WO2005102347A1

WO2005102347A1 - Calcium sensitisers for treating symptoms of venomous bites and stings

Info

Publication number: WO2005102347A1
Application number: PCT/FI2005/000192
Authority: WO
Inventors: Alexandre Mebazaa; Fekri Abroug
Original assignee: Orion Corporation
Priority date: 2004-04-23
Filing date: 2005-04-22
Publication date: 2005-11-03

Abstract

Calcium sensitising agents, such as levosimendan or its active metabolite, are useful for treating or preventing symptoms of venomous bites and stings due to terrestrial and aquatic animals, e.g. scorpions and snakes.

Description

CALCIUM SENSITISERS FOR TREATING SYMPTOMS OF VENOMOUS BITES AND STINGS

Technical field

The present invention relates to the use of calcium sensitising agents for treating or preventing symptoms of venomous bites and stings due to terrestrial and aquatic animals, e.g. scorpions and snakes.

Background of the invention

Snake and scorpion envenomation is frequently encountered throughout the world, for example in Tunisia alone 30 000 - 40 000 patients/year. This induces a high morbidity (admission in emergency units, hospitalisation), an increased hospital cost and a high mortality mostly in young and healthy patients. Many North African authorities consider scorpion envenomation as a health priority.

In North African countries, the clinical features of snake and scorpion envenomation are roughly similar. The clinical spectrum ranges from an isolated localized pain to the most severe stage characterised by sudden rise in plasma catecholamines and the occurrence of severe systemic symptoms such as circulatory failure and pulmonary edema. Although hemodynamic profile following scorpion envenomation is now better defined, the physiopathology of these alterations is still unclear. In addition, no treatment has so far achieved the level of clinical evidence to be routinely recommended for severely envenomated patients. Indeed, a recent clinical study has shown that the anti-venom administration did not alter morbidity or mortality (Abrough F. et al., Lancet, 1999; 354: 906-909). Because of the dissatisfaction with the current treatments, improved methods for the treatment of venomous bites and stings would be highly desired.

Summary of the invention It has now been found that agents capable of increasing calcium sensitivity of contractile proteins of cardiac muscle, i.e. calcium sensitising agents, are particularly useful in the treatment or prevention of symptoms of venomous bites and stings due to terrestrial and aquatic animals, such as scorpions and snakes. Astonishingly, the compounds of the invention were able to almost completely block the paramount hemodynamic effects caused by scorpion venom in mammals and the sudden rise of catecholamines in plasma.

Thus, the present invention provides a method for the treatment or prevention of symptoms of venomous bites and stings in a mammal, said method comprising administering to a mammal in need thereof an effective amount of a calcium sensitising agent.

The present invention also provides the use of a calcium sensitising agent in the manufacture of a medicament for the treatment or prevention of symptoms of venomous bites and stings.

The preferred calcium sensitising agents are levosimendan and its active metabolite (R)-N-[4-(l,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]- acetamide (II) or any of their pharmaceutically acceptable salts. Brief description of the drawings

Fig. 1 shows cardiac output (1/min) in 2 dogs treated according to protocol 1 (solid line), administration of levosimendan 10 minutes after scorpion venom infusion, and in 3 dogs treated according to protocol 2 (dotted line), administration of levosimendan 15 min before scorpion venom infusion.

Fig. 2 shows plasma concentration of epinephrine (nmol/1) in 2 dogs treated according to protocol 1 (solid line), administration of levosimendan 10 minutes after scorpion venom infusion, and in 2 dogs treated according to protocol 2 (dotted line), administration of levosimendan 15 min before scorpion venom infusion.

Detailed description of the invention The method of the invention employs calcium sensitising agents as the active ingredients. The term "calcium sensitising agent" means here an agent that increase calcium sensitivity of contractile proteins of cardiac muscle. Calcium sensitising agents increase the contractile force generated for a given amount of free calcium in the cytoplasm. This results in enhanced myocardial contractility in the heart. The terms "bite" and "sting" refer to an injury caused by the venom of an animal (biotoxin) introduced into an individual. The term "venom" refers to a poison, more specifically, a toxic substance normally secreted by an animal. As used herein, the term "venom" does not include substances which are considered to be pure neurotoxins. Examples of animals which are known to secrete venom include, but are not limited to, certain types of snakes, scorpions, spiders, bees and jellyfish.

The present invention provides a method for the treatment or prevention of the symptoms of venomous bites and stings which comprises administering to a mammal an amount of calcium sensitising agent effective to reduce, inhibit or prevent symptoms of venomous bites and stings in a mammal, including man. The administration of a calcium sensitising agent can be enteral, e.g. oral or rectal; parenteral, e.g. intravenous; or transdermal.

One preferred embodiment of the invention provides a method for the treatment or prevention of symptoms caused by venomous bites of a snake, said method comprising administering to a mammal in need thereof an effective amount of a calcium sensitising agent.

Another preferred embodiment of the invention provides a method for the treatment or prevention of symptoms caused by venomous stings of a scorpion, said method comprising administering to a mammal in need thereof an effective amount of a calcium sensitising agent.

Well known calcium sensitising agents useful in the present invention include levosimendan and its active metabolite. Levosimendan is currently used in the treatment of decompensated heart failure.

Levosimendan, which is (-)-enantiomer of [[4-(l,4,5,6-tetrahydro-4-methyl-6- oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile, and the method for its preparation is described in EP 565546 Bl . The hemodynamic effects of levosimendan in man are described in Sundberg, S. et al., Am. J. Cardiol., 1995; 75: 1061-1066 and in Lilleberg, J. et al., J. Cardiovasc. Pharmacol., 26(SupplJ), S63- S69, 1995. Clinical studies have confirmed the beneficial effects of levosimendan in heart failure patients. Levosimendan has an active metabolite, (R)-N-[4-(l,4,5,6-tetrahydro-4- methyl-6-oxo-3-pyridazinyl)phenyl]acetamide (II), which is present in human following administration of levosimendan. The effects of the active metabolite (II) are similar to levosimendan. The use of the active metabolite (II) for increasing calcium sensitivity of contractile proteins in the cardiac muscle has been described in WO 99/66932.

Other calcium sensitising agents useful in the present invention have been described e.g. in patent publications EP 383449 and WO 01/68611. A calcium sensitising agent useful in the invention can be identified using the procedure described below in the experimental part.

The preferred calcium sensitising agent used in the present invention is levosimendan or its active metabolite (II) or any of their pharmaceutically acceptable salts. Levosimendan is particularly preferred.

Compounds of the invention are formulated into dosage forms suitable for the treatment of venomous bites and stings using the principles known in the art. It is given to a patient as such or preferably in combination with suitable pharmaceutical excipients in the form of tablets, dragees, capsules, suppositories, emulsions, suspen- sions or solutions whereby the contents of the active compound in the formulation is from about 0.5 to 100 % per weight. Choosing suitable ingredients for the composition is a routine for those of ordinary skill in the art. It is evident that suitable carriers, solvents, gel forming ingredients, dispersion forming ingredients, antioxidants, colours, sweeteners, wetting compounds, release controlling components and other ingredients normally used in this field of technology may be also used.

Bites and stings are quasi-exclusively related to subcutaneous administration of the venom. Envenomation is therefore local before being systemic when the venom will subsequently diffuse into the blood stream. It was found that the compounds of the invention prevented the signs of envenomation more effectively if they were present in the blood before the arrival of the venom in the blood stream. Therefore, the compound of the invention should be administered as early as possible after the bite or sting attack. Thus, intravenous administration of the compounds of the invention, e.g. by bolus injection, is preferred. The effective amount of a calcium sensitising agent to be administered to a subject depends on the substance used, the route of administration, age, weight and the condition of the patient.

Levosimendan, for example, can be administered intravenously using the infusion rate typically from about 0.01 to 10 μg/kg/min, more typically from about 0.02 to 5 μg/kg/min. For the intravenous treatment of venomous bites and stings an intravenous bolus of 10 - 200 μg/kg optionally followed by infusion of 0J - 3 μg/kg/min may be needed. The active metabolite (II) can be administered intravenously using an infusion rate, which is from about 0.001 to 1 μg/kg/min, preferably from about 0.005 to 0.5 μg/kg/min.

Levosimendan is administered orally to man generally in daily dose from about 0J to 20 mg, preferably from 0.2 to 15 mg, more preferably from 0.5 to 10 mg.

The oral daily dose of the active metabolite (II) in man is generally within the range of 0.05 - 10 mg. Formulations suitable for intravenous administration such as injection or infusion formulation, comprise sterile isotonic solutions of levosimendan or its active metabolite (II) and vehicle, preferably aqueous solutions. Typically an intravenous infusion solution comprises from about 0.01 to 0J mg/ml of levosimendan or its active metabolite (II).

For oral administration in tablet form, suitable carriers and excipients include e.g. lactose, corn starch, magnesium stearate, calcium phosphate and talc. For oral administration in capsule form, useful carriers and excipients include e.g. lactose, corn starch, magnesium stearate and talc. Tablets can be prepared by mixing the active ingredient with the carriers and excipients and compressing the powdery mixture into tablets. Capsules can be prepared by mixing the active ingredient with the carriers and excipients and placing the powdery mixture in capsules, e.g. hard gelatin capsules. For example, a tablet or a capsule comprises from about 0J to 10 mg, more typically 0.2 to 5 mg, of levosimendan or its active metabolite (II).

Examples Pharmaceutical examples.

Example 1. Concentrate solution for intravenous infusion

(a) levosimendan 2.5 mg/ml (b) Kollidon PF 12 10 mg/ml (c) citric acid 2 mg/ml (d) dehydrated ethanol ad 1 ml (785 mg)

The concentrate solution was prepared by dissolving citric acid, Kollidon PF121 and levosimendan to dehydrated ethanol in the sterilized preparation vessel under stirring. The resulting bulk solution was filtered through a sterile filter (0.22 μm). The sterile filtered bulk solution was then aseptically filled into 8 ml and 10 ml injection vials (with 5 ml and 10 ml filling volumes) and closed with rubber closures. The concentrate solution for intravenous infusion is diluted with an aqueous vehicle before use. Typically the concentrate solution is diluted with aqueous isotonic vehicles, such as 5 % glucose solution or 0.9 % NaCl solution so as to obtain an aqueous intravenous solution, wherein the amount of levosimendan is generally within the range of about 0.001 - 1.0 mg/ml, preferably about 0.01 - 0J mg/ml.

Example 2. Oral capsule

Hard gelatin capsule size 3 Levosimendan 2.0 mg Lactose 198 mg The pharmaceutical preparation in the form of a capsule was prepared by mixing levosimendan with lactose and placing the powdery mixture in hard gelatin capsule.

Experiments

Experiment 1. Calcium sensitizing effect in skinned cardiac fiber

The compounds acting as a calcium sensitiser can be identified using the following procedure.

Left ventricular papillary muscle of the guinea-pig was dissected and rinsed in ice-cold Tyrode solution. Thereafter the papillary muscle was immersed into a solution containing (mM): Potassium acetate 74.7, EGTA 10, MgSO4 5.4, ATP-Na2 4, DTE 1,

MOPS 20, pH 7.0 (by 1 M KOH). Subsequently, the papillary muscle was sonicated at 10 Watt for 60 sec in this ice-cold solution. The distance between ultrasound probe and the papillary muscle was 10 mm. The fibers (< 200 μm in diameter) were dissected from surface of sonicated papillary muscles. Moreover, the sonicated and dissected fibers were kept for 30 min in a "skinning" solution (ice-cold) containing saponin (250 μg/ml) in addition to the other constituents. Continuous magnetic stirring was used during this treatment.

The fibers which were further dissected (< 100 μm in diameter) were then mounted horizontally with a glue (cellulose acetate in acetone) between a steel-rod extension of isometric force transducer (AME-801 strain gauge, Horten Electronics, Norway) and a glass rod attached to a micro-manipulator. The force transducer was connected to an amplifier. The fibers were kept in the "relaxing" solution containing (mM): imidazole 30, ATP-Na2 10, NaN3 5, EGTA 5, MgCl2 12.5, and 350 U creatin kinase. The temperature of the solution was 22°C and the pH was set to 6.7 by 1 M KOH. The ionic strength was adjusted with 1 M KC1 to correspond that of the "activating" solution. The composition of the "activating" solution was the same as that of the "relaxing" solution except that it contained also CaCl2- The fibers were induced to contract in desired free pCas (-log[Ca²⁺j) which were obtained by properly mixing of the "relaxing" and "activating" solutions. Tension produced by a fiber at pCa 4.8 was taken as maximum response. At the beginning of the experiment the fiber was stretched as described above. The calcium sensitising effect of a test compound can be calculated as a percent of force change compared to control. Experiment 2. Effect in scoφion envenomation model

Scoφion venom, namely Androctonus australis, known to induce death in humans, was intravenously administered to anesthetized dogs. It induced clinical signs similar to what is observed in humans: increased blood pressure, decreased cardiac output, pulmonary edema and increased plasma catecholamine levels.

Levosimendan (0.2 μg/kg/min infusion over 4 hours) was tested in dogs following two protocols: protocol 1, administration of levosimendan 10 minutes after scoφion venom infusion; protocol 2, administration of levosimendan 15 min before scoφion venom infusion. Figure 1 shows cardiac output for 2 dogs in protocol 1 group (solid line) and for 3 dogs in protocol 2 group (dotted line). Figure 2 shows plasma concentration of epinephrine for 2 dogs in protocol 1 group (solid line) and for 2 dogs in protocol 2 group (dotted line).

Interestingly, in protocol 2 (n=5 dogs), levosimendan totally or partially prevented venom-induced hemodynamic alterations and the sudden rise in catecholamine release. Accordingly, levosimendan is able to prevent signs of scoφion envenomation if it is present in the blood before the venom arrives in the circulation.

Claims

1. A method for the treatment or prevention of symptoms of venomous bites and stings in a mammal, said method comprising administering to a mammal in need thereof an effective amount of a calcium sensitising agent.

2. A method of claim 1 wherein the calcium sensitising agent is levosimendan or its active metabolite (II) or any of their pharmaceutically acceptable salts.

3. A method of claim 1 wherein the venomous bite or sting is caused by a snake or a scoφion.

4. The use of a calcium sensitising agent in the manufacture of a medicament for the treatment or prevention of symptoms of venomous bites and stings.