EP1532140A1 - Thienylhydrazone possedant des proprietes analogues a celles de la digitale (effets inotropes positifs) - Google Patents

Thienylhydrazone possedant des proprietes analogues a celles de la digitale (effets inotropes positifs)

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Publication number
EP1532140A1
EP1532140A1 EP00941596A EP00941596A EP1532140A1 EP 1532140 A1 EP1532140 A1 EP 1532140A1 EP 00941596 A EP00941596 A EP 00941596A EP 00941596 A EP00941596 A EP 00941596A EP 1532140 A1 EP1532140 A1 EP 1532140A1
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EP
European Patent Office
Prior art keywords
lassbio
compound
muscle
tension
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00941596A
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German (de)
English (en)
Inventor
Edson X. Albuquerque
Eliezer J. De Barreiro
Roberto Takashi Sudo
Yasco Aracava
Magner Monteiro Cintra
Paulo De Assis Melo
Francois Germain Noel
Gisele Zapata Sudo
Claudio Lucia Martins Da Silva
Newton Goncalves De Castro
Patricia Dias Fernandes
Carlos Alberto Manssour Fraga
Ana Luisa Palhares De Miranda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidade Federal do Rio de Janeiro UFRJ
University of Maryland at Baltimore
Original Assignee
Universidade Federal do Rio de Janeiro UFRJ
University of Maryland at Baltimore
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Application filed by Universidade Federal do Rio de Janeiro UFRJ, University of Maryland at Baltimore filed Critical Universidade Federal do Rio de Janeiro UFRJ
Publication of EP1532140A1 publication Critical patent/EP1532140A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure

Definitions

  • the invention relates to chemical compounds exhibiting digitalis-like properties and activity, and to methods of making and using this compound.
  • the invention further includes use of these compounds in the treatment of cardiac disease and muscle fatigue.
  • tne invention relates to pharmaceutical compositions containing these compounds.
  • the invention further relates to methods of synthesis of these compounds.
  • digitalis-derived drugs or digitalis glycosides
  • the mechanism of action of digitalis-derived drugs, or digitalis glycosides is to selectively inhibit active transport of K + and Na + , in cardiac muscle, which increases the rate of Ca 2+ cycling. This has the effect of increasing the velocity and extent of shortening of cardiac muscle by increasing the availability of Ca 2+ to interact with contractile proteins.
  • Digitalis glycosides also affect the sympathetic nervous system and reduce neurohumeral activation. Examples of currently marketed digitalis glycosides are Lanoxin® and Lanoxicaps® made by Glaxo- Wellcome.
  • Digitalis glycosides share the property of being toxic immediately above their therapeutic range. Toxic effects of these drugs include: arrhythmias, ECG effects such as increased blood pressure and heart rate, pulmonary congestion, delirium, fatigue, disturbance of color vision, anorexia, nausea, and vomiting.
  • the drugs are cardiotoxic and neurotoxic because of their effect on the sympathetic nervous system.
  • Digoxin is also metabolized by intestinal flora and antibiotic treatment can increase drug bioavailability, causing an overdose.
  • the treatment for overdose is to bind circulating drug with antibodies to digitalis glycosides.
  • the cost of this treatment can be in excess of $3,000 per incident.
  • An example of such an antibody is Digibind® made by
  • Digibind® is a protein derived from sheep and carries its own risks.
  • Congestive heart failure is an important cause of mortality and morbidity in the U.S.: over 2.5 million patients are currently diagnosed.
  • Compounds derived from digitalis which are in the class of cardiac glycosides, are the primary drugs used in the treatment of congestive heart failure, particularly systolic dysfunction.
  • digitalis ameliorates congestive heart failure by producing a positive inotropic effect.
  • a positive inotropic agent strengthens the contractility of muscular tissue.
  • Digitalis-derived drugs have the defect of being cardio-toxic and neuro-toxic at doses just above their therapeutic range (Hardman, J.G. and Limbird, L.E., The Pharmaceutical Basis of Therapeutics, 9 th ed. McGraw-Hill New York, 1996, chapter 34).
  • the invention includes the novel chemical compound having the formula (I)
  • each of Ri and R 2 is hydrogen, alkyl of 1 to 6 carbon atoms, phenyl and substituted phenyl.
  • the invention further includes the synthesis of a novel compound that, like digitalis, produces a positive isotropic effect on cardiac and skeletal muscle. Like digitalis, it has utility in the treatment of congestive heart failure. Unlike digitalis, it does not have toxic properties near its effective therapeutic range and therefor the invention has a medical advantage over that class of drugs. The invention has further utility in the treatment of muscle fatigue in pathological states.
  • the invention compound(s) act(s) as a calcium sensitizer in heart and skeletal muscle. It delays and shortens fatigue of skeletal muscle and thus also has utility in the treatment of muscle fatigue. Muscle fatigue is a symptom of certain pathological states such as: major injury, cancer, HIV infection, sepsis, Crohn's disease, ulcerative colitis, and athletic over-training.
  • Figure 1 Table 1. Histopathological Study of Tissues and Organs of Rats Injected with BASSBio-294. Table 2. Toxicity Study of LASSBio-294 Injected in Mice. Weight of mice in grams. Table 3. Blood Cell Analysis in Mice Treated with LASSBIO-294.
  • the first column shows the numbers done in control experiments (DMSO) or in 25 ⁇ M of compound 294.
  • the last line is the ratio between the values obtained in compound 294 and the control experiments in Ringer plus DMSO.
  • FIG. 1 The papillary muscle, and bundles of atrial and ventricular cells obtained from rat hearts were dissected and set in an aerated chamber to enable recording of isometric tension.
  • LASSBio-294 was added to the chamber in a cumulative manner, first
  • Figure 4. Hearts of adult rats were quickly removed and placed in an aorta retrograde perfiision system (modified Langendorrff) for measurement of electrocardiogram (ECG).
  • ECG electrocardiogram
  • FIG. 1 Recordings of twitch tension in isolated hearts. Control (top), LASSBio- 294 (middle) and wash (bottom).
  • Figure 6 Recording of left intraventricular pressure and arterial pressure in dogs anesthetized and ventilated normally.
  • Figure 8 Contracture induced by caffeine in relation to loading of the sarcoplasmic reticulum with a solution of pCa 6.6.
  • Figure 9 The contractual responses were obtained during exposure to 20 mM caffeine after loading of the sarcoplasmic reticulum with pCA 6.6 in the absence and the presence of LASSBio-294.
  • Figure 13 Bundles of left ventricular muscles were treated with Triton X-100 and exposed to increasing concentrations of Ca 2+ . Muscle tension expressed as % of the maximal response induced by 0.5 mM CaCl 2 is graphed as a function of pCa.
  • FIG. 14 Isolated human skeletal muscle fiber from the vastus later alis (sarcolemmal membrane-free) was exposed to 0.5 mM of CaCl 2 to enable maximal muscle tension (Po). The histogram show the effect of LASSBio-294 at concentrations of 25, 50 and 100 ⁇ M on induced tension in human fibers.
  • FIG. 15 Effect of 12.5 ⁇ M of compound 294 on force development in a single muscle fiber stimulated at different frequencies. The frequency of stimulation in Hz is indicated under each trace. Top panel, stimulating cycle done in Ringer without compound 294. Middle panel, stimulating cycle done 17 min after the fiber was bathed with compound 294. Bottom panel, second cycle of stimulation done 17 min after compound 294 was washed out with Ringer. Force calibration bars are 50 mg for twitches and 10 Hz simulations and 100 mg for 30, 60, and 90 Hz.
  • Figure 16 Histogram showing the effect of 12.5 ⁇ M compound 294 and after washing out LASSBio-294 on fractional twitch tension (T ⁇ /T 0 ). T x represents the twitch tension obtained when the fiber was bathed with the solutions indicated above each column. The order of the columns are the order in which the simulating cycles were performed.
  • FIG. 19 Same as Fig. 17, but with 50 ⁇ M of LASSBio-294.
  • the force calibration bar is the same for all the stimulation frequencies.
  • FIG. 20 Effect of 12.5 ⁇ M LASSBio-294 on peak twitch tension and maximal 10 Hz tetanic tension. The measurements were taken after the fibers had been in each solution for 40 min without being stimulated. Each column represents the ratio of the corresponding tension (T x ) divided by the corresponding control tension (T 0 ) which was taken as 1.
  • FIG. 21 A Effect of 12.5 (circles) and 25 (dots) ⁇ M of LASSBio-294 on fractional tension potentiation at different frequencies of stimulation. Fractional tension is expressed as the ratio of the maximal force obtained at each different frequency (T # 94 ) divided by the maximal force obtained in Ringer plus DMSO at each corresponding different frequency (TDMSO)-
  • FIG. 21B Histogram showing the effect of 12.5 ⁇ M LASSBio-294 and wash out of the compound on twitch tension ratio (Tx/To). Twitch tension obtained during the first cycle of stimulation in Ringer and DMSO was taken as one (To). Tx represents the twitch tension obtained when the fiber was bathed with the solutions indicated above each column. The order of the columns is the order in which the stimulating cycles were done.
  • FIG 22 Comparison of the time course of twitch tension obtained in Ringer plus DMSO (dots) with the time course of twitch tension elicited in Ringer plus 25 ⁇ M of L AS SBio-294 (squares).
  • FIG 23 Relationship between fractional twitch tension and LASSBio-294 concentration. Peak twitch tensions (T n ) were measured during the second stimulation cycle after the cells were in different LASSBio-294 concentrations and ratio against peak twitch tension (T 0 ) in Ringer without LASSBio-294 which was taken as 1.
  • Figure 24 Time parameters measured during the twitch tension time course. T p is the time to reach peak force. T 0 5 is the relaxation time it takes for tension to decline from peak tension to 50% of the peak tension. T 0.8 is the relaxation time it takes for tension to 80% of the peak tension.
  • FIG. 25 Effect of LASSBio-294 on the time course of fatigue development.
  • Top trace control experiment the fiber was bathed with Ringer plus DMSO.
  • Bottom trace the fiber was bathed with Ringer plus 12.5 ⁇ M of LASSBio-294.
  • the fibers were stimulated with the parameters described in methods.
  • the first trace is the tension elicited with a 10 Hz stimulation frequency.
  • Force and time calibration bars are 50 mg and 1 sec for the 10 Hz stimulation frequencies and 100 mg and 1 min for the repetitive stimulation.
  • FIG 26 Summary of the time course of the index of fatigue development elicited in fibers bathed with Ringer only (green dots), Ringer plus DMSO (squares) and Ringer plus 12.5 ⁇ M of LASSBio-294 (red dots).
  • t R toM SO and t ⁇ M indicate the times it takes for tetanic tension to start declining (fatigue) after the beginning of the repetitive stimulation when the fibers were bathed with Ringer only, Ringer plus DMSO and Ringer plus 12.5 ⁇ M respectively. Abscissas are expressed either as the number of tetanic stimulations or the time of stimulation.
  • Figure 27 Time course of the index of fatigue development with different concentrations of compound 294 as indicated. The control curve, done in Ringer only, is the average of three experiments; each of the other correspond to only one fiber.
  • Figure 28 Time (in min) for tetanic force to decrease (fatigue) to 50% of the original tetanic force during repetitive stimulations as in Fig. 17. Left column in 50 ⁇ M of DMSO only ( ⁇ 6 in). Right column in 50 ⁇ M of LASSBio-294 ( ⁇ 9 min).
  • Figure 29 Time of recovery to 80% of the pre-fatigue tetanic force. Left column in 50 ⁇ M of DMSO only ( ⁇ 90 min). Right column in 50 ⁇ M of LASSBio-294 ( ⁇ 10 min).
  • Figure 31 Records of contractions of rat gastrocnemius muscles induced by stimulation of the sciatic nerve and of arterial pressure. For control, contraction was induced by intravenous injection of DMSA (0.04 ml). LASSBio-294 was administered in
  • a compound of the invention like digitalis, produces a positive isotropic effect on cardiac and skeletal muscle. Like digitalis, it has utility in the treatment of congestive heart failure. Unlike digitalis, it does not have toxic properties at its effective therapeutic dosage levels; and, therefor, the invention provides a medical advantage over that class of drugs.
  • a compound of the invention has further utility in the treatment of muscle fatigue in pathological states.
  • the invention acts as a calcium sensitizer in heart and skeletal muscle and it delays and shortens fatigue of skeletal muscle and thus also has utility in the treatment of muscle fatigue. Muscle fatigue is a symptom of certain pathological states such as: major injury, cancer, HIV infection, sepsis, Crohn's disease, ulcerative colitis, and athletic over-training.
  • the invention includes the novel chemical compound having the formula (I)
  • Each of Ri and R 2 is a substituent selected from the following in any combination: hydrogen., alkyl of 1 to 6 carbon atoms phenyl, and substituted phenyl.
  • at least one of Ri or R 2 is hydrogen.
  • each of Ri and R 2 I hydrogen; and then the compound has formula (II):
  • the compound of formula (II) is 3, 4-methylenedioxybenzoyl-2-thienylhydrazone and has been designated LASSBio-294.
  • the invention further includes pharmaceutically acceptable salts of the compounds of formula (I) and (II).
  • Such salts can include the acetate, citrate, phosphate, fumarate, benzoate, tartrate, succinate, chlorate, sulfate, butyrate, stearate, palmitate, lactate, methylate, and carbonate salts. These salt forms can be prepared by reacting the compound with appropriate acids under standard conditions.
  • a compound of formula I can be a potent, positive inotropic agent for cardiac and skeletal muscle. It is within the scope of the invention to use the invention compound(s) to increase the strength of heart muscle contraction.
  • a compound of formula II can be used, like cardiac glycosides, to treat congestive heart failure.
  • the invention encompasses the effects of the compound on skeletal muscle; and its use in the therapeutic treatment of muscle fatigue. Muscle fatigue is a serious complication of certain pathological states.
  • the invention includes the novel chemical compound having the formula (I) .
  • Compounds of formula (I) may contain substitution on the 2 and/or 5 and/or 6 position of the benzoyl moiety and/or on the thienyl moiety in the 2 and/or on the 3 and/or 4 position(s) of the thienyl moiety.
  • R 3 , Rt, R 5 , W_, R , and R 8 may be hydrogen, alkyl of 1 to 6 carbon atoms, phenyl [unsubsituted or substituted], amino [secondary, tertiary or quaternary amino] , nitro-, ester [RCOO-], acid [-COOH , alcohol [-OH] or ether [R.O-], to form compounds of the following structure (III): (III)
  • each of R3-R8 is as defined above.
  • compositions of the invention comprise a compound of formula (I), formula (II) and/or of formula (III).
  • the dose range for the compound of formula (II), based on in vitro and in vivo tests, is one that produces between levels of 100 nM and 500 ⁇ M in plasma, a more effective dose produces between levels of l ⁇ M and 100 ⁇ M, the most effective dose produces between levels of 20-50 ⁇ M in plasma. This plasma concentration can be achieved in several ways depending on the therapeutic requirements of the patient.
  • a parenteral form can be used for intravenous and intramuscular infusion. This can be supplied as either a powder or a concentrate to be used as a solution at the time of dosing or as an injectable, sterile solution.
  • the diluent could be water, saline, or a lipid based diluent containing ethanol and buffers such as citrate to stabilize pH, and preservatives such as sodium benzoate and methylparaben, as required.
  • the diluent could further contain such other excipients and could contain pharmaceutically acceptable carriers as may be desirable such as a protein carrier, including serum albumen.
  • the invention also encompasses the use of solvents such as dimethyl sulfoxide (DMSO), alcohol, ethylene glycol or polyethylene glycol. Such solvents can be used alone or in combination.
  • DMSO dimethyl sulfoxide
  • a nasal spray is also encompassed by the invention and could readily be compounded by one of skill in the art using such diluents and inactive ingredients as are commonly used. Solutions can be emulsions or micro-emulsions containing an oily phase, an aqueous phase, and optionally a surfactant.
  • the oily phase can contain one or more of the following: carboxylic acid esters, fatty acids, fatty esters, glyceryl derivatives such as glyceryl behenate, short, medium and long chain triglycerides, and others.
  • Surfactants that may be used with the invention to produce a pharmaceutically acceptable formulation include polyoxyethylene sorbitan esters, ethyleneoxide propylene oxide block co- polymers, polyglycolized glycerides, sucrose esters, polyoxyethylene laurel esters, and others.
  • compositions of the invention in tablet form.
  • inactive ingredients such as cellulose, microcrystalhne cellulose, corn starch, lactose, starch, silica, dextrose and stearic acid, and such additional ingredients as dis-integrants, including carboxy methyl cellulose, soy polysaccharides, pre-gelatinized starches, and polyethylene glycol and lubricants to achieve a pharmaceutical preparation that can be readily manufactured.
  • lubricants can include polyethylene glycol, leucine, glycerol behenate, magnesium stearate, or calcium stearate.
  • the pharmaceutical composition of the invention can be used as a hard or soft gelatin capsule in combination with suitable inactive ingredients such as lactose, cornstarch, microcrystalline cellulose, soy polysaccharides, calcium phosphate dihydrate, calcium sulfate, lactose, sucrose, sorbitol, or suitable liquids or gels.
  • suitable inactive ingredients such as lactose, cornstarch, microcrystalline cellulose, soy polysaccharides, calcium phosphate dihydrate, calcium sulfate, lactose, sucrose, sorbitol, or suitable liquids or gels.
  • suitable inactive ingredients such as lactose, cornstarch, microcrystalline cellulose, soy polysaccharides, calcium phosphate dihydrate, calcium sulfate, lactose, sucrose, sorbitol, or suitable liquids or gels.
  • the tablet or capsule could readily be coated. Such a coating could be an enteric coating to provide for intestinal release of the drug, or a neutral coating to improve
  • compositions of the invention can also be provided as an elixir for pediatric and geriatric dosing.
  • Such an elixir could readily by formulated by one of skill in the art and could contain water, ethanol, solvents and surfactants as well as a preservative, such as methyl paraben, citric acid, and coloring and flavoring ingredients, as desired.
  • a rectal suppository is also with in the scope of the invention and could be readily compounded using standard methods.
  • Such a suppository could contain waxes, oils, lipids or gelling agent to produce a stable formulation which melts at body temperature. It could contain such solubilizers, surfactants, and stabilizers as might be required.
  • LASSBio294 a pro-drug of LASSBio294.
  • a carrier molecule for example a dipeptide, tripeptide, or any molecule absorbed in the intestine via transporter-mediated transport., so as to increase the bioavailability of the drug in oral formulation.
  • LASSBio-294 has a positive effect on contractility of cardiac muscle. It increases the isometric tension achieved by isolated bundles of muscle. Ventricular, papillary and atrial cardiac muscle bundles all achieved increased tension up to a 2-fold over control, when treated with LASSBio-294 at concentrations of up to 200uM. This test predicts that the invention will have positive inotropic effects in human cardiac muscle.
  • Predictive tests can also be performed on isolated hearts of animals. Testing intraventricular and arterial pressure in isolated dog hearts, under pre-load, is an animal model for human congestive heart disease, as is well known in the art. This is called Langendorff s method. It allows examination of therapeutic strategies to treat congestive heart disease. When dog hearts were so tested, LASSBio-294 treated hearts achieved a 50 % increase in intraventricular and aortic pressure compared to control or wash out after treatment. The electro-cardiogram (ECG) of isolated rat heart, without pre-load, showed no change after treatment with LASSBio-294. When LASSBio-294 was tested on intact, anesthetized dogs with normal cardiac function and no pre-load, there was no change in pressure after injection of LASSBio-294. These tests predict that the invention has utility as a medicament in the treatment of congestive heart failure (Curtis, M.J., (1998) Cardiovasc. Res. 39, 194-21, incorporated expressly by reference herein).
  • LASSBio-294 Another predictive test can be performed on isolated cardiac muscle fibers.
  • the mechanism of action of LASSBio-294 was determined by examining the sarcoplasmic reticulum of cardiac muscle fibers. Tests showed that treatment with LASSBio-294 increases uptake of Ca 2+ . Further tests demonstrated that LASSBio-294 increases the storage of Ca 2+ by the sarcoplasmic reticulum. Thus, the mechanism of action of LASSBio-294 is different from cardiac glycosides, which act by inhibiting active transport of Na + and K + . LASSBio-294 increases bioavailability by increasing the storage of calcium in the sarcoplasmic reticulum (SR); it does not change the sensitivity of muscle fibers to calcium. The tests were performed on human skeletal as well as rat cardiac muscle fibers.
  • LASSBio-294 will achieve its therapeutic effects in human cardiac and skeletal muscle (Weir, W.G. and Hess, P., ( ⁇ 984 )J. Gen. Physiol., 83, 395).
  • LASSBio-294 treated skeletal muscle fibers showed increased resistance to muscle fatigue compared to control fibers. Neuromuscular transmission was unaffected by LASSBio-294. Supportive tests were also successfully performed in amphibians. These tests predict a second therapeutic use of LASSBio-294 in the treatment of muscle fatigue (Albuquerque, E.X., Daly, J.W., Warnick, J.E. (1988) Ion Channels, 1, 95; Gallant, E.M., Godt, R.E., and Gronert, G.A.,. (1980) J. Pharmacol. Exp. Ther. 213, 91).
  • LASSBio-294 has very low toxic effects. This was tested in whole mouse, rat, and dog models (Greaves, P. (1998) Exp. Toxicol. Pathol. 50, 283). These models are commonly used to predict toxicity in humans (Chou, W.L., Robbie G., Chung, S.M., Wu, T.C., and Ma, C. (1998) Pharm. Res. 15, 1474). Major tissue types and organs were unaffected by LASSBio-294 given in doses greater than those which showed inotropic activity, and animal weights and blood cell counts were unaffected. The LD 50 in dogs is 1.5g/kg, an amount more then 1000 fold greater than the effective dose. These tests predict LASSBio-294 can be dosed in humans to achieve therapeutic plasma levels without significant risk of toxicity. Definitions
  • Calcium sensitizer An agent that increases the Ca 2+ and/or the amount of Ca 2+ available.
  • Congestive heart failure Heart failure in which the heart is unable to maintain an adequate circulation of blood in the bodily tissues or to pump out the venous blood returned to it by the veins.
  • LangendorfP s method The experimental method using perfiision of the isolated mammalian heart by carrying fluid under pressure into the sectioned aorta, and thus into the coronary system.
  • Muscle fatigue Temporary loss of power to respond induced in a muscle by continued stimulation. This symptom is found in patients with HIV infection, cancer, major injuries, sepsis, Crohn's disease, ulcerative colitis, chronic fatigue syndrome, and to some extent in over-trained athletes.
  • Positive inotropic agent an agent that strengthens the contractility of muscular tissue.
  • Pharmaceutically or therapeutically acceptable carrier a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
  • Safrole (4-allyl-l, 2-methyldioxybenzene) as starting material or reagent.
  • Safrole is the principal constituent of sassafras oil, from which it is readily isolated. It is also available from commercial sources.
  • LASSBio-294 is synthesized from safrole following the following scheme.
  • Toxicity a. Table 1 shows a histo-pathological table of rat tissues and organs examined after the animal had been treated with LASSB-294. The table shows that there were no pathological changes in the tissues and organs examined after LASSBio-294 injection.
  • Table 2 shows weights of mice in grams after injection with LASSBio294, compared to saline and solvent vehicle controls. Six rats were examined in each group, measurements were made after 1-15 days of treatment. The data show that there is no change in weight in any group during the test period.
  • mice were injected with LASSBio-294 and their blood examined for cell changes. Experimental groups received 2 or 10 mg/kg of drug.
  • Table shows no significant change in blood cell values for any group. Hematocrit levels, leucocytes and hemoglobin were not significantly changed by LASSBio-294 (Table 3). d. Table 4 lists blood chemistry analysis of mice treated with LASSBio-294. Experimental groups received 2 or 10 mg/kg of drug. Controls were saline and solvent. The data show no significant change in blood chemistry values for any group. The low values of glucose found could be due to an artifact of the technique, glucose being a byproduct of hemolysis. In male and female mice, subjected to long-term treatment with the compound, tests of hemoglobin, erythrocytes and blood biochemistry again showed values similar to control.
  • LASSBio-294 To test for possible systemic toxicological changes induced by LASSBio-294, animals were divided into four groups, control (saline), solvent (dimethylsulphoxide (DMSO)/polyethylene glycol (PEG), LASSBio-294 2 mg/kg, and LSSBio-294 10 mg/kg. Animals were injected daily for more than 14 days. The results show that LASSBio-294 at concentrations that produce significant positive inotropic effect on the heart muscle produces no significant histopathological changes in the organ and tissues studied. EXAMPLE 2
  • Isometric Tension in Cardiac Muscle a The test shown in Figure 2 measured the effect of LASSBio-294 on the isometric tension of papillary, atrial, and ventricular bundles of rat cardiac muscle. Chart traces are of muscle tension for each muscle group. All muscle groups show an increase in tension after 10 ⁇ M and 50 ⁇ M of LASSBio-294.
  • Figure 3 contains a graphic representation of the effect of LASSBio-294 on isometric tension of papillary, atrial, and ventricular bundles of rat cardiac muscle.
  • LASSBio-294 at concentrations of 0 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, and 500 ⁇ M was used. Isometric tension is expressed as a percent of control that was measured prior to addition of the test solution. Isometric tension increased for each concentration up to 200 ⁇ M.
  • Figure 3 shows the accumulated results of nine tests for each concentration.
  • the papillary muscle, and bundles of atrial and ventricular cells obtained from rat hearts were dissected and set in an aerated verticle chamber to enable recording of isometric tension.
  • LASSBio-294 was added to the chamber in a cumulative manner. Recordings made after allowing 5 min. for equilibration.
  • Isolated Hearts a. Recordings of electrocardiogram (ECG) of isolated rat heart are shown in Figure 4 which compare control, LASSBio-294 treatment, 10 uM, 50 ⁇ M and 100 ⁇ M and post- treatment wash. No treatment had any effect on ECG. These data indicates that LASSBio-294 does not cause abnormal ECG even at dose levels that show increased isometric tension.
  • Figure 5 illustrates a test of change in isometric tension induced by LASSBio-294 in isolated hearts under preload. Concentration of LASSBio-294 in the bathing solution is 50 ⁇ M, which is half of the highest concentration tested in the ECG tests described in Figure 4.
  • Hearts were treated under a pre-load of 1 g; control was compared to LASSBio- 294 and wash. LASSBio-294 treated hearts demonstrated an increase in isometric tension compared to control. This effect was lost in the wash out period.
  • the pre-load tension test is considered to be an animal model of congestive heart disease. Comparing the effect of drugs with this test is predictive of therapeutic effect of treatment in human congestive heart disease.
  • Figure 6 illustrates a test that measured pressure from the hearts of intact dogs during LASSBio-294 application. Recordings were made of left intraventricular pressure and arterial pressure. These measures show no change with LASSBio-294 application indicating that, in dogs without congestive heart failure, the compound had no significant inotropic effect.
  • Dogs were anesthetized and normally ventilated. Recordings were made before and after application of LASSBio-294, lmg kg "1 min "1 for 13 min.
  • FIG. 7 illustrates a test that demonstrates increased uptake of Ca 2+ by the sarcoplasmic reticulum (SR) of LASSBio-294 treated fibers after caffeine induced contraction.
  • SR sarcoplasmic reticulum
  • Figure 7 Graphic representation of the tests in Figure 7 showing the effect of LASSBio-294 on uptake of Ca 2+ by sarcoplasmic reticulum in isolated cardiac muscle with sarcolemma removed. Uptake follows contracture induced by caffeine measured as a function of loading time, shown in Figure 8.
  • Figure 9 illustrates the effect of LASSBio-294 on tension in isolated cardiac muscle with sarcolemma removed was measured.
  • FIG. 9 is a graphic representation of the effect of increasing concentrations of LASSBio-294 between 0 and 20 ⁇ M. Response to caffeine increases until lO ⁇ M LASSBio-294 level. d. This test demonstrates the effect of increasing concentrations of caffeine from 0.05 to 10 mM on contractile tension.
  • Figure 10 shows data comparing control to 100 ⁇ M
  • LASSBio-294 shows a graphic representation of data in Figure 10 showing isometric tension elicited by caffeine. Contraction of LASSBio-294 treated fibers is increased over control at all concentrations.
  • Figure 12 illustrates the effect of LASSBio-294 on sensitivity of cardiac fibers to calcium. Calcium levels can induce isometric twitch. At each level of pCa the isometric twitch response of fibers was greater in the presence of LASSBio-294 than in control.
  • Figure 13 provides a graphic representation of data in Figure 12.
  • Figure 14 illustrates that LASSBio-294 induces the release of Ca 2+ from the sarcoplasmic reticulum of isolated human skeletal muscle fibers with sarcolemmal membrane removed.
  • the histogram shows effect of LASSBio-294 at concentrations of 25 ⁇ M, 50 ⁇ M, and 100 ⁇ M on induced tension in human muscle fibers.
  • Isolated muscle fiber from the vastus lateralis was exposed to 0.5 mM of CaCl 2 to enable maximal muscle tension.
  • the contractile response to caffeine was observed after loading of the sarcoplasmic reticulum (SR) with a solution of pCa of 7.0 for 3 min., LASSBio-294 evoked a contraction in the human by liberating Ca from the SR.
  • SR sarcoplasmic reticulum
  • FIG. 15 illustrates the measurement of the effect of LASSBio-294 on force development in a single muscle fiber stimulated at different frequencies. Control is Ringer's solution without compound; following is stimulation after wash with plain Ringer's solution.
  • FIG. 16 The histogram in Figure 16 shows effect of 12.5 ⁇ M LASSBio-294 on fractional twitch tension with 10 Hz stimulation. T x /T 0 reflects twitch tension after bathing, T x , divided by initial tension in Ringer's solution alone, T 0 . Bars are labeled and are placed right to left in the histogram, in the order performed. c.
  • Figure 20 is a histogram of T x /T 0 , 12.5 uM LASSBio-294 compared to DMSO.
  • Figure 21 A shows the effect of two different doses of LASSBio-294 on fractional tension potentiation.
  • Figure 21B shows a histogram effect of 12.5 ⁇ M LASSBio-294 on twitch tension ratio, at 10 Hz, 30 Hz, 60 Hz, and 90 Hz.
  • Figure 22 illustrates a comparison of time course of twitch tension in control vs. LASSBio-294.
  • Figure 23 is a graphic representation of the relationship between fractional twitch tension and LASSBio-294 concentration from 0 to 100 ⁇ M.
  • Figure 24 shows time parameters measured during twitch tension time course. Table 5 gives the time parameters experimentally measured in vehicle control and LASSBio-294 12.5 ⁇ M.
  • Figure 25 is a graphic representation of effect of LASSBio-294 on time course of fatigue development, comparing Ringer's solution, vehicle control and LASSBio-294 12.5 ⁇ M. m.
  • Figure 26 shows data giving the time course of the index of fatigue development.
  • Figure 27 is a graphic representation of the time for tetanic force to decrease to 50% of original tetanic force comparing Ringer's solution to 12.5, 25, and 50 ⁇ M of LASSBio-294.
  • Figure 28 is a graphic representation of the time required tetanic force to decrease (fatigue) to 50% of the pre-fatigue tetanic force.
  • Figure 29 is a graphic representation of the time to recovery to 80% of the pre-fatigue tetanic force.
  • the tests in amphibians were performed in isolated single muscle cells freshly isolated from either the semitendinous or the tibialis anterior muscles of the frog, rana pipiens.
  • the isolated muscle cells were left resting for at least half an hour in Ringer's solution.
  • the fibers were then stimulated with single, low-voltage, electric shocks. If fibers gave brisk twitches and had no signs of membrane damage, they were used. Otherwise, they were discarded. Healthy fibers were transferred to the experimental chamber, which consisted of a 0.3 ml narrow channel where the solutions could be changed several times within five seconds.
  • One tendon of the fiber was gripped with a small clamp and the other tendon was attached to a hook of an Ekhart;s type force transducer.
  • the stimulating electrode consisted of platinum wires placed to each side of the fiber.
  • the muscle fibers were stretched 1.3 times their slack length to reach approximately an average sarcomere length of 1.6 ⁇ m.
  • the fibers were stimulated with single electric pulses of 0.5 msec duration and variable voltage. The voltage was increased until the threshold for contraction was reached. This voltage was then increased by 50% and the experimental protocol started.
  • a stimulating protocol was used consisting of a series of single twitches elicited every 3 sec. followed by different frequencies of tetanic stimulation of 10 Hz, 30 Hz, and 60 Hz. The fibers rested for three minutes between each tetanic stimulation. When the whole series of stimulations were repeated, the fibers rested for 10 min. between each series of twitches and tetanic stimulations. Fatigue was induced by repetitive cycles of electrical stimulation.
  • Each cycle consisted of a train of electric shocks delivered at 60 Hz for 0.8 sec followed by a single twitch after 2.2 sec and repeated every 4.15 sec.
  • a fatigue index at different times of stimulation by taking the ratio of maximum tetanic tension produced during every 3rd tetanus to the tension output in the 1st tetanus i.e. T n /T ⁇ .
  • the first series of tests were done with LASSBio-294 in normal Ringer and then in Ringer's solution plus LASSBio-294. Solutions: The Ringer's solution contained, in mM: NaCl, 115; KC1, 2.5; CaCl 2 , 1.8; MgCl 2,
  • a stock solution of 50 mM LASSBio-294 was dissolved in Ringer's solution to produce a 100 ⁇ M solution by diluting 40 ⁇ l of the original 50 mM LASSBio-294 in 20 ml of Ringer's solution. From this 100 ⁇ M 294 compound solution final dilutions were prepared; e.g. for a 12.5 ⁇ M LASSBio-294 solution, 1.25 ml of the 100 ⁇ M solution was dissolved in 10 ml of normal Ringer's solution.
  • Figure 30 illustrates a test to measure muscle tension stimulation, comparing
  • LASSBio-294 treated with vehicle control. LASSBio-294 has no effect on the neuromuscular junction.
  • Figure 31 illustrates a test to measure muscle tension after neural stimulation, comparing LASSBio-294 to vehicle control. LASSBio-294 has no effect on neuromuscular transmission.
  • the compound did not affect either Ca 2+ -ATPase or Na + /K + -ATPase extracted from heart muscle or gastrocnemius muscle of the rat, as well as binding was not affected by the compounds. Determination of phosphodiesterase direct effect was also negative.
  • Cyclic GMP as well as cyclic AMP, are degraded by phosphodiesterase isoenzymes (PDE), which have been classified into at least seven isoenzymes families, according to the nucleotide preferentially hydrolyzed and to
  • PDE 1 Ca -calmodulin dependent
  • PDE 2 cyclic GMP-stimulated
  • PDE 3 cyclic GNP-inhibited
  • PDE 4 cyclic AMP-specific PDE
  • PDE 5 cyclic GMP-specific PDE
  • LASSBio-294 100 ⁇ M relaxed noradrenaline-precontracted aortic rings (Fig. 32), although more slowly than the acetylcholine- and IBMX-induced relaxation. However, in endothelium-denuded aorta this relaxation was abolished, indicating an endothelial contribution to its effect (Fig. 33).
  • NOS nitric oxide synthase
  • Fig. 34 This treatment caused no difference in the relaxation induced by LASSBio-294 (Fig. 34), suggesting no direct role for NO.
  • LASSBio-294 relaxed intact aortic rings in a concentration-dependent manner, however its effect was less pronounced than either endothelium dependent and independent relaxation produced by acetylcholine and IBMX (a non-specific phosphodiesterase PDE inhibitor), respectively. Furthermore its effect was abolished by the removal of the endothelial cells. Delpy and Gouville (1996) recently reported that DMPPO, an inhibitor of PDE 5, also lost its effect after the removal of endothelium. As this relaxant effect could be due to the activation of 1-arginine/NO pathway we investigated the influence of the NOS inhibitor on LASSBio-294 effect.
  • 1-NAME treatment caused no difference in the relaxation elicited by LASSBio-294 indicating that basal vascular NO was not directly involved in this response, despite the endothelial dependence for the vaso-relaxant effect. Furthermore when the tissue was treated with 1-NAME plus indomethacin, the relaxant effect induced by LASSBio-294 was the same as observed in the presence of 1-NAME alone, discarding a direct contribution of PGI .
  • LASSBio-294 did not inhibit directly particulate PDE 3 and 4 iso-forms present in rabbit and rat heart, respectively. As a conclusion, the inotropic agent LASSBio-294 has vasodilator activity. A non-limiting explanation is that LASSBio 294 works by increasing cyclic GNP and/or cyclic AMP.
  • Aortic rings Male wistar rats (300-350 g) were anaesthetized with ether and killed by cervical dislocation. The thoracic aorta was quickly removed, placed in physiological solution, cleaned of fat and connective tissue, and cut into 3-mm rings. In some experiments endothelium was mechanically removed by gently rubbing inverted rings on a cotton surface moistened with physiological solution.
  • the rings were fixed in an organ bath chamber filled with physiological solution (composition (mM): NaCl 122, KC1 5, NaHCO 3 15, glucose 11.5, MgCl 2 1.25, CaCl 2 1.25 and KH 2 PO 4 1.25) aerated with O 2 /CO , maintained at 37°C, and left to equilibrate for 60 min during which the physiological solution was changed twice. All experiments were carried out under an initial tension of 20 mN, and the developed active tension was measured isometrically using a Grass Transducer (FT03). Data were acquired and analyzed by Chart 3.4/s software (MacLab, USA).
  • the contraction was induced by 1 ⁇ M Noradrenaline (NOR) and when it reached a plateau the relaxant drugs (or solvent - time-matched control) were added.
  • the rings contracted with 1 ⁇ M NOR that relaxed 40 - 50% in response to l ⁇ M acetylcholine were considered with intact endothelium.
  • LASSBio-294 and ODQ were dissolved in 100% dimethyl sulphoxide (DMSO).
  • Noradrenalin, 1-NAME, IBMX, methylene blue, acetylcholine and indomethacin were purchased from SIGMA (USA). All drugs but indomethacin were dissolved in water in the day of the experiment. Indomethacin was dissolved in 5% sodium carbonate.

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Abstract

L'invention concerne un composé représenté par la formule (I), dans laquelle R1 est sélectionné dans le groupe constitué par hydrogène, alkyle doté de 1 à 6 atomes de carbone, phényle non substitué et phényle substitué ; R2 est sélectionné dans le groupe constitué par H, alcène, phénol non substitué et phényle substitué ; et des sels pharmaceutiquement acceptables de ce composé qui possèdent des propriétés analogues à celles de la digitale. L'invention concerne en outre un nouveau procédé de synthèse de 3,4-méthylènedioxybenzoyl-2-thiénylhydrazone (LASSBio-294). LASSBio-294 a un effet positif inotrope sur les muscles cardiaque et squelettiques. L'invention est utile dans le traitement d'insuffisance cardiaque congestive et de fatigue musculaire. Elle ne comporte pas les effets toxiques observés chez des glycosides de digitale.
EP00941596A 1999-06-21 2000-06-21 Thienylhydrazone possedant des proprietes analogues a celles de la digitale (effets inotropes positifs) Withdrawn EP1532140A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14035299P 1999-06-21 1999-06-21
US140352P 1999-06-21
PCT/US2000/017024 WO2000078754A1 (fr) 1999-06-21 2000-06-21 Thienylhydrazone possedant des proprietes analogues a celles de la digitale (effets inotropes positifs)

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EP1532140A1 true EP1532140A1 (fr) 2005-05-25

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EP (1) EP1532140A1 (fr)
AU (1) AU5628700A (fr)
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Publication number Priority date Publication date Assignee Title
BRPI0403363A (pt) * 2004-08-20 2006-05-02 Univ Rio De Janeiro relaxantes musculares seletivos e composições farmacêuticas
BRPI0601885A (pt) * 2006-05-15 2008-03-25 Univ Rio De Janeiro composição farmacêutica antiinflamatória e analgésica contendo derivados n-acilidrazÈnicos do safrol, uso, e processo para sua preparação
EP2151239B1 (fr) * 2007-01-31 2012-04-25 Guangzhou Zhongwei Biotechnology Ltd Poudre de piperphentonamine hydrochlorique lyophilisée pour injections, son procédé de préparation et ses utilisations
CN103304561B (zh) * 2013-06-21 2016-06-01 天津药物研究院 一类具有抗血栓作用的化合物

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Publication number Priority date Publication date Assignee Title
US4410540A (en) * 1981-11-04 1983-10-18 Merrell Dow Pharmaceuticals Inc. Cardiotonic 4-aroylimidazolidin-2-ones
CA1266268A (fr) * 1985-10-15 1990-02-27 J. Martin Grisar Aroylthiazolones cardiotoniques

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Title
See references of WO0078754A1 *

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CA2384525A1 (fr) 2000-12-28

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