD₅₀ in laboratory animals (mice, rats, guinea pigs, rabbits, hamsters, etc.), for example, as described by Theodor Wieland, "Peptides of poisonous Amanita mushrooms" Springer- Verlag, Berlin, 1986, p. 45. Toxic phalloidin analogs and derivatives have a very high affinity for F-actin, irreversibly bind to actin, or have an LD₅₀ which is similar to, or up to 15 times greater than, the LD₅₀ of phalloidin. High affinity for actin leads to strong stabilization of the actin filament. As described hereinbelow, the ability of some phalloidin derivatives to modify skeletal muscle tension was found to be qualitatively correlated with their ability to structurally stabilize F-actin. Surprisingly, dethiophalloidin, a derivative of phalloidin that is a poor F-actin stabilizer in skeletal muscle was found to enhance cardiac muscle contraction (Example 2). Thus, preferred phalloidin analogs or derivatives bind to actin more weakly than does phalloidin. Preferably, the phalloidin analogs or derivatives of the invention have at least about 1%, preferably at least about 10% or 50%, and more preferably at least about 100%, or greater than 200%, of the biological activity of dethiophalloidin, e.g., the ability to enhance cardiac muscle contraction. The biological activity of a phalloidin analog or derivative on muscle, skeletal or cardiac, is determined by methods well known to the art. See, for example, Examples 1-2 hereinbelow, Bukatina et al., J. Mol. Cell. Cardiol, 27:1311-1315, 1995; Bukatina and Fuchs, J. Muscl. Res. & Cell. Motil, 15:29-36, 1994; Bukatina et al., Histochem., 81:89-94, 1985; Bukatina et al., J. Muscl. Res. & Cell. Motil, 17:365-371, 1996. Thus, the activity of an analog or derivative may be characterized by in vitro assays, such as the contractile force of cardiac muscle under normal or stress conditions, relaxation times in an explanted heart, or actin binding, or by in vivo assays such as left ventricle ejection fraction, oxygen consumption or exercise tolerance in a mammal, in the presence or absence of the analog or derivative.

A preferred phalloidin analog or derivative is a compound of formula ©:

wherein:

(1) each of R¹ and R° is H, OH, (C,-C₄)alkyl, O(C,-C₄)alkyl, halo, halo(C_r

C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(R")(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₂₂)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, or a residue of a sugar;

(2) the bonds represented by are present or one is absent; (3) R is H, (C,-C₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(Rⁿ)(R¹²); (4) R² is H or (C,-C₄)alkyl; (5) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl; (6) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy) (preferrably, R⁴ is cis to the carboxy substituent at the 2-position of the pyrrolidine ring);

(7) R⁵ is H, OH, acetoxy, halo or OTs;

(8) each of R⁶ and R⁷ is absent or is H, (C,-C₄)alkyl, acetyl, or phenyl when the C N bond is absent;

(9) R¹⁰ is H, (C,-C₄)alkyl, CO₂H, CO₂(C,-C₄)alkyl, or C(O)N(R' ^!)(R¹²);

(10) X is H, -CH₂-, C(=O), C(=S), C(=NR¹⁵), S(O),.₂, O, S, or Se, wherein R¹⁵ is hydrogen, (C_rC₆)alkyl, hydroxy, (C,-C₄)alkoxy, tosyl, or mesyl;

(11) R⁸ is absent, or is H or (C C₄)alkyl when the X Z bond is absent; (12) when the X Z bond is present and X is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O),_.2, then Z is O, S, S(O),.₂, Se or NR¹⁶, wherein R¹⁶ is hydrogen, (C_r

C₆)alkyl, hydroxy, (C_rC₄)alkoxy, tosyl, or mesyl; when the X Z bond is present and X is O, S, S(O),.₂, Se or NR¹⁶, then Z is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O) ₂,; or when the X Z bond is absent then Z is H or (C_rC₄)alkyl;

(13) x is 0, 1 or 2;

(14) Y is H, (C_rC₄)alkyl, optionally substituted by N(R' ')(R¹²), acetoxy, acetyl, 2-methyl-l,3-dithiolan-2-yl optionally 4-substituted with CH₂N(Rⁿ)(R¹²) or CO₂Rⁿ; or is C(R¹³)(R¹⁴)(OH) wherein R¹³ is H, (C,-C₄)alkyl, CO₂H or CH₂OQ wherein Q is H, acetyl, or Ts (tosyl) and R¹⁴ is (C,-C₄)alkyl, halo(C_rC₄)alkyl, haloacetyl, CO₂H, CH₂OQ wherein Q is as defined above, or is C(O)(C₁₂-C₂₂)alkyl, a peptide or protein, a residue of a sugar (e.g. rhamnose, glucose, xylose, galactose, etc.), or in combination with R⁶ is -CH₂-O- and R⁶ is absent or is OR" when the C N bond is absent; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (I) is not phalloidin. For example, when R, R°, R¹ and R² are H, X is S, Z is CH₂, R³ is CH₃, R⁴ and R⁵ are OH, R⁶, R⁷ and R⁸ are absent, Y is C(CH₃)(OH)(CH₂OH), then R¹⁰ is not CH₃. A preferred compound of formula (I) is dethiophalloidin and analogs and derivatives thereof. Another preferred compound of formula (I) is secophalloidin and analogs and derivatives thereof.

As used herein, a "derivative" of a therapeutic agent of the invention includes modifications that do not substantially effect the biological activity of the therapeutic agent. By "substantially effect" is meant that the activity is quantitatively different but qualitatively the same. For example, a, or other modifications which may improve the derivative of phalloidin may comprise for example, bound peptides, polypeptides, phospholipids, steroidal moieties, fatty acid moieties, covalently linked carbohydrates solubility, absorption or biological half life of the derivative. The modifications may also decrease the toxicity of the analog or derivative, or eliminate or attenuate any undesirable side effect of the analog or derivative.

Another preferred phalloidin analog or derivative is a compound of formula (II):

wherein:

(1) each of R¹ and R° is H, OH, CH₃O, halo, CN, NO₂, CF₃, CO₂H, CO₂(C_r C₄)alkyl or N(R^π)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, CH₃, formyl, or acetyl; (4) R² is H or CH₃;

(5) R³ is CH₃, iso-propyl, or iso-butyl;

(6) R⁴ is H or OH;

(7) R⁵ is H or OH:

(8) R⁶ is absent or together with R¹⁰ is a covalent bond; (9) R⁷ is absent or is COCH₃ or R¹ * when the C N bond is absent ;

(10) X is H, S, O, Se or S(O)_1-2;

(11) Z is C(O) or -CH₂-, or H or (C_rC₄)alkyl when the X Z bond is absent;

(12) R⁸ is absent or is H or (C_rC₄)alkyl when the X Z bond is absent;

(13) R¹⁰ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl;

(14) R¹³ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (II) is not phalloidin. A preferred compound of formula (II) is dethiophalloidin and analogs and derivatives thereof. Another preferred phalloidin analog is a compound of formula (III):

wherein the bond represented by C — N is absent or present, R is -(C(OR⁴)(CH₃)(CH₂OR⁵) or haloacetyl, wherein R⁴ is H or together with Z is a covalent bond; R⁵ is H, a peptide or protein, a sugar residue or C(O)R' wherein R' is (C_]2-C₂₂)alkyl, optionally comprising 1-3 double bonds, Z is absent or together with R⁴ is a covalent bond, Y is absent or is H when the C — N bond is absent, and X is - H H-, SO, O, or Se; R³ is H or an acetyl, or a pharmaceutically acceptable salt thereof.

Yet another preferred analog or derivative of the invention is a compound of formula (IN):

wherein:

(1) R^a, R^b, and R^g are individually any naturally or unnaturally occurring alpha amino acid, or lysine coupled through its ε-amino group to a steroidal or fatty acid moiety; (2) x is 0, 1 or 2;

(3) each of R¹ and R° is H, OH, (C,-C₄)alkyl, O(C_rC₄)alkyl, halo, halo(C_r

C₄)alkyl, CΝ, ΝO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, or a residue of a sugar; (4) the bond represented by is present or absent; (5) R is H, (C_rC₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(R' ')(R¹²);

(6) R² is H or (C,-C₄)alkyl;

(7) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl;

(8) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy); (9) X is H, O, S, Se, or S(O),_₂;

(10) R⁸ is absent or is H or (C_rC₄)alkyl when the X Z bond is absent; and

(11) Z is C(O) or -CH₂-, or is H or (C,-C₄)alkyl when the X Z bond is absent.

Preferably, R^a and R^b are individually valine, leucine, isoleucine, alanine, serine, methionine, phenylalanine, norleucine, glycine or lysine. Preferably, R^ε is valine, leucine, isoleucine, serine, methionine, phenylalanine, norleucine, glycine or lysine. The invention also provides a method to enhance cardiac muscle contraction in a mammal, comprising the administration of an amount of at least one non-toxic phalloidin derivative or analog effective to enhance cardiac muscle contraction in said mammal. Preferably, the phalloidin analog or derivative which is administered is a compound of formula (I), a compound of formula (II), a compound of formula (III), or a compound of formula (IN), or a combination thereof.

The invention also provides novel phalloidin analogs or derivatives of formula (I), (II), (III), or (IN), as well as salts thereof, and derivatives thereof that comprise bound peptides, polypeptides, phospholipids, steroidal moieties, fatty acid moieties, or covalently linked carbohydrates.

Some phalloidin analogs or derivatives may be useful as intermediates for preparing other phalloidin analogs or derivatives. The invention also provides intermediates (e.g. compounds of formula (I), (II), (III), or (IN)) that are useful for preparing other phalloidin analogs or derivatives.

Brief Description of the Figures

Figure 1 depicts the dose-dependent effects of dethiophalloidin (DTPH) on cardiac muscle force. The lowest trace indicates pCa values. Single arrows indicate solution changes. Double arrows indicate transfer of the preparation to a new well.

Figure 2 depicts the response of DTPH at different pCa.

Figure 3 depicts the absence of effect of DTPH on passive, relaxed cardiac muscle.

Figure 4 depicts the absence of effect of DTPH on rigor force cardiac muscle.

Figure 5 depicts a representative trace of the force of the effect of DTPH on cardiac muscle. Figure 6 depicts a graph of force versus pCa. A) Absolute force versus pCa. B) Relative force versus pCa. Points indicate mean ± SE.

Figure 7 depicts a graph of force versus pCa. A) Absolute force versus pCa. B) Relative force versus pCa. Points indicate mean ± SE. This data was obtained from a muscle from a different animal than shown in Figure 5. In contrast to the data shown in Figure 5, the experiment was conducted at pH 6.8.

Figure 8 depicts the prevention of the DTPH-induced cardiac muscle force enhancement by pretreatment with phalloidin (PH) (80 μM).

Figure 9 depicts the effect of PH administration (40 μM) on muscle force elevated by DTPH. Figure 10 depicts the effect of (S)-phalloidin-sulfoxide A on muscle isometric force at full activation (A) and at partial activation (B).

Figure 11 depicts the dose-dependent effect of secophalloidin at maximal calcium activation.

Figure 12 depicts the relationship between the action of secophalloidin (80 μM) and phalloidin (80 μM). In each activation episode, pCa was 5.89, which corresponds to about half maximal activation (see Figure 6B). (S)- phalloidin-sulfoxide A was added at 40 μM.

Figure 13 depicts exemplary and preferred amino acid substitutions. Detailed Description of the Invention

The present invention provides a method to prevent or treat congestive heart failure in a mammal which employs a phalloidin-based cardiac inotrope having specificity for cardiac muscle. Phalloidin, an agent which modulates the contractile process, i.e., the interaction of actin filaments with myosin in muscle, by binding to actin, is one of several toxic compounds produced by the mushroom Amanita phalloides (Weiland, T., Peptides of Poisonous Amanita Mushrooms. New York, Springer, 1986). In binding tightly to F-actin, phalloidin stabilizes the F-actin filament and inhibits filament depolymerization. Phalloidin is highly toxic to cells, such as liver cells, where rapid actin depolymerization and polymerization is required for cellular function. However, in muscle cells, phalloidin does not disrupt cellular function but instead modulates the contractile process (Bukatina and Morozov, Biophysics, 24:527-531 (1979); Son'kin et al., Biophysics, 28:892-899 (1983); Bukatina et al., Histochemistry, 81 :301-304(1984); Alievskaya et al., Biophysics, 32:105-109 (1987); Boels and Pfϊtzer, J. Muscle Res., Cell Motil, 13:71-80 (1992); Bukatina and Fuchs, J. Muscle Res. Cell Motil, 15:29- 36 (1994); Bukatina et al., J. Mol Cell. Cardiol, 27:1311-1315 (1995); Bukatina et al., J. Muscle Res. Cell Motil, 17:365-371 (1996); Bukatina, Biophysics, 41:97-104 (1996)).

Moreover, this modulation varies qualitatively with muscle type (Bukatina, Biophysics, 41:97-104 (1996)). For example, cardiac muscle response to phalloidin differs from that of other muscles, both with respect to the shape of the response and the time scale with which it evolves. In all types of skeletal muscle that have been studied (fast and slow muscles from evolutionarily distant animals) and in smooth muscle, the main effect of phalloidin is a decrease in tension. In mammalian skeletal muscles under specific conditions, the initial decrease is followed by an increase in tension. Unexpectedly, cardiac muscle responds uniquely with only an increase in tension. Moreover, whereas the phalloidin- induced force changes develop over tens of minutes in skeletal and smooth muscle, the response in cardiac is completed more quickly, i.e., in two to three minutes.

The observed muscle-type specific differences in response to phalloidin are believed to be due to the differences in thin filament composition. Both skeletal and smooth muscle thin filaments contain proteins located along the entire filament length (nebulin and caldesmon, respectively) that are not expressed in cardiac muscle (Small et al., Eur. J. Biochem., 208:559-572 (1992)). Moreover, there is evidence that phalloidin binds very slowly in skeletal muscle but binds much more quickly in cardiac muscle (Ao and Lehrer, Biophys. J., 66:A194, 1994). Furthermore, phalloidin and nebulin bind to the same site on the actin filament, and the binding of phalloidin causes unzipping of nebulin from the actin filament. This slow process is believed to be the rate limiting step in phalloidin binding in skeletal muscle. Thus, derivatives and analogs of phalloidin were analyzed for their ability to enhance cardiac muscle contraction.

I. Identification of Therapeutic Agents Falling Within the Scope of the Invention

Agents useful in the practice of the methods of the invention include agents that increase heart muscle contractility, e.g., phalloidin analogs and derivatives, preferably by reversibly binding to cardiac actin. When administered to a subject in need of treatment, the agents of the invention are substantially free of natural contaminants which associate with the agent either in vivo (in a prokaryotic or eukaryotic) host, or in vitro (as a result of a chemical synthesis). An agent is said to be "substantially free of natural contaminants" if it has been substantially purified from materials with which it is normally and naturally found before such purification. Examples of natural contaminants include, but are not limited to, non- actin-binding polypeptides or peptides, carbohydrates, glycosylated peptides, lipids, membranes and the like. Thus, preparations containing the agents of the invention may include quantities of contaminants which do not interfere with the desired therapeutic effect of the agent upon administration thereof and do not harm the animal as the result of the administration.

A preferred therapeutic agent is an analog or derivative of phalloidin. Phalloidin analogs and derivatives falling within the scope of the invention include pharmaceutically useful, or "non-toxic," acyclic and cyclic phalloidin analogs or derivatives which enhance cardiac muscle contraction by increasing the sensitivity, i.e., response, of cardiac muscle to Ca²⁺. These phalloidin analogs and derivatives include all phalloidin analogs and derivatives that bind to thin filament actin of normal rabbit skeletal muscle more weakly than phalloidin. Phalloidin analogs and derivatives of the invention include, but are not limited to dethiophalloidin, secophalloidin, N-acetyl-secophalloidin, and the phalloidin analogs and derivatives described in Weiland (see Tables 16 and 18 in Peptides of the Amanita Mushrooms, Springer Nerlag, 1986, both of which Tables are specifically incorporated by reference herein) and Shepro et al. (U.S. Patent No. 5,278,143, Table 1 of which is specifically incorporated by reference herein), the disclosures of which are incorporated by reference herein.

Also included within the scope of the invention are amino acid analogs of phalloidin which have at least one amino acid substitution, deletion or addition relative to phalloidin. Amino acid substitutions include substitutions which utilize the D rather than L form, as well as other well known amino acid analogs. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, garnma-carboxyglutamate; hippuric acid, octahydroindole-2- carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methyl-alanine, para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine.

One or more of the residues of the analog can be altered, so long as the analog is biologically active. Preferably, the analog has at least about 10% of the biological activity of the corresponding non-toxic phalloidin. Conservative amino acid substitutions are preferred—that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative substitutions are shown in Figure 13 under the heading of exemplary substitutions. More preferred substitutions are under the heading of preferred substitutions. After the substitutions are introduced, the amino acid analogs are screened for biological activity.

Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the generally cyclic nature of the phalloidins, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic; trp, tyr, phe. Preferably, the substitution increases the hydrophobicity of the analog so as to increase its bioavailability.

The invention also envisions phalloidin analogs with non- conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another. For example, preferred amino acid analogs of phalloidin include a compound of formula (IN):

wherein:

(I) R^a, R^b, and R^g are individually any naturally or unnaturally occurring alpha amino acid, or lysine coupled through its ε-amino group to a steroidal or fatty acid moiety; (2) x is 0, 1 or 2;

(3) each of R¹ and R° is H, OH, (C,-C₄)alkyl, O(C_rC₄)alkyl, halo, halo(C_r C₄)alkyl, CΝ, ΝO₂, CF₃, CO₂H, CO₂(C_rC₄)alkyl or N(R")(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, or a residue of a sugar; (4) the bond represented by is present or absent;

(5) R is H, (C,-C₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(R> >)(R¹²);

(6) R² is H or (C,-C₄)alkyl;

(7) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl;

(8) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy); (9) X is H, O, S, Se, or S(O),.₂;

(10) R⁸ is absent, or is H or (C_rC₄)alkyl when the X Z bond is absent; and

(I I) Z is C(O) or -CH₂-, or is H or (C,-C₄)alkyl when the X Z bond is absent.

Preferably, R^a and R^b are individually valine, leucine, isoleucine, alanine, serine, methionine, phenylalanine, norleucine, glycine or lysine. Preferably, R^g is valine, leucine, isoleucine, serine, methionine, phenylalanine, norleucine, glycine or lysine. Another preferred phalloidin analog or derivative is a compound of formula (I):

wherein: (1) each of R¹ and R° is H, OH, (C_rC₄)alkyl, O(C,-C₄)alkyl, halo, halo(C_r C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C_rC₄)alkyl or N(R^U)(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₄)alkyl, benzyl or phenyl

(2) the bonds represented by are present or one is absent;

(3) R is H, (C,-C₅)alkyl, acetyl, benzyl, or CH₂C(O)N(Rⁿ)(R¹²); (4) R² is H or (C,-C₄)alkyl;

(5) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl (R³ is preferrably H, (C,-

C₄)alkyl or benzyl);

(6) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy); (7) R⁵ is H, OH, acetoxy, halo or OTs; (8) each of R⁶ and R⁷ is absent or is H, (C,-C₄)alkyl, acetyl, or phenyl when the

C N bond is absent;

(9) R¹⁰ is H, (C,-C₄)alkyl, CO₂H or C(O)N(Rⁿ)(R¹²) (R¹⁰ is preferrably H, (C,- C₄)alkyl, or C(O)N(Rⁿ)(R¹²));

(10) X is H, O, S, Se, or S(O),.₂; (11) R⁸ is absent, or is H or (C_rC₄)alkyl when the X Z bond is absent;

(12) Z is C(O) or -CH₂-, or is H or (C , -C₄)alkyl when the X Z bond is absent;

(13) x is 0, 1 or 2;

(14) Y is H, (C_rC₄)alkyl, optionally substituted by N(Rⁿ)(R¹²), acetoxy, acetyl, 2-methyl- 1 ,3-dithiolan-2-yl optionally 4-substituted with CH₂N(R' ')(R¹²) or

CO₂R^π; or is C(R¹³)(R¹⁴)(OH) wherein R¹³ is H, (C_rC₄)alkyl, CO₂H or CH₂OQ wherein Q is H, acetyl, or Ts (tosyl) (R¹³ is preferrably H, (C_r C₄)alkyl, or CH₂OQ) and R¹⁴ is halo(C_rC₄)alkyl, haloacetyl, CO₂H, or CH₂OQ (R¹⁴ is preferrably halo(C_rC₄)alkyl, CO₂H, or CH₂OQ); wherein Q is as defined above, or is C(O)(C₁₂-C₂₂)alkyl, a peptide or protein, a residue of a sugar, or when the C N bond is absent, in combination with R⁶ is

-CH₂-O- and R⁶ is absent, or is OR¹¹ (preferrably Q is H, acetyl, Ts, C(O)(C₁₂-C₂₂)alkyl optionally comprising 1-3 double bonds, a peptide or protein, a residue of a sugar, haloacetyl, or when the C N bond is absent, in combination with R⁶ is -CH₂-O- and R⁶ is absent, or R⁶ is OR¹¹); or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (I) is not phalloidin. When Y is a peptide or protein, it is preferred that the peptide or protein is a fragment of an antibody, or an antibody, which binds to a molecule which is specific to the heart, e.g., cardiac muscle. When Y is haloacetyl, a peptide or protein molecule can be readily added at this position, by methods well known to the art. Also preferably, a compound of formula (I) includes wherein:

(1) each of R° and R¹ is H, (C₂-C₄)alkyl, O(C₂-C₄)alkyl, halo(C,-C₄)alkyl, CO₂(C₃-C₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is (C₃-C₄)alkyl, benzyl or phenyl;

(2) the bonds represented by are present or one is absent;

(3) R is H, (C₂-C₅)alkyl, benzyl, or CH₂C(O)N(R¹ (R¹²);

(4) R² is H or (C₂-C₄)alkyl;

(5) R³ is H, (C,-C₄)alkyl or benzyl; (6) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy);

(7) R⁵ is H, OH, acetoxy, halo or OTs;

(8) each of Re and R⁷ is absent or is H, (C,-C₄)alkyl, COCH₃, or phenyl when the C N bond is absent; (9) R¹⁰ is H, (C_rC₄)alkyl or C(O)N(R' ')(R¹²);

(10) X is H, S, or S(O),.₂;

(11) R⁸ is absent, or is H or (C_rC₄)alkyl when the X Z bond is absent;

(12) Z is -CH₂- or is H or (C,-C₄)alkyl when the X Z bond is absent;

(13) x is 0, 1 or 2; (14) Y is H, (C_rC₄)alkyl, optionally substituted by N(R^U)(R¹²), acetoxy, acetyl, 2-methyl-l,3-dithiolan-2-yl optionally 4-substituted with CH₂N(R^π)(R¹²) or CO₂Rⁿ; or is C(R^I3)(R¹⁴)(OH) wherein R¹³ is H, (C,-C₄)alkyl or CH₂OQ wherein Q is H, Ac or Ts (tosyl) and R¹⁴ is halo(C_rC₄)alkyl, CO₂H or CH₂OQ wherein Q is as defined above, or when the C N bond is absent in combination with R⁶ is -CH₂-O- and R⁶ is absent or R⁶ is OR¹¹; or a pharmaceutically acceptable salt thereof, with the proviso that the compound of formula (I) is not phalloidin.

wherein:

(1) each of R¹ and R° is H, OH, CH₃O, halo, CN, NO₂, CF₃, CO₂H, CO₂(C_r C₄)alkyl or N(R^π)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(O)(C_!-C₂₂)alkyl, a peptide or protein, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, CH₃, formyl, or acetyl;

(4) R² is H or CH₃;

(5) R³ is CH₃, iso-propyl, or iso-butyl; (6) R⁴ is H or OH;

(7) R⁵ is H or OH:

(8) R⁶ is absent or together with R¹⁰ is a covalent bond;

(9) R⁷ is absent or is COCH₃ or Rⁿ when the C N bond is absent;

(10) X is H, S, O, Se or S(O),.₂; (11) Z is C(O) or -CH₂-, or H or (C,-C₄)alkyl when the X Z bond is absent;

(12) R⁸ is absent, or is H or (C,-C₄)alkyl when the X Z bond is absent; (13) R¹⁰ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl; and

(14) R¹³ is H; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (II) is not phalloidin. When R¹⁰ or R¹³ is a peptide or protein, it is preferred that the peptide or protein is a fragment of an antibody, or an antibody, which binds to a molecule which is specific to the heart, e.g., cardiac muscle.

Exemplary phalloidin analogs include a compound of formula (III):

wherein the bond represented by C — N is absent or present, R is -(C(OR⁴)(CH₃)(CH₂OR⁵), or haloacetyl wherein R⁴ is H or together with Z is a covalent bond; R⁵ is H, a peptide or protein, a sugar residue or C(O)R' wherein R' is (C₁₂-C₂₂)alkyl, optionally comprising 1-3 double bonds, Z is absent or together with R⁴ is a covalent bond, Y is absent, or is H when the C — N bond is absent, and X is -H H-, SO, O, Se; R³ is H or an acetyl, or a pharmaceutically acceptable salt thereof. When R⁵ is a peptide or protein, it is preferred that the peptide or protein is a fragment of an antibody, or an antibody, which binds to a molecule which is specific to the heart.

Preferably, the therapeutic agents of the invention bind cardiac actin more strongly than skeletal actin, improve cardiac contractility over a wide range of Ca²⁺ concentrations, relax smooth muscle, normalize smooth muscle function or any combination thereof. Preferred therapeutic agents of the invention lower intracellular Ca²⁺ levels. It is also preferred that the therapeutic agents of the invention do not increase MvO2, i.e., the consumption of oxygen by the heart. Preferred therapeutic agents have a LD₅₀ which is less than the LD₅₀ of phalloidin. The LD₅₀ of phalloidin is species specific (see, Faulstich, In: Chemistry of Peptides and Proteins, Noelter et al (eds.), Walter de Gruyter, Berlin, Germany (1982), pp. 279-288).

II. Preparation of the Phalloidin Analogs and Derivatives of the Invention The phalloidin analogs or derivatives can be prepared by methods well known to the art, including derivatizing or otherwise modifying phalloidin (available from Sigma Chemical Co., St. Louis, MO). For example, see Weiland et al. (Derivate. Liebigs Ann. Chem., 577:215-233, 1952); Weiland (Peptides of the Amanita Mushrooms, Springer Nerlag, 1986); Kobayashi et al. (Eur. J. Chem., 232: 726-736, 1995); Wulf et al. (Proc. Natl. Acad. Sci. USA, 76: 498-4502, 1979); Weiland et al. (Derivate. Liebigs Ann. Chem., 1983:1533-1540, 1983); Weiland et al. (Pharmacol. Rev., 11:87-107, 1959); Weiland et al. (Crit. Rev. Biochem., 5:185- 260, (1978). Raymond et al. (Eur. J. Pharmacol, 138:217, 1987).

The bicyclic ring system can be converted to a monocyclic ring system by two types of reaction: by cleavage of a peptide bond and by removal of the sulfur bridge.

Secocompounds. Since the peptide bond between the γ -hydroxy lated side chain-7 and amino acid No. 1 is preferentially split on treatment with mild acids (water-free trifluoroacetic acid, a few hours at room temperature), monocyclic secocompounds can be obtained. The alteration of the shape of the bicyclic peptide as a consequence of the opening of one ring can be observed readily by circular dichroism measurements. All of the bicyclic parent compounds show a characteristic curve with positive Cotton effects around 240 and 300 nm and a negative effect in the short wavelength region. Dethiocompounds. The sulfur atom forming the thia bridge in the bicyclic peptides can be removed hydrogenolytically using Raney nickel. The removal of the thia bridge, like seco-formation, causes a major change in the molecular shape as evident from the CD spectra. The Role of the Side Chains. The influence of the chemical nature of the side chains of phalloidins on the toxicity and, as measured so far, on the affinity to F-actin has been studied by chemical modification of side chains and by comparing synthetic analogues.

Chemical Modifications. Functional groups subjected to modification include the hydroxyl groups in side chains 2, 4, and 7, the carboxyl group in side chain-2 of the acidic phalloidins and the indole NH of the tryptathionine moiety. The side chain No. 7 of γ,δ-dihydroxyleucine of phalloidin, with its glycol unit, is subject to oxidation by periodate. The sulfur atom of the tryptathionine residue can undergo oxidation by peroxyacetic acid and intramolecular alkylation.

Reaction with the Hydroxyl Groups. The phalloidins possess primary and secondary OH-groups that differ in their reactivity, and a tertiary group that is virtually non-reactive, except in lactone formation. In phalloidin (and in phallacidin), the most reactive is the γ-hydroxyl group of side chain-7. It has been selectively acetylated (Faulstich and Wieland, Eur. J. Biochem. 22:70-86, 1971) and tosylated (Wieland and Rehbinder, Liebigs Ann. Chem, 670:149-157, 1963). The monoacyl products can be toxic, providing evidence that side chain-7 is not involved in binding of the molecule to F-actin. Since the diacetyl-phalloidin is toxic, the OH-group of the D-threonine residue, but not that of the hydroxyproline residue, can be acetylated. The latter OH participates in binding to the target protein, for its absence leads to lack of toxicity. In ditosyl-phalloidin, the second tosyl residue may have the essential hydroxyl of α/7ohydroxy proline. The triacylated derivatives and also the tribromo derivative, obtained from a tritosyl compound by a Finkelstein reaction (KBr in acetone), are non-toxic. Alkylation at the indole-N can be achieved by reaction of the sodium compound with the corresponding alkyl iodides (Faulstich and Wieland, supra, 1971) to obtain the N-methyl, N-ethyl, N-«-propyl, N-n-pentyl, N-tert-butyl, and carbamidomethyl compounds. Among these, only the N-methyl derivative cannot be distinguished chromatographically from the parent phalloidin, since the R_F values of the N-methyl derivative were nearly identical in all solvents. For identification of this derivative, UN spectroscopy (γ_max = 290 nm, no shoulders) and the color reaction (with cinnamaldehyde HC) can be used, the later yielding a blue color for phalloidin and a violet one for all Ν-alkylated derivatives. The carboxyl group of the acidic phalloidins in side chain No. 2 is not essential for binding to F-actin, since it can be replaced by methyl, as in the neutral phalloidins, or may be esterified by diazoalkanes. Through aminolysis of the ester by ammonia, methylamine or dimethylamine, the corresponding amides whose toxicity decreased according to the above sequence were obtained (Faulstich et al., Liebigs Ann. Chem, 1975:2324-2330, 1975).

By the reaction of the methyl ester with excess 1,2-diaminoethane, a 2-aminoethylamide of phallacidin was prepared that, on reaction with 4-chloro-7- nitro-benz-2-oxa-l,3-diazole, yields a fluorescent derivative of phallacidin, NPD- PHC, that still binds to F-actin (Barak et al., Proc. Natl. Acad. Sci. USA, 77:980- 9841980; Barak and Yocum, Anal. Biochem., 110:31-38, 1981; U.S. Patent No. 4,387,088). A non-toxic conjugate of phallacidin with bovine serum albumin has been described by Wieland and Buku, FEBSLett., 4:341-342, 1969.

The side chain No. 7 of phalloidins can be modified by a variety of chemical reactions of monotosyl phalloidin. The O-tosyl group of δ⁷ O-tosyl phalloidin (Wieland and Rehbinder, supra, 1963) can be displaced by various nucleophiles, such as ammonia, methylamine, aniline, aminofluorescein and hydrogen sulfide (HS ), to yield the corresponding δ-substituted phalloin derivatives (Wieland et al., Liebigs Ann. Chem., 1983:1533-1540, 1983). δ-Aminophalloin can be used to introduce various acyl residues into the phalloidin skeleton, without destroying its binding property to F-actin. Thus, reaction of aminophalloin with, e.g., iodoacetyl chloride afforded a iodoacetylamino-phalloin. A photolabile radioactive affinity label can be introduced by acylating with the conjugate of [³H]- containing β-alanine with the photolabile carbene-generating 4-(l-azi-2,2,2- trifluoroethyl)-benzoic acid of Nassal (Liebigs Ann. Chem., 1983:1510-1523, 1983). Fluorescent probes FLPHD (Wieland et al., Liebigs Ann. Chem., 1980:416-424, 1980) and RHPHN (Wieland et al., supra, 1983), respectively, were successfully applied for the visualization of F-actin in a variety of cells, δ- Aminophalloin is only weakly toxic, as it is also the N-methyl compound. As an intermediate in all reactions of the monotosylate with nucleophilic (basic) substances, an epoxide is formed that could be obtained as a pure substance (Wieland and Rehbinder, supra, 1963). On thiolysis with the hydrogen sulfide anion, HS^", the δ-mercapto derivative is formed and isolated as its disulfide. By using isotopic hydrogen sulfide, a radioactively labeled phalloidin is accessible.

Thioether-Trans-crosslinking. An intramolecular reaction of the δ- tosylate of phalloidin occurs on exposure to strong bases like methoxide ions. In this case, a product is formed whose Cotton effect in CD shows an opposite direction (negative) to that of phalloidin. In the compound, a dehydroalanine moiety is detected by addition of cysteamine, forming 4-thialysine. The most probable mechanism of its formation is an alkylation of the sulfur by the δ-tosylated side chain No. 7 and subsequent β-elimination of the new sulfur bridge from the intermediate sulfonium ion (Wieland et al., J. Am. Chem. Soc, 105:6193-6195, 1983). Periodate Oxidation of Side Chain No. 7. On oxidation with periodate, the γ,δ-dihydroxyleucine side chain forms formaldehyde and an acetonyl side chain (Wieland and Schδpf, Liebigs Ann. Chem., 626:174-184, 1959), resulting in "ketophalloidin" (α-aminolevulinic acid-7-phalloidin). The analogous oxidation of the γ,δ,δ-trihydroxyleucin side chain of phallisin with periodate, yields, after elimination of 2 equivalents of formaldehyde, a carboxylic group.

By reduction with NaBH₄, the carbonyl group of ketophalloidin was converted to a secondary alcohol (Wieland and Rehbinder, supra, 1963); demethylphallom (α-amino-γ-hydroxyvaleric acid-7-phalloidin) can be obtained in a radioactively labeled form by using [³H]-Na boranate (Puchinger and Wieland, Liebigs Ann. Chem., 725:238-240, 1969).

By reaction with ethane- 1,2-dithiol, ketophalloidin forms a dithiolane derivative. This reaction can be used for the introduction of [³²S], and for the attachment of an amino-containing side chain using l-amino-2,3-dimercaptopropane (Wieland et al., supra, 1980). The amino group of the side chain of dithiolane can be used for the introduction of an iodoacetyl residue and serve as a link to fluorescent moieties by reaction with fluorescein- or rhodamine-isothiocyanate (Faulstich et al, Exp. Cell Res., 144:73-82, 1983). The dithiolane is desulfurized hydrogenolytically without cleaving the tryptathionine bridge, by careful treatment with Raney nickel, thus yielding norphalloin (norvaline-7-phalloidin) (Wieland and Jeck, Liebigs Ann. Chem., 713:196-200, 1968). The first total synthesis of a bioactive phalloidin was carried out with norvaline (Fahrenholz et al., Liebigs Ann. Chem., 743:83-94, 1971). Sulfoxides and Sulfone. In contrast to the amatoxins, which are (R)- sulfoxides, S-oxygenated phalloidins have never been found in nature. By oxidation with peroxy acids, phalloidin yielded two diastereoisomeric sulfoxides (R- and S-) and a sulfone, which were separated by chromatography (Faulstich et al., Liebigs Ann. Chem., 713:186-195, 1968). One sulfoxide (B), formed in lesser amount than the other (A). The sulfone of phalloidin can be generated by further oxidation of the sulfoxides. Correspondingly, the affinities to F-actin of the respective compounds are, in percent of that of phalloidin, as follows: 10(B):l(A):40(sulfone). The absolute configurations of the phalloidin-sulfoxides A and B have been established by comparing the ORD curves with that of one crystallized diastereomer of the model compound mentioned above (Wieland et al, Liebigs Ann. Chem., 1974:1570- 1579, 1974). The X-ray structural analysis of the dextro-rotatory sulfoxide reveal its (^-configuration.

Synthetic Analogues. After norphalloin had been obtained by total synthesis, as the first of the biologically active phalloidin analogs, the role of the 4- cw-hydroxy-L-proline (αtVo-hydroxyproline) in position 4 was investigated by the synthesis of a norphalloin analogue with trøns-4-hydroxy-L-proline or L-proline, respectively, in the same position (Faulstich et al., Liebigs Ann. Chem., 1973:50-58, 1973). The same was true for Gly³-norphalloin. In a second study, it was established that the D-threonyl residue in norphalloin can be replaced by D-α- aminobutyric acid without loss of toxicity of the analogue (Heber et al., Int. J. Peptide Protein Res., 6:381-389, 1974). Heber also described the synthesis of a norphalloin, containing an alanine in the 5 -position, whose methyl group is tri- deuterated. The total synthesis of phalloin, Leu⁷-phalloin, a homologue of norphalloin, and prophalloin (Pro⁴-phalloin) has been reported (Munekata et al., Liebigs Ann. Chem., 1977:1758-1765, 1977; Munekata et sλ., Liebigs Ann. Chem., 1978:776-784, 1978). Additional analogues can be prepared by a semi-synthetic route described by Munekata et al., In: Peptides: Proc. 5th Am. Peptide Symp., Goodman and Meienhofer (eds.), pp.243-245, 1977. Exchange of alanine-1 in phalloidin for various amino acids yielded several analogues. The preparation of two other analogues, the D-tf-aminobutyric acid-2, L-lysine-7 analogue, which binds to F-actin with an affinity of about 20% that of demethylphalloin, and the D- Ala², Leu⁷-analogue, are described in Wieland et al., Biochem., 22:1264-1271, 1983. The phalloidin precursor, derivative or analog can also be synthesized by the solid phase peptide synthetic method. This established and widely used method, including the experimental procedures, is described in the following references: Stewart et al., Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc, 85:2149 (1963); Meienhofer in Hormonal Proteins and Peptides, ed.; CH. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; and Bavaay and Merrifield, The Peptides, eds. E. Gross and F. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285.

To prepare cyclic forms of the phalloidin analogs or derivatives of the invention, conventional peptide synthetic methods can be employed. In general, the cyclization reaction may take place at any position using the acyclic precursor where an amino group and an activated carboxyl group are available for reaction. For example, an amino reactive group is reversibly protected. The amino protective group is then removed and proteinated. The deproteination of the reactive amino group in highly dilute basic solution, in the presence of an activated carboxyl group, leads to cyclization. Alternatively, the addition of base in dilute solution, or the addition of a dehydrating agent, with the amino group in proteinated form, in the presence of an activated carboxyl group, leads to cyclization.

The phalloidin analogs or derivatives may be purified by fractionation on immunoaffmity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography, and the like. Moreover, once purified, derivatives and chemically derived variants of the purified phalloidin analog or derivative, e.g., a compound of formula (I), can be readily prepared. For example, amides of phalloidin analogs of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. A preferred method for amide formation at the C-terminal carboxyl group is to cleave the phalloidin analog from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.

Salts of carboxyl groups of the phalloidin analog may be prepared in the usual manner by contacting the analog with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like. N-acyl derivatives of an amino group of the present phalloidin analogs or derivatives may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O- acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired. In addition, the amino acids of the phalloidin analogs can be modified by substituting one or two conservative amino acid substitutions for the positions specified, including substitutions which utilize the D rather than L form. Conservative amino acid substitutions are preferred— that is, for example, hydroxyproline-aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.

Acid addition salts of the phalloidin analogs may be prepared by contacting the analog with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the analogs may also be prepared by any of the usual methods known in the art.

πi. Dosages. Formulations and Routes of Administration of the Therapeutic Agents Administration of a therapeutic agent of the invention in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

One or more suitable unit dosage forms comprising the agents of the invention, which, as discussed below, may be optionally be formulated for sustained release, can be administered by a variety of routes including oral, or parenteral, including by rectal, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal routes. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system. When the agent of the invention is prepared for oral administration, it is preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations comprise from 0.1 to 99.9%) by weight of the formulation. By "pharmaceutically acceptable" it is meant the carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum. The active ingredient may also be presented as a bolus, electuary or paste.

Pharmaceutical formulations containing the agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. For example, the agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC, and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

For example, tablets or caplets containing the agents of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxypropyl methylcellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like. Hard or soft gelatin capsules containing the agents of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, the enteric coated caplets or tablets of the agents of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum. Coatings may also reduce the immunogenicity of the agents. Particularly useful coatings include phospholipids, including liposomal encapsulation and coatings of bound polyethylene glycol and/or polyethylene glycol derivatives. The agents of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes. The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, dispersions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the agents of the invention may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the prior art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, C_rC₄ alkyl esters of short- chain acids, preferably ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol", isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They can also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.

It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes and colorings. Also, other active ingredients may be added, whether for the conditions described or some other condition.

For example, among antioxidants, t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol and its derivatives may be mentioned. The galenical forms chiefly conditioned for topical application take the form of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, or alternatively the form of aerosol formulations in spray or foam form or alternatively in the form of a cake of soap. Additionally, the agents of the invention are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes.

The therapeutic agents of the invention can be delivered via patches for transdermal administration. See U.S. Patent No. 5,560,922 for examples of patches suitable for transdermal delivery of a therapeutic agent. Patches for transdermal delivery can comprise a backing layer and a polymer matrix which has dispersed or dissolved therein a therapeutic agent, along with one or more skin permeation enhancers. The backing layer can be made of any suitable material which is impermeable to the therapeutic agent. The backing layer serves as a protective cover for the matrix layer and provides also a support function. The backing can be formed so that it is essentially the same size layer as the polymer matrix or it can be of larger dimension so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can extend outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means. Alternatively, the polymer matrix can contain, or be formulated of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. Examples of materials suitable for making the backing layer are films of high and low density polyethylene, polypropylene, polyurethane, polyvinylchloride, polyesters such as poly(ethylene phthalate), metal foils, metal foil laminates of such suitable polymer films, and the like. Preferably, the materials used for the backing layer are laminates of such polymer films with a metal foil such as aluminum foil. In such laminates, a polymer film of the laminate will usually be in contact with the adhesive polymer matrix.

The backing layer can be any appropriate thickness which will provide the desired protective and support functions. A suitable thickness will be from about 10 to about 200 microns. Generally, those polymers used to form the biologically acceptable adhesive polymer layer are those capable of forming shaped bodies, thin walls or coatings through which therapeutic agents can pass at a controlled rate. Suitable polymers are biologically and pharmaceutically compatible, nonallergenic and insoluble in and compatible with body fluids or tissues with which the device is contacted. The use of soluble polymers is to be avoided since dissolution or erosion of the matrix by skin moisture would affect the release rate of the therapeutic agents as well as the capability of the dosage unit to remain in place for convenience of removal.

Exemplary materials for fabricating the adhesive polymer layer include polyethylene, polypropylene, polyurethane, ethylene/propylene copolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetate copolymers, silicone elastomers, especially the medical-grade polydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, crosslinked polymethacrylate polymers (hydro- gel), polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber, epichlorohydrin rubbers, ethylenvinyl alcohol copolymers, ethylene- vinyloxyethanol copolymers; silicone copolymers, for example, polysiloxane- polycarbonate copolymers, polysiloxanepolyethylene oxide copolymers, polysiloxane-polymethacrylate copolymers, polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylene copolymers), polysiloxane-alkylenesilane copolymers (e.g., polysiloxane-ethylenesilane copolymers), and the like; cellulose polymers, for example methyl or ethyl cellulose, hydroxy propyl methyl cellulose, and cellulose esters; polycarbonates; polytetrafluoroethylene; and the like. Preferably, a biologically acceptable adhesive polymer matrix should be selected from polymers with glass transition temperatures below room temperature. The polymer may, but need not necessarily, have a degree of crystal- linity at room temperature. Cross-linking monomeric units or sites can be incorporated into such polymers. For example, cross-linking monomers can be incorporated into polyacrylate polymers, which provide sites for cross-linking the matrix after dispersing the therapeutic agent into the polymer. Known cross-linking monomers for polyacrylate polymers include polymethacrylic esters of polyols such as butylene diacrylate and dimethacrylate, trimethylol propane trimethacrylate and the like. Other monomers which provide such sites include allyl acrylate, allyl methacrylate, diallyl maleate and the like.

Preferably, a plasticizer and or humectant is dispersed within the adhesive polymer matrix. Water-soluble polyols are generally suitable for this purpose. Incorporation of a humectant in the formulation allows the dosage unit to absorb moisture on the surface of skin which in turn helps to reduce skin irritation and to prevent the adhesive polymer layer of the delivery system from failing.

Therapeutic agents released from a transdermal delivery system must be capable of penetrating each layer of skin. In order to increase the rate of permeation of a therapeutic agent, a transdermal drug delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum corneum, which provides the most resistance to the penetration of molecules. The fabrication of patches for transdermal delivery of therapeutic agents is well known to the art. Suitable transdermal delivery systems are also disclosed, for example, in U.S. Patent No. 4,788,603, U.S. Patent No. 4,931,279, U.S. Patent No. 4,668,506, and U.S. Patent No. 4,713,224.

The local delivery of the therapeutic agents of the invention can also be by a variety of techniques which administer the agent at or near the site of disease. Examples of site-specific or targeted local delivery techniques are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, such as an infusion or indwelling catheter, e.g., a needle infusion catheter, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct applications.

For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. Conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active ingredients can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051 ,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-25% by weight.

The agents of the invention may also be formulated so as to be suitable for administration by inhalation or insufflation or for nasal, intraocular or other topical (including buccal and sub-lingual) administration. For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic agents of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluorornefhane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler. For intra-nasal administration, the therapeutic agent may be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.

The therapeutic agent may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier. The formulations and compositions described herein may also contain other active ingredients such as antimicrobial agents, or preservatives. Furthermore, the agents of the invention may also be used in combination with other therapeutic agents, for example, other cardiac inotropes, anti-inflammatory agents, diuretics, vasodilators, anti-arrythmics, anti-coagulants, Ca²⁺ channel blockers, β- blockers, ACE inhibitors, and the like. Moreover, the administration of a combination of a phalloidin analog or derivative with other agents, e.g., other cardiac inotropes, may have a synergistic effect.

The examples of the local delivery of the agents of the invention are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, such as an infusion or indwelling catheter, e.g., a needle infusion catheter, or other implantable devices. Catheters which may be useful in the practice of the invention include catheters such as those disclosed in Just et al. (U.S. Patent No. 5,232,444), Abusio et al. (U.S. Patent No. 5,213,576), Shapland et al. (U.S. Patent No. 5,282,785), Racchini et al. (U.S. Patent No. 5,458,568) and Shaffer et al. (U.S. Patent No. 5,049,132), the disclosures of which are incorporated by reference herein.

Local delivery by an implant describes the surgical placement of a matrix that contains the agents of the invention. The implanted matrix releases the analog or derivative by diffusion, chemical reaction, or solvent activators. Lange, Science. 249, 1527 (1990).

Local delivery by site specific carriers describes attaching the agents of the invention to a carrier which will direct the therapeutic agent to the target site, e.g., to a vessel of the heart. Examples of this delivery technique includes the use of carriers such as a protein ligand, e.g., a monoclonal antibody or antibody fragment. Lange, Science. 242,1527 (1990). See, for example, WO 94/16706 for methods useful to link or couple, preferably covalently link or couple, peptide or protein ligands to other macromolecules. Phalloidin analogs or derivatives, e.g., a compound of formula (I), are preferably intravenously administered at doses of about 0.01-100.0 μM/L blood, more preferably at doses of about 0.01-50.0 μM/L blood, and even more preferably at doses of about 0.1-10.0 μM/L blood, although other dosages may provide beneficial results. Alternatively, the phalloidin analogs or derivatives can be administered at about 0.01-100 mg/kg/day, preferably at about 0.01-50 mg/kg/day, and more preferably at about 0.1-10 mg/kg/day, although other dosages may provide beneficial results. For local administration, e.g., topical, a compound of formula (I) is preferably administered at about 2-20, more preferably 1.5-15, and even more preferably 1 - 10 μg of analog or derivative per gram of topical carrier.

The Method of the Invention

The invention provides a method of treating a mammal having, or preventing, a disease characterized by reduced or decreased heart muscle contractility, e.g., congestive heart failure, ischemic myocardial infarction, hypertension, myocarditis, epicarditis, endocarditis, pulmonary edema, atrial or ventricle arrhythmias, and other conditions classified as cardiomyopathies, or acute heart failure related to septic shock. One embodiment of the invention employs the systemic administration of a compound of formula (I) including a pharmaceutically acceptable salt thereof, or a combination thereof, in an amount effective to increase or elevate heart muscle contractility in a diseased heart. Preferably, the compound of formula (I) useful in the practice of the invention is administered continually over a preselected period of time or administered in a series of spaced doses, i.e., intermittently, for a period of time as a preventative measure. It will be recognized by those skilled in the art that therapeutically/prophylactically effective dosages of these analogs and derivatives and compositions will be dependent on several factors. Those skilled practitioners trained to deliver drugs at therapeutically or prophylactically effective dosages (e.g., by monitoring drug levels and observing clinical effects in patients) will determine the optimal dosage for an individual patient based on experience and professional judgment.

The invention will be further described by the following Examples.

Example 1

Cardiac Muscle Preparation Cardiac muscle bundles were dissected from the left ventricular free- wall of fresh bovine heart obtained from the local abbatoir (Washington State University Quality Meat Laboratory). Dissection was performed in a 4°C cold room to produce oriented muscle bundles about 1 to 2 cm in length and 1 mm in diameter. Muscle bundles were tied to a plastic frame and incubated for 6 hours in a skinning solution (80 mM KC1, 5 mM MgCl₂, 20 mM MOPS (pH 7.0), 5 mM EGTA, 2 mM DTT, 1% Triton XI 00) and then further incubated in storage solution (80 mM KC1, 1 mM MgCl₂, 20 mM phosphate buffer (pH 7.0), 2.5 mM DTT, 50% glycerol) with continuous agitation for 24 hours at 4°C. Thereafter, muscle bundles were stored in storage solution at -20 °C.

Just prior to testing, thin muscle preparations of 100-250 μm in diameter were teased apart under a dissecting microscope at room temperature. The ends of these fibers were glued to stainless steel hooks; one hook attached to a AE 801 force transducer and the other hook attached to a length-adjustment mechanism. The glued fiber was lowered into a temperature-controlled (20°C) 150 μl well containing rigor solution (90 mM KC1, 5 mM MgCl₂, 100 mM MOPS (pH 7.0), 2 mM EGTA) and slightly (about 5 %) stretched. The apparatus was mounted on a Tiyoda inverted microscope. A video image of the muscle fiber was displayed on a TN monitor using a video camera through a X40 objective lens. Total magnification from fiber to TV monitor was approximately 10⁴. The system was calibrated with a stage micrometer. Length of muscle sarcomeres was determined in rigor solution by measuring the length of a row of 10 sarcomeres on the TV monitor. The average sarcomere length value was based on 5 measurements made along a 2 mm length of the fiber. Fibers used in these experiments had sarcomere lengths in the range of 2.1 - 2.3 μm.

After sarcomere length measurement, muscle preparations in the well were bathed with 100 μl relaxing solution and, after several minutes of equilibration, the muscle fibers were activated. Activation was by addition of aliquots of 0.1 M CaCl₂to bring the pCa to the desired value. As a rule this was done in several steps because it was observed that stepwise addition of Ca²⁺ provided more stable tension values. pCa values were calculated with constants given by Fabiato and Fabiato (J. Physiol, 75:463-505, 1979) with the exception that the absolute CaEGTA stability constant was taken to be 7.9 x 10'° M^~' (as described by Bukatina et al., J. Mus. Res. & Cell Motil, 17:365-371, 1996).

Each preparation underwent several episodes of activation followed by relaxation. The first episode of activation was one of a brief period (approximately 1 minute) of maximal activation (pCa 4.5). This allowed evaluation of the contractile viability of the preparation. This maximal activation episode was terminated by quickly changing the bathing solution to relaxing solution. Quick solution changes were achieved by draining the well through a special port connected to a vacuum line and quickly refilling the cell with a new solution. During the entire period of activation and relaxation, solutions were intensively agitated with a vibrating stainless steel wire to ensure uniform distribution of substances within the bathing solution and minimize diffusion gradients.

To study the muscle response to phalloidin analogs or derivatives, phalloidin analogs or derivatives were applied as dried preparations to muscle preparations when the muscle preparations were developing force during Ca²⁺ activation. Alternatively, to determine their influence on the force-pCa curve, the phalloidin analogs or derivatives were added as dried preparations to the relaxing solution several minutes before step-wise Ca²⁺ activation.

Stock solutions of phalloidin derivatives were prepared as follows: dethiophalloidin (37.5, 20, 7.5, 3.75 mM), phalloidin sulfoxide-A (10 mM) and phalloidin (20 and 10 mM) in ethyl alcohol, and secophalloidin (10 mM) in water. These stock solutions were stored in a freezer at -20 °C. At the time they were to be administered to muscle preparations, 0.4 μl (or 0.8 μl) of a stock solution was dried on the end of thin narrow sheet of parafilm in the air. To add into solution, the parafilm sheet was lowered into the solution in the cell just near the vibrating wire for several seconds. This procedure can cause transient changes in tension, which is a mechanical artifact, as seen in the experimental data described below.

Example 2 Micromolar Concentrations of Dethiophalloidin Are Effective in Enhancing Cardiac Muscle Contraction at All Levels of Ca²⁺- Activation Following an initial maximal activation episode (pCa 4.5) and relaxation, a muscle preparation was partially activated with pCa 6.20 and then dethiophalloidin was added in several steps to achieve 15, 30, 60, and 90 μM concentrations (Figure 1), which led to a marked increase in tension. The major increase in tension (80%) was reached with 15 μM dethiophalloidin. At a dethiophalloidin concentration of 90 μM, saturation had clearly been reached (Figure 1). Moreover, no additional increases in force were observed when the dethiophalloidin concentration was increased to 160 μM (see second activation episode in Figure 2). From data such as this, the apparent dissociation constant for dethiophalloidin binding to cardiac myofilaments was derived. Assuming the percentage of force enhancement to be proportional to the percentage of saturation of myofilaments with dethiophalloidin, the apparent equilibrium constant for dethiophalloidin binding is derived from K= [DTPH] (1-X)/X, where X is the fraction of force enhancement at a given [DTPH]. From the results of 7 experiments similar to that shown in Figure 1, K = 5 μM (SE ± 1 μM ). To work at a saturating concentration of dethiophalloidin, 80 μM dethiophalloidin was employed for the remaining experiments. The amount of force enhancement by dethiophalloidin varied with the level of Ca²⁺-activation. When 80 μM of dethiophalloidin was added to maximal Ca²⁺-activated fibers (1st activation episode in Figure 2), the increase in force was approximately 15%. This contrasts with force enhancement during partial activation at pCa 6.20 (second activation episode in Figure 2) where the increase in force with dethiphalloidin addition was 180%. However, dethiophalloidin does not enhance or cause force development in cardiac muscle fibers when pCa < 8.0.

The results shown in Figure 3 were obtained after the muscle fiber was first maximally activated and relaxed with relaxing solution (pCa < 8.0) and then, while relaxed, stretched until passive force was approximately 1/2 maximal activated force. In these relaxed, passive conditions there was no strong-binding between actin and myosin and the addition of dethiophalloidin did not change the expression of passive force. Furthermore, when muscle fibers were induced to go into rigor after withdrawal of ATP (pCa < 8.0), which created maximum strong binding between actin and myosin, the addition of dethiophalloidin did not change the level of rigor force (second force episode in Figure 4). These results show that dethiophalloidin acts only on Ca²⁺-activated crossbridge force.

Dethiophalloidin also increases Ca²⁺-sensitivity and maximum Ca²⁺- activated force in cardiac muscle fibers. Recordation of force obtained in several sequential activation episodes are shown in Figure 5. This data shows that the muscle preparation was sufficiently stable to be repeatedly activated and relaxed. If force is plotted versus pCa (Figures 6 and 7) during sequential activation, a force- pCa curve results. Figures 6 and 7 represent data from fibers derived two different animals. The graphs in panel A of Figures 6 and 7 show that dethiophalloidin increased maximum Ca²⁺-activated force by 10% and shifted the force-pCa curve to the left, i.e., greater force is achieved for the same level of activating Ca²⁺. This shift reflects an increase in Ca²⁺-sensitivity. The graphs in panel B of Figures 6 and 7 show force-pCa curves which were normalized to their maximum value so that the increase in Ca²⁺-sensitivity with dethiophalloidin can be easily quantified. From panel B, the Ca²⁺-sensitivity increase can be calculated as the length of the segment between two curves at the level of half maximal activation. There were 0.33 pCa units of increase at half-maximal activation with dethiophalloidin treatment in both fibers.

A measure of the strength of enhanced activation by dethiophalloidin can be obtained by comparing the pCa units of left shift with the pCa units required to change activation from 25 to 75% of maximal activation under control (untreated) conditions. A strong Ca²⁺ sensitizing effect would be one in which the left shift is as large as the change in pCa required to go from 25 to 75% of maximal activation. For dethiophalloidin, 0.33 pCa units of left shift compares with 0.3 pCa units to change activation from 25 to 75% of maximal. Thus, dethiophalloidin is a strong Ca²⁺-sensitizer with sensitizing effects that are as large as may be tolerated so as to avoid excessive activation at low pCa.

Example 3 Relationship Between Actions of Phalloidin and Dethiophalloidin To study the relation between the mechanism of action of dethiophalloidin and that of phalloidin, two experimental protocols were performed. The first protocol was to examine the influence of pre-treatment of the muscle preparation with phalloidin on the response to dethiophalloidin. Given that phalloidin binds extremely firmly to actin, pre-treatment with phalloidin would result in all phalloidin-binding sites being irreversibly occupied by phalloidin. If dethiophalloidin exerts its contraction enhancing effects after binding to the phalloidin binding site, then pretreatment with phalloidin would suppress the dethiophalloidin effects.

The results of a phalloidin pretreatment experiment are shown in Figure 8. The muscle fiber is maximally activated (first activation episode), relaxed and, then maximally activated again when it is treated with phalloidin (second activation episode). Phalloidin caused a small increase in maximally activated force in agreement with earlier observations (Bukatina et al., J. Mol Cell. Cardiol, 27:1311-1315 (1995)). After several washings with relaxing solution, the fiber is partially activated (pCa 6.2) and treated with dethiophalloidin (third activation episode). There was no force enhancement with dethiophalloidin treatment. The results shown in Figure 8 were compared with the results shown in Figure 2, where treatment of the partially activated fiber with dethiophalloidin produced a large increase in force. Clearly, pretreatment with phalloidin completely inhibited the activating effects of dethiophalloidin. Thus, the binding sites for phalloidin and dethiophalloidin are overlapping.

In the second protocol, maximal Ca²⁺-activated force was enhanced by the addition of dethiophalloidin and then phalloidin was added (Figure 9). Because of the great difference in strength of binding of phalloidin and dethiophalloidin to F-actin, it was expected that dethiophalloidin would be displaced by phalloidin. Indeed, the fiber responded to the phalloidin addition by losing some of the enhanced force induced by dethiophalloidin. This loss of force was not complete because phalloidin itself has a small force-enhancing effect in maximal activated fibers (Figure 8). Therefore, dethiophalloidin is a stronger activator of contraction than phalloidin even though dethiophalloidin binds more weakly to the thin filament. Furthermore, the effects of dethiophalloidin on muscle are reversible, unlike the effects of phalloidin on muscle.

Example 4

Other Phalloidin Derivatives Also Enhance Ca²⁺- Activation Other weak actin-binding phalloidin derivatives, (S)-phalloidin- sulfoxide A (Figure 10) and secophalloidin (Figures 11 and 12), were also found to enhance Ca²⁺-activated force in cardiac muscle preparations. Similar to dethiophalloidin, both of these other derivatives have stronger effects at partial activation than at full activation. Data shown in Figure 10 indicated that the effect of 40 mM (S)-phalloidin-sulfoxide A at maximal Ca²⁺-activation was to increase force about 4 % whereas it increased force about 60 % at partial activation (pCa 6.2). This compared with the force increase caused by 80 mM secophalloidin which was 14 % (Figure 11) at maximal activation and about 40 % at half activation (first activation episode in Figure 12). Therefore, both of these phalloidin derivatives can increase cardiac myofilament Ca²⁺ sensitivity.

Both (S)-phalloidin-sulfoxide A and secophalloidin seem to be less effective cardiac muscle activators than dethiophalloidin; the apparent dissociation constant for secophalloidin was 50-70 mM, an order of magnitude greater than dethiophalloidin. But the effect of secophalloidin at saturating concentrations was greater than that of dethiophalloidin. Interestingly, secophalloidin exhibited an unusual property. The force-enhancing effects of secophalloidin persisted even after pre-treatment with phalloidin: incubation with phalloidin during a first activation did not prevent response to secophalloidin in a second activation (Figure 12). To further investigate this observation, secophalloidin was washed out and the muscle preparation was maximally activated for a third time. During the third activation, dethiophalloidin, (S)-phalloidin-sulfoxide A, and secophalloidin were sequentially added. Neither dethiophalloidin nor (S)-phalloidin-sulfoxide A caused any response, indicating that all phalloidin binding sites were occupied with phalloidin. However, secophalloidin induced a large increase in force. Thus, while the dethiophalloidin and (S)-phalloidin-sulfoxide A binding sites overlap with phalloidin sites, secophalloidin exerts its force-enhancement effects at least partially at sites that do not overlap with the phalloidin binding site.

All publications and patents are incorporated by reference herein, as though individually incorporated by reference, as long as they are not inconsistent with the present disclosure. The invention is not limited to the exact details shown and described, for it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention defined by the claims.

Claims

WHAT IS CLAIMED IS:

1. A method to treat a condition characterized by reduced heart muscle contractile strength, comprising: administering to a mammal in need thereof an effective amount of at least one agent that reversibly binds to cardiac actin.

2. A method to enhance cardiac muscle contraction in a mammal, comprising: the administration of an effective amount of at least one agent that reversibly binds to cardiac actin.

3. The method of claim 1 or 2 wherein the condition is congestive heart failure, systolic congestive heart failure or cardiomyopathy.

4. The method of claim 1 or 2 wherein the agent is a phallotoxin.

5. The method of claim 4 wherein the agent is a non-toxic analog or derivative of phalloidin.

6. The method of claim 1 or 2 wherein the agent is a Ca²⁺ sensitizing agent.

7. A method to treat a condition characterized by reduced heart muscle contractile strength, comprising: admimstering to a mammal in need thereof an effective amount of a compound of formula (I):

wherein:

(1) each of R¹ and R┬░ is H, OH, (C,-C₄)alkyl, O(C,-C₄)alkyl, halo, halo(C_r C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₂₂)alkyl, benzyl, phenyl, C(O)(C_rC₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, (C_rC₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(R^╧Ç)(R¹²);

(4) R² is H or (C,-C₄)alkyl;

(5) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl;

(6) R⁴ is H, OH, halo, acetoxy or OTs;

(7) R⁵ is H, OH, acetoxy, halo or OTs;

(8) each of R⁶ and R⁷ is absent or is H, (C_rC₄)alkyl, acetyl, or phenyl when the C N bond is absent;

(9) R¹⁰ is H, (C,-C₄)alkyl, CO₂H, CO₂(C,-C₄)alkyl, or C(O)N(R^u)(R¹²);

(10) X is H, -CH₂-, C(=O), C(=S), C(=NR¹⁵), S(O),.₂, O, S, or Se, wherein R¹⁵ is hydrogen, (C_rC₆)alkyl, hydroxy, (C_rC₄)alkoxy, tosyl, or mesyl;

(11) R⁸ is absent, or is H or (C C₄)alkyl when the X Z bond is absent; (12) when the X Z bond is present and X is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O)╬╣.₂, then Z is O, S, S(O),.₂, Se or NR¹⁶, wherein R¹⁶ is hydrogen, (C,-

C₆)alkyl, hydroxy, (C_rC₄)alkoxy, tosyl, or mesyl; when the X Z bond is present and X is O, S, S(O)_1-2, Se or NR¹⁶, then Z is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O),.₂,; or when the X Z bond is absent then Z is H or

(C_rC₄)alkyl;

(13) x is 0, 1 or 2;

(14) Y is H, (C_rC₄)alkyl, optionally substituted by N(R^! ')(R¹²), acetoxy, acetyl, 2-methyl-l,3-dithiolan-2-yl optionally 4-substituted with CH₂N(R^╧Ç)(R¹²) or CO₂Rⁿ; or is C(R¹³)(R¹⁴)(OH) wherein R¹³ is H, (C_rC₄)alkyl, CO₂H or

CH₂OQ wherein Q is H, acetyl, or Ts (tosyl) and R¹⁴ is (C C₄)alkyl, halo(C_rC₄)alkyl, haloacetyl, CO₂H, CH₂OQ wherein Q is as defined above, or is C(O)(C₁₂-C₂₂)alkyl, a peptide or protein, a residue of a sugar (e.g. rhamnose, glucose, xylose, galactose, etc.), or in combination with R⁶ is -CH₂-O- and R⁶ is absent or is OR¹¹ when the C N bond is absent; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (I) is not phalloidin.

8. A method to treat a condition characterized by reduced heart muscle contractile strength, comprising: administering to a mammal in need thereof an effective amount of a compound of formula (II):

wherein:

(1) each of R¹ and R┬░ is H, OH, CH₃O, halo, CN, NO₂, CF₃, CO₂H, CO₂(C_r C₄)alkyl or N(R^U)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(╬╕╧çC╬╣-C₂₂)a╬╣kyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, CH₃, formyl, or acetyl;

(4) R² is H or CH₃;

(5) R³ is CH₃, iso-propyl, or iso-butyl;

(6) R⁴ is H or OH;

(7) R⁵ is H or OH:

(8) R⁶ is absent or together with R¹⁰ is a covalent bond;

(9) R⁷ is absent or is COCH₃ or R¹¹ when the C N bond is absent ;

(10) X is H, S, O, Se or S(O),.₂; (11) Z is C(O) or -CH₂-, or H or (C_rC₄)alkyl when the X Z bond is absent;

(12) R⁸ is absent or is H or (C,-C₄)alkyl when the X Z bond is absent; (13) R¹⁰ is H, a peptide or protein, a monosaccharide, C(O)(C_I2-C₂₂)alkyl, or haloacetyl;

(14) R¹³ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (II) is not phalloidin.

9. A method to treat a condition characterized by reduced heart muscle contractile strength, comprising: administering to a mammal in need thereof an effective amount of a compound of formula (III):

wherein the bond represented by C ΓÇö N is absent or present, R is -(C(OR⁴)(CH₃)(CH₂OR⁵) or haloacetyl, wherein R⁴ is H or together with Z is a covalent bond; R⁵ is H, a peptide or protein, a steroidal moiety, a fatty acid moiety, a sugar residue or C(O)R' wherein R' is (C₁₂-C₂₂)alkyl, optionally comprising 1-3 double bonds, Z is absent or together with R⁴ is a covalent bond, Y is absent or is H when the C ΓÇö N bond is absent, and X is -H H-, SO, O, or Se; R³ is H or an acetyl, or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (III) is not phalloidin.

10. A method to treat a condition characterized by reduced heart muscle contractile strength, comprising: administering to a mammal in need thereof an effective amount of a compound of formula (IV):

wherein:

(1) R^a, R^b, and R^╬╡ are individually any naturally or unnaturally occurring alpha amino acid, or lysine coupled through its ╬╡-amino group to a steroidal or fatty acid moiety;

(2) x is 0, 1 or 2; (3) each of R¹ and R┬░ is H, OH, (C,-C₄)alkyl, O(C_rC₄)alkyl, halo, halo(C_r C₄)alkyl, C╬¥, ╬¥O₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(R^U)(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₄)alkyl, benzyl, phenyl, C(O)(C_rC₂₂)alkyl, a peptide or protein, or a residue of a sugar; (4) the bond represented by is present or absent; (5) R is H, (C,-C₅)alkyl, (C_rC₅)alkanoyl, benzyl, or CH₂C(O)N(Rⁿ)(R¹²);

(6) R² is H or (C,-C₄)alkyl;

(7) R³ is H, (C_rC₄)alkyl, CO₂H, CH₂OH or benzyl;

(8) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy);

(9) X is H, O, S, Se, or S(O),.₂; (10) R⁸ is absent or is H or (C╬╣-C₄)alkyl when the X Z bond is absent; and (11) Z is C(O) or -CH₂-, or is H or (C_rC₄)alkyl when the X Z bond is absent; or a pharmaceutically acceptable salt thereof.

11. The method of any one of claims 1 to 10 wherein the administration is 5 systemic.

12. The method of any one of claims 1 to 10 wherein the administration is local.

13. The method of any one of claims 1 to 10 wherein the agent increases the o strength of Ca²⁺-activated crossbridges.

14. The method of any one of claims 1 to 10 wherein dethiophalloidin, secophalloidin, (S)-phalloidin-sulfoxide A, N-acetyl secophalloidin, or a combination thereof, is administered. 5

15. The method of any one of claims 1 to 10 wherein the agent is parenterally administered in combination with a pharmaceutically acceptable carrier.

16. The method of claim 15 wherein the administration is by injection or 0 infusion.

17. The method of claim 15 wherein the carrier is a liquid carrier.

18. The method of claim 16 wherein the compound is administered by catheter. 5

19. The method of claim 15 wherein the compound is administered by inhalation or insufflation.

20. The method of claim 15 wherein the compound is administered topically.

21. The method of any one of claims 1 to 10 wherein the compound is administered orally.

22. The method of claim 7 or 10 wherein x is 1.

23. The method of claim 7 or 8 wherein R⁵ is OH.

24. The method of any one of claims 7, 8, or 10 wherein R⁴ is OH.

25. The method of claim 7 or 8 wherein R³ is CH₃ or iso-propyl.

26. The method of any one of claims 7, 8, or 10 wherein R² is H.

27. The method of claim 1 or 2 wherein the agent is not levosimendan, EMD 57033, CGP 48506, sumazol, pimobendan, MCI 154 or EMD 53998.

28. A non-toxic phalloidin derivative or analog for use in medical treatment, provided said medical treatment is not the treatment of inflammatory interleukin-2 mediated edema.

29. Claim 28 wherein the medical treatment is not the treatment of noninflammatory interleukin-2 mediated edema.

30. Claim 28 wherein the non-toxic phalloidin derivative or analog is a compound of formula (I) :

wherein:

(I) each of R¹ and R┬░ is H, OH, (C,-C₄)alkyl, O(C,-C₄)alkyl, halo, halo(C,- C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(R^╧Ç)(R¹²) wherein each of R^u and R¹² is H, (C,-C₂₂)alkyl, benzyl, phenyl, C(O)(C_rC₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2 the bonds represented by are present or one is absent; (3 R is H, (C_rC₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(Rⁿ)(R¹²); ( R² is H or (C,-C₄)alkyl; (5 R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl; (6 R⁴ is H, OH, halo, acetoxy or OTs; (7 R⁵ is H, OH, acetoxy, halo or OTs; (S each of R⁶ and R⁷ is absent or is H, (C.,-C₄)alkyl, acetyl, or phenyl when the

C N bond is absent; (9) R¹⁰ is H, (C,-C₄)alkyl, CO₂H, CO₂(C,-C₄)alkyl, or C(O)N(R^╧Ç)(R¹²);

(10) X is H, -CH₂-, C(=O), C(=S), C(=NR¹⁵), S(O),.₂, 0, S, or Se, wherein R¹⁵ is hydrogen, (C_rC₆)alkyl, hydroxy, (C,-C₄)alkoxy, tosyl, or mesyl;

(I I) R⁸ is absent, or is H or (C,-C₄)alkyl when the X Z bond is absent; (12) when the X Z bond is present and X is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O)_1-2, then Z is O, S, S(O),_₂, Se or NR¹⁶, wherein R¹⁶ is hydrogen, (C_r

C₆)alkyl, hydroxy, (Cj-C alkoxy, tosyl, or mesyl; when the X Z bond is present and X is O, S, S(O),.₂, Se or NR¹⁶, then Z is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O)_!.₂,; or when the X Z bond is absent then Z is H or

(C,-C₄)alkyl;

(13) x is 0, 1 or 2;

(14) Y is H, (C_rC₄)alkyl, optionally substituted by N(Rⁿ)(R¹²), acetoxy, acetyl, 2-methyl-l,3-dithiolan-2-yl optionally 4-substituted with CH₂N(R^H)(R¹²) or CO₂Rⁿ; or is C(R¹³)(R¹⁴)(OH) wherein R¹³ is H, (C,-C₄)alkyl, CO₂H or

CH₂OQ wherein Q is H, acetyl, or Ts (tosyl) and R¹⁴ is (C,-C₄)alkyl, halo(C╬╣-C₄)alkyl, haloacetyl, CO₂H, CH₂OQ wherein Q is as defined above, or is C(O)(C₁₂-C₂₂)alkyl, a peptide or protein, a residue of a sugar (e.g. rhamnose, glucose, xylose, galactose, etc.), or in combination with R⁶ is -CH₂-O- and R⁶ is absent or is OR¹¹ when the C N bond is absent; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (I) is not phalloidin;

a compound of formula (II):

wherein:

(1) each of R¹ and R┬░ is H, OH, CH₃O, halo, CN, NO₂, CF₃, CO₂H, CO₂(C_r C₄)alkyl or N(R^U)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(O)(C_rC₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, CH₃, formyl, or acetyl;

(4) R² is H or CH₃;

(5) R³ is CH₃, iso-propyl, or iso-butyl; (6) R⁴ is H or OH;

(7) R⁵ is H or OH:

(8) R⁶ is absent or together with R¹⁰ is a covalent bond;

(9) R⁷ is absent or is COCH₃ or R^╧Ç when the C N bond is absent ;

(12) R⁸ is absent or is H or (C_rC₄)alkyl when the X Z bond is absent; (13) R¹⁰ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl;

(14) R¹³ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (II) is not phalloidin;

a compound of formula (III):

wherein the bond represented by C ΓÇö N is absent or present, R is -(C(OR⁴)(CH₃)(CH₂OR⁵) or haloacetyl, wherein R⁴ is H or together with Z is a covalent bond; R⁵ is H, a peptide or protein, a steroidal moiety, a fatty acid moiety, a sugar residue or C(O)R' wherein R' is (C₁₂-C₂₂)alkyl, optionally comprising 1-3 double bonds, Z is absent or together with R⁴ is a covalent bond, Y is absent or is H when the C ΓÇö N bond is absent, and X is -H H-, SO, O, or Se; R³ is H or an acetyl, or a pharmaceutically acceptable salt thereof;

or a compound of formula (IN):

wherein:

(I) R^a, R^b, and R^╬╡ are individually any naturally or unnaturally occurring alpha amino acid, or lysine coupled through its ╬╡-amino group to a steroidal or fatty acid moiety; (2) x is 0, 1 or 2;

(3) each of R¹ and R┬░ is H, OH, (C_rC₄)alkyl, O(C_rC₄)alkyl, halo, halo(C_r C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(R^╧Ç)(R¹²) wherein each of R" and R¹² is H, (C_rC₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, or a residue of a sugar; (4) the bond represented by is present or absent;

(5) R is H, (C,-C₅)alkyl, (C_rC₅)alkanoyl, benzyl, or CH₂C(O)N(R¹ ')(R¹²);

(6) R² is H or (C_rC₄)alkyl;

(7) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl;

(8) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy); (9) X is H, O, S, Se, or S(O)_1-2;

(10) R⁸ is absent or is H or (C_rC₄)alkyl when the X Z bond is absent; and

(I I) Z is C(O) or -CH₂-, or is H or (C_rC₄)alkyl when the X Z bond is absent; or a pharmaceutically acceptable salt thereof.

31. The use of a non-toxic phalloidin derivative or analog for the preparation of a medicament for treating congestive heart failure, systolic congestive heart failure or cardiomyopathy.

32. A non-toxic phalloidin derivative or analog for use in medical treatment, provided said phalloidin derivative or analog is not a compound of formula (N):

wherein:

R¹ is C(CH₃)OH-CH₂OH, C(CH₃)OH-CH_3> C(CH₃)OH-CH₂-Otos , 10 C(CH₃)OH-CH₂OAc, CO-CH₃, CH₂-CH₃ or C(OH) (CH₂OH)₂ , R² is CH₃, R³ is C(H)OH-CH₃(threo), and R⁴ is OH(allo), when R⁵ is H; or

R¹ is C(CH₃)OH-CH₂OH, R² is CH₃, R³ is C(H)OH-CH₃(threo), R⁴ is OH(allo) and R⁵ is H when S is replaced with -H H-; or

R¹ is C(CH₃)OH-CH₂OH, R² is CH₃, R³ is C(H)OH-CH₃(threo), R⁴ is 15 OH(allo), and R⁵ is H, when the peptide bond in P⁷ is hydrolytically split; or

R¹ is C(CH₃)OH-CH₂OH, R² is CH(CH₃)₂, R³ is C(H)OH-CO₂H(erythro), and R⁴ is OH(allo) when R₅ is H; or

R¹ is C(CH₃)OH-CH₂OAc, R² is CH₃, R³ is C(H)OH-CH₃(threo) or C(H) OAc CH₃, and R⁴ is Oac(allo), or OH(allo), when R⁵ is H; or R¹ is C(CH₃)OH-CH₂-Otos , R² is CH₃, R³ is C(H)Otos-CH₃(threo) and R⁴ is OH(allo) when R⁵ is H;

R¹ is C(CH₃)OH-CH₂-Otos , R² is CH₃, R³ is C(H)OH-CH₃(threo) and R⁴ is Otos(allo) when R⁵ is H R¹ is C(CH₃)OH-CH₂- Otos , R² is CH₃, R³ is C(H)Otos -CH₃(threo) and R⁴ is Otos (allo); when R⁵ is H;

R¹ is C(CH₃)OH-CH₂-Br, R² is CH₃, R³ is C(H)Br-CH₃(threo) and R⁴ is Br(allo) when R⁵ is H;

R¹ is C(CH₃)OH-CH₂OH, R² is CH₃, R³ is C(H)OH-CH₃(threo), and R⁴ is OH(allo), when R⁵ is CH₃ , C₂H₅, n-C₃H_7╬╣┬½-C₅H_u, ┬½-C₄H₉ or CH₂CONH₂; or

R¹ is CH₂-CH₃, R² is CH₃, R³ is C(H)OH-CH₃(threo), R⁴ is OH(allo), and R⁵ is H, when peptide bond at P⁸ is broken and COOisobutyl is substituted on N and NH₂ is substituted on Co; or when open at P⁸ and between threonine and cysteine.

33. Claim 32 wherein R³ is not 1-hydroxyethyl, (carboxy)(hydroxy)methyl, (methoxycarboyl)(hydroxy)methyl, (aminocarbonyl)(hydroxy)methyl, (methylaminocarbonyl)(hydroxy)methyl, or (dimethylaminocarbonyl)- (hydroxy)methyl.

34. Claim 33 wherein the phalloidin derivative or analog is compound of formula (I):

wherein:

(1) each of R¹ and R┬░ is H, OH, (C,-C₄)alkyl, O(C,-C₄)alkyl, halo, halo(C,- C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(R^U)(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₂₂)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, (C,-C₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(Rⁿ)(R¹²);

(4) R² is H or (C_rC₄)alkyl;

(5) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl;

(6) R⁴ is H, OH, halo, acetoxy or OTs;

(7) R⁵ is H, OH, acetoxy, halo or OTs;

(11) R⁸ is absent, or is H or (C,-C₄)alkyl when the X Z bond is absent; (12) when the X Z bond is present and X is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O),.₂, then Z is O, S, S(O)_!.₂, Se or NR¹⁶, wherein R¹⁶ is hydrogen, (C_r

(C_rC₄)alkyl;

(13) x is 0, 1 or 2;

(14) Y is H, (C,-C₄)alkyl, optionally substituted by N(R^U)(R¹²), acetoxy, acetyl, 2-methyl-l,3-dithiolan-2-yl optionally 4-substituted with CH₂N(R^U)(R¹²) or CO_jR¹¹ ; or is C(R¹³)(R¹⁴)(OH) wherein R¹³ is H, (C,-C₄)alkyl, CO₂H or

CH₂OQ wherein Q is H, acetyl, or Ts (tosyl) and R¹⁴ is (C,-C₄)alkyl, halo(C,-C₄)alkyl, haloacetyl, CO₂H, CH₂OQ wherein Q is as defined above, or is C(╬╕╧çC₁₂-C₂₂)alkyl, a peptide or protein, a residue of a sugar (e.g. rhamnose, glucose, xylose, galactose, etc.), or in combination with R⁶ is -CH₂-O- and R⁶ is absent or is OR¹¹ when the C N bond is absent; or a pharmaceutically acceptable salt thereof; with the proviso that the compound of formula (I) is not phalloidin;

a compound of formula (II):

wherein:

(1) each of R¹ and R┬░ is H, OH, CH₃O, halo, CN, NO₂, CF₃, CO₂H, CO₂(C_r C₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, CH₃, formyl, or acetyl;

(4) R² is H or CH₃;

(5) R³ is CH₃, iso-propyl, or iso-butyl; (6) R⁴ is H or OH;

(7) R⁵ is H or OH:

(8) R⁶ is absent or together with R¹⁰ is a covalent bond;

(9) R⁷ is absent or is COCH₃ or R" when the C ΓÇö -N bond is absent ;

(12) R⁸ is absent or is H or (C,-C₄)alkyl when the X Z bond is absent; (13) R¹⁰ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl;

(14) R¹³ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl; or a pharmaceutically acceptable salt thereof; with the proviso that the

5 compound of formula (II) is not phalloidin;

a compound of formula (III):

wherein the bond represented by C ΓÇö N is absent or present, R is l o -(C(OR⁴)(CH₃)(CH₂OR⁵) or haloacetyl, wherein R⁴ is H or together with Z is a covalent bond; R⁵ is H, a peptide or protein, a steroidal moiety, a fatty acid moiety, a sugar residue or C(O)R' wherein R' is (C₁₂-C₂₂)alkyl, optionally comprising 1-3 double bonds, Z is absent or together with R⁴ is a covalent bond, Y is absent or is H when the C ΓÇö N bond is absent, and X is -H H-, SO, O, or Se; R³ is H or an acetyl, 15 or a pharmaceutically acceptable salt thereof;

or a compound of formula (IV):

wherein:

(3) each of R¹ and R┬░ is H, OH, (C,-C₄)alkyl, O(C,-C₄)alkyl, halo, halo(C_r C₄)alkyl, C╬¥, ╬¥O₂, CF₃, CO₂H, CO₂(C_rC₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, or a residue of a sugar; (4) the bond represented by is present or absent;

(5) R is H, (C_rC₅)alkyl, (C,-C₅)alkanoyl, benzyl, or CH₂C(O)N(R^╧Ç)(R¹²);

(6) R² is H or (C,-C₄)alkyl;

(7) R³ is H, (C,-C₄)alkyl, CO₂H, CH₂OH or benzyl;

(10) R⁸ is absent or is H or (C_rC₄)alkyl when the X Z bond is absent; and

35. Claim 28 or 32 wherein the medical treatment is the treatment of congestive heart failure, systolic congestive heart failure or cardiomyopathy.

36. A phalloidin derivative or analog provided said phalloidin derivative or 5 analog is not a compound of formula (N):

wherein:

R¹ is C(CH₃)OH-CH₂OH, C(CH₃)OH-CH_3, C(CH₃)OH-CH₂-Otos , 10 C(CH₃)OH-CH₂OAc, CO-CH₃, CH₂-CH₃ or C(OH) (CH₂OH)₂ , R² is CH₃, R³ is C(H)OH-CH₃(threo), and R⁴ is OH(allo), when R⁵ is H; or

R¹ is C(CH₃)OH-CH₂OH, R² is CH₃, R³ is C(H)OH-CH₃(threo), and R⁴ is OH(allo), when R⁵ is CH₃ , C₂H₅, n-C₃H_7╬╣┬½-C₅H_n, ┬½-C₄H₉ or CH₂CONH₂; or

37. Claim 32 wherein R³ is not 1 -hydroxy ethyl, (carboxy)(hydroxy)methyl, (methoxycarboyl)(hydroxy)methyl, (aminocarbonyl)(hydroxy)methyl, (methylaminocarbonyl)(hydroxy)methyl, or (dimethylaminocarbonyl)- (hydroxy)methyl.

38. The phalloidin derivative or analog of claim 37 which is a compound of formula (I):

wherein:

(I) each of R¹ and R┬░ is H, OH, (C_rC₄)alkyl, O(C_rC₄)alkyl, halo, halo(C_r C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is H, (C_rC₂₂)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent; (3) R is H, (C,-C₅)alkyl, (C_rC₅)alkanoyl, benzyl, or CH₂C(O)N(R^u)(R¹²); (4) R² is H or (C,-C₄)alkyl; (5) R³ is H, (C_rC₄)alkyl, CO₂H, CH₂OH or benzyl; (6) R⁴ is H, OH, halo, acetoxy or OTs; (7) R⁵ is H, OH, acetoxy, halo or OTs; (8) each of R⁶ and R⁷ is absent or is H, (C,-C₄)alkyl, acetyl, or phenyl when the

C N bond is absent; (9) R¹⁰ is H, (C,-C₄)alkyl, CO₂H, CO₂(C,-C₄)alkyl, or C(O)N(Rⁿ)(R¹²);

(10) X is H, -CH₂-, C(=O), C(=S), C(=NR¹⁵), S(O),.₂, O, S, or Se, wherein R¹⁵ is hydrogen, (C,-C₆)alkyl, hydroxy, (C_rC₄)alkoxy, tosyl, or mesyl;

(I I) R⁸ is absent, or is H or (C_rC₄)alkyl when the X Z bond is absent; (12) when the X- ΓÇö Z bond is present and X is -CH₂-, C(=O), C(=S), C(=NR¹⁵), or S(O),.₂, then Z is O, S, S(O),.₂, Se or NR¹⁶, wherein R¹⁶ is hydrogen, (C_r

C₆)alkyl, hydroxy, (C_rC₄)alkoxy, tosyl, or mesyl; when the X Z bond is present and X is O, S, S(O),.₂, Se or NR¹⁶, then Z is -CH₂-, C(=O), C(=S),

C(=NR¹⁵), or S(O),.₂₎; or when the X Z bond is absent then Z is H or

(C_rC₄)alkyl;

(13) x is 0, 1 or 2;

(14) Y is H, (C_rC₄)alkyl, optionally substituted by N(R^╧Ç)(R¹²), acetoxy, acetyl, 2-methyl-l,3-dithiolan-2-yl optionally 4-substituted with CH₂N(R^╧Ç)(R¹²) or CO₂Rⁿ; or is C(R¹³)(R¹⁴)(OH) wherein R¹³ is H, (C_rC₄)alkyl, CO₂H or CH₂OQ wherein Q is H, acetyl, or Ts (tosyl) and R¹⁴ is (C_rC₄)alkyl, halo(C,-C₄)alkyl, haloacetyl, CO₂H, CH₂OQ wherein Q is as defined above, or is C(O)(C₁₂-C₂₂)alkyl, a peptide or protein, a residue of a sugar (e.g. rharnnose, glucose, xylose, galactose, etc.), or in combination with R⁶ is

-CH₂-O- and R⁶ is absent or is OR¹¹ when the C N bond is absent; or a pharmaceutically acceptable salt thereof;

a compound of formula (II):

wherein:

(1) each of R¹ and R┬░ is H, OH, CH₃O, halo, CN, NO₂, CF₃, CO₂H, CO₂(C_r C₄)alkyl or N(Rⁿ)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(O)(C╬╣-C₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(2) the bonds represented by are present or one is absent;

(3) R is H, CH₃, formyl, or acetyl;

(4) R² is H or CH₃;

(5) R³ is CH₃, iso-propyl, or iso-butyl; (6) R⁴ is H or OH;

(7) R⁵ is H or OH:

(8) R⁶ is absent or together with R¹⁰ is a covalent bond;

(9) R⁷ is absent or is COCH₃ or R¹¹ when the C N bond is absent ;

(12) R⁸ is absent or is H or (C,-C₄)alkyl when the X Z bond is absent;

(13) R¹⁰ is H, a peptide or protein, a monosaccharide, C(O)(C_I2-C₂₂)alkyl, or haloacetyl;

(14) R¹³ is H, a peptide or protein, a monosaccharide, C(O)(C₁₂-C₂₂)alkyl, or haloacetyl; or a pharmaceutically acceptable salt thereof;

a compound of formula (III):

or a compound of formula (IV):

wherein:

(I) R^a, R^b, and R^g are individually any naturally or unnaturally occurring alpha amino acid, or lysine coupled through its ╬╡-amino group to a steroidal or fatty acid moiety; (2) x is 0, 1 or 2;

(3) each of R¹ and R┬░ is H, OH, (C,-C₄)alkyl, O(C,-C₄)alkyl, halo, halo(C_r C₄)alkyl, CN, NO₂, CF₃, CO₂H, CO₂(C,-C₄)alkyl or N(R^╧Ç)(R¹²) wherein each of R¹¹ and R¹² is H, (C,-C₄)alkyl, benzyl, phenyl, C(O)(C,-C₂₂)alkyl, a peptide or protein, a steroidal moiety, a fatty acid moiety, or a residue of a sugar;

(4) the bond represented by is present or absent;

(5) R is H, (C,-C₅)alkyl, (C_rC₅)alkanoyl, benzyl, or CH₂C(O)N(Rⁿ)(R¹²);

(6) R² is H or (C_rC₄)alkyl;

(7) R³ is H, (C_rC₄)alkyl, CO₂H, CH₂OH or benzyl; (8) R⁴ is H, OH, halo, acetoxy or OTs (tosyloxy);

(9) X is H, O, S, Se, or S(O),.₂;

(10) R⁸ is absent or is H or (C_rC₄)alkyl when the X Z bond is absent; and