Patent Application
20130345159
A methanolic extract of Geum japonicum, denoted “EGJ,” has been shown to have activity in promoting regeneration of myocardium. Cheng et al., PLoS One 4(2): e4461 (2009). That activity was attributed to an EGJ component, a cardiac glycoside called “cardiogenin” (C36H58O11), which is (2α,3β,4α)-2,3,19,23-tetrahydroxy-urs-12-en-28-oic acid β-D-glucopyranosyl ester. The chemical structure ascribed to cardiogenin possesses 16 chiral centers, giving rise to the theoretical possibility of many stereoisomers.
In this context, Cheng et al. described a procedure in a manner that suggests the obtention of single stereoisomer of cardiogenin. There was insufficient detail provided, however, for the practicable isolation of a cardiogenin composition characterized by least 90% purity, and Cheng et al. did not themselves describe the purity of “isolated” cardiogenin. Pursuant to the Cheng methodology, therefore, it was unknown whether and to what extent impurities existed in the resultant composition.
Against this background of the conventional technology, the present inventors discovered that “cardiogenin” extracted and purified from EGJ, using the method described by Cheng et al. (2009), actually comprises two, closely eluting isomers of the same mass. Accordingly, the inventors developed an approach for separating the previously unrecognized cardiogenin major isomer, which was found to be active, from the minor isomer, which is inactive. Via this approach, the inventors succeeded in extracting from EGJ a cardiogenin major-isomer composition that is substantially free of the minor isomer (hereafter, “isolated cardiogenin major isomer”).
Thus, the inventive methodology provides an isolated cardiogenin major isomer having at least 98% (a/a) HPLC purity at 210 nm (hereafter, “substantial purity”). Substantial purity would be achieved by crystallizing the isolated cardiogenin major isomer to separate the impurities. In this context, “a/a” denotes the percent area of a peak of interest in a chromatogram to the total area of all other peaks in the chromatogram at a specific wavelength. The a/a value serves here as the unit measure of optical purity for cardiogenin isomer.
The present invention comprehends the major and the minor cardiogenin isomers, the corresponding aglycone thereof, and related compositions, as well as methodology for making and using them. In accordance with one of its aspects, therefore, the invention provides isolated cardiogenin major isomer, which can be described, for example, in terms of the formula:
In accordance with another of its aspects, the present invention provides aglycone of the isolated cardiogenin major isomer with at least 92% purity by HPLC. The aglycone can be described, for example, in terms of the formula:
The cardiogenin major isomer preferably is present in substantial purity. An illustrative embodiment of this state is the compound with a HPLC purity of 98.97% (a/a) at 210 nm. In a further embodiment, a pharmaceutical composition is provided that comprises the isolated cardiogenin major isomer and/or its corresponding aglycone, as well as a pharmaceutically acceptable carrier.
The invention also provides a methodology for isolating the major isomer of cardiogenin. The inventive methodology comprises (A) obtaining an extract from the methanol extract of Geum japonicum and (B) subjecting the extract to chiral phase chromatography or supercritical fluid chromatography, whereby the major isomer is obtainable in isolated form. An embodiment involving chiral phase chromatography can entail, for instance, the use of a Chiralpak® IC™ column, a product of Chiral Technologies, Inc. (West Chester, Pa.). The inventive methodology for isolating the cardiogenin major isomer also may comprise crystallizing the composition.
Another aspect the invention relates to an improvement on the chromatographic procedures of Cheng et al. (2009), comprising (A) precipitating and filtering an methanolic/water solution of Geum japonicum to remove of unwanted solids, (B) phase-separative extracting the methanol/water solution with dichloromethane and tert-butyl methyl ether (TBME), and then (C) extracting with n-butanol. The improved chromatographic procedures of the invention also may comprise subjecting a composition comprised of the major isomer of cardiogenin to low-pressure adsorption chromatography (Diaion HP-20 and silica gel), using an optimized mass ratio of resin to material load (typically, about 15:1) and a step gradient of methanol/water, followed by low pressure silica gel chromatography using an optimized mass ratio of resin to material load (about 20:1) and a step gradient of dichloromethane/methanol. In another embodiment, the improvement over Cheng et al. (2009) further comprises subjecting a major isomer-containing composition to high-pressure, reverse-phase chromatography (HPRC), employing aqueous buffer and methanol mobile phases in a gradient program. In this regard, the HPRC can involve using a Luna® C18 (2) column, a product of Phenomenex, Inc. (Torrance, Calif.).
Myocardial infarction due to coronary artery disease is one of the leading causes of premature death. One solution is to replace the infarcted heart tissue with regenerated myocardium from endogenous progenitor pools or exogenously introduced stem cells. A methanolic extract of Geum japonicum was shown by Cheng et al. to have such potential.
As noted above, although Cheng et al. described their procedure in a manner suggesting the obtention of a single stereoisomer of cardiogenin, there was insufficient detail for the practicable isolation of a cardiogenin composition characterized by at least 95-98% (a/a) HPLC purity at 210 nm, a typical regulatory expectation for modern, small-molecule pharmaceutical substances. Moreover, Cheng et al. did not describe the purity of “isolated” cardiogenin. Pursuant to the Cheng methodology, it was unknown whether impurities existed in the resultant composition and, if they did exist, the extent of such impurities.
During an evaluation of the conventional method of extracting cardiogenin from EGJ, an HPLC assay method was developed to analyze the purity of the extracted cardiogenin, with an expectation that the extracted composition would comprise a single stereoisomer of cardiogenin. Surprisingly, using a HPLC assay method that was improved over that employed by Cheng et al., the present inventors observed that the conventional method of extracting cardiogenin instead yields a mixture of two closely eluting isomers with identical mass. Subsequently, both LC-MS and 1H-NMR spectra analyses were performed, each method independently confirmed the discovery that the “cardiogenin” produced via Cheng's method actually comprises two cardiogenin isomers. The LC-MS results are shown in
The inventors also found find that, following the Cheng methodology, the HPLC purity of the isolated cardiogenin major isomer was only about 75.73% (a/a) at 210 nm, with about 16.87% of the HPLC impurities attributable to the minor isomer. Moreover, the isolated cardiogenin major isomer was found to be biologically active, the impurities to be biologically inactive.
To obtain isolated cardiogenin major isomer with substantial purity, a method was developed to remove impurities, including the previously unrecognized minor isomer of cardiogenin. The extraction procedure taught by Cheng et al. (2009) utilizes chloroform, ethyl acetate and finally n-butanol phase separative extraction against water. The n-butanol phase has been retained for further purification and other organic phases were discarded. It has been found that considerable loss of cardiogenin occurs with the ethyl acetate extraction, and the inventors have determined that chloroform cannot be used on an industrial manufacturing scale, given toxicity risks.
Accordingly, an optimization of the extraction process was undertaken. First, the EGJ extraction process was improved by introduction of a methanol re-slurry of EGJ, followed by filtration, concentration and dilution with water and a second Celite-aided filtration. These steps remove from the EGJ extract approximately 50% solids, which are undesired materials, and help to reduce subsequent emulsion formation upon the ensuing organic phase separation. The filter cake is devoid of cardiogenin when the filtration solids are analyzed. Next chloroform extraction of the aqueous/methanol filtrate was replaced with dichloromethane to avoid using highly toxic chloroform, a solvent that presents both an operator safety risk and an environmental risk. An extraction with TBME was added, since this extraction had been shown to remove impurities that elute during HPLC purification at retention times close to that of cardiogenin, without appreciably removing cardiogenin from the methanol/water filtrate. The aqueous methanolic phase was converted io a saturated sodium chloride solution and extracted with n-butanol, which removed cardiogenin from the aqueous/methanol phase with good overall recovery of cardiogenin.
The chromatographic separations taught by Cheng et al. (2009) haven been incorporated, including low-pressure Diaion HP-20 adsorption chromatography, followed by low-pressure normal phase silica and finally high pressure reverse phase chromatography, to increase the overall purity of the isomeric mixture. The Diaion and normal-phase silica chromatography are conducted generally as taught by Cheng et al., although conditions are optimized. In particular, Diaion chromatography has been optimized by definition of the optimal mass ratio of resin to material load (15:1) and by the use of a step gradient starting with 20% MeOH/water, increasing in 10% increments to 80% MeOH/water. Silica gel chromatography has been optimized by definition of the optimal mass ratio of resin to material load (20:1), with replacement of chloroform with dichloromethane in the mobile phase, for operator and environmental safety considerations described above, and the use of a stepwise gradient of dichloromethane/methanol ranging from dichloromethane/methanol 90%:10% to dichloromethane/methanol 80%:20%.
The process has been improved further by the introduction of either chiral phase chromatography or supercritical fluid chromatography, after the high pressure reverse phase chromatography, to allow separation of the two closely eluting enantiomers of cardiogenin. Finally the separated major isomer of cardiogenin was crystallized from methanol/water to increase its purity, removing low level impurities seen throughout the elution profile of the HPLC purity method. In accordance with the method, the invention provides the major isomer of cardiogenin in at least 98% HPLC purity with an overall yield from EGJ to cardiogenin exceeding that reported by Cheng et al.
In this regard, the category of suitable reverse phase chromatography techniques encompasses any chromatographic method that uses a non-polar stationary phase. Polar compounds are eluted first while non-polar compounds are retained. The column can be octadecyl carbon chain (C18)-bonded silica. The eluent can be a mixture of ACN and 20 mM NH4OAc (pH=7). The sample then is dissolved at 47 g/L in MeOH:Buffer=50:50 (v:v). The temperature for the elution can be room temperature. A mobile phase gradient transitions in a linear fashion from 80% 20 mM ammonium acetate/acetonitrile to 65% 20 mM ammonium acetate/acetonitrile. The flow rate of the column can be 15 mL/min. on an ID=2 cm column. The presence of cardiogenin can be detected by HPLC at 210 nm. As can be calculated from the data shown in
The two cardiogenin isomers are separated by chiral phase chromatography. In this regards, category of chiral phase chromatography techniques encompasses any column chromatography in which the stationary phase contains a single enantiomer of a chiral compound, rather than being achiral. Chiral stationary phase selection is critical to achieve adequate separation. The two isomers of cardiogenin elute from the column at different times because of their transiently different solubility characteristics when bound to the chiral column stationary phase. The column can be a Chiralpak® IC™ column. The eluent can be a mixture of IPA:MTBE=50:50 (v:v). The sample can be dissolved in IPA/MTBA=50:50 (v:v) or neat IPA. The temperature for the elution can be room temperature. The flow rate of the column can be 1 mL/min on an analytical column or higher flow rate on semipreparative columns using an isocratic mobile phase of IPA:MTBE=50:50 (v:v). The presence of cardiogenin can be detected by HPLC at 210 nm.
The two cardiogenin isomers also can be separated by supercritical fluid chromatography, as described, for example, by Anton & Berger, S
The purity of the isolated cardiogenin major isomer can be increased further by crystallization. In this regard, the “crystallization” category encompasses any method for forming solid crystals of the cardiogenin major isomer, typically by precipitating from a solution, melt, or gas. In a preferred embodiment, the cardiogenin major isomer is dissolved in 10 volumes of methanol at 40° C. Then 40 volumes of water are added slowly, at the same temperature, over a period of about 50 minutes, during which crystallization of the cardiogenin major isomer occurs.
Pursuant to the above-described methodology, cardiogenin major isomer can be isolated from EGJ with a HPLC purity of 98.97% (a/a) at 210 nm and potency by NMR assay of 95.50% (w/w). The isolated cardiogenin major isomer is a stable compound when stored in either MTBE/IPA=50:50 or as solid. It also is resistant to thermal stress at 40° C.
The structure of the cardiogenin major isomer is depicted below:
The corresponding aglycone can be converted from the isolated cardiogenin major isomer, a polycyclic glycoside, by hydrolysis of the ester linkage that connects the sugar moiety to the polycyclic core of the molecule. Such hydrolysis can be accomplished by either of two approaches:
(a) Acid-catalyzed hydrolysis, which involves the use of a dilute aqueous solution of a mineral acid to effect cleavage of the ester. The resultant products are the sugar and the free carboxylic acid form of cardiogenin.
(b) Base-catalyzed hydrolysis (saponification), in which a base such as sodium hydroxide or potassium hydroxide is used to hydrolyze the ester. Typically, such hydrolysis is carried out in an aqueous medium, or a solvent system is employed that is a mixture of water and an appropriate alcohol. The product obtained from base-catalyzed hydrolysis is the salt form of the carboxylic acid group of cardiogenin. This salt can readily be converted to free acid, using a mineral acid.
The cardiogenin major isomer and the corresponding aglycone can be used to induce or to enhance cardiogenic differentiation, both in vitro and in vivo. This utility is evidenced by the fact that MSCs cultured in the presence of a composition comprising the cardiogenin major isomer or its aglycone exhibit substantially enhanced differentiation into cardiomyocytes. In addition, beating cardiomyocytes differentiate from MSCs when the latter are co-cultured with cardiomyocytes in the presence of the cardiogenin major isomer or its aglycone.
The isolated cardiogenin major isomer and/or its corresponding aglycone can be administered, alone or with other compounds having similar or different biological activities. For instance, the compounds and pharmaceutical compositions of the invention may be administered in a combination therapy, i.e., either simultaneously in single or separate dosage forms or in separate dosage forms within hours or days of each other. Examples of such combination therapies include administering the compound of cardiogenin major isomer and/or its corresponding aglycone, with other agents used to treat stroke, skeletal muscle degeneration, wound healing, or cardiac problems.
In one embodiment, therefore, the invention provides a pharmaceutical composition comprising the compound of cardiogenin major isomer, the hydrolytic free acid product thereof (i.e., aglycone), or a pharmaceutically acceptable salt of the free acid, as well as a solvate, tautomer, polymorph, hydrate, structural derivative or prodrug thereof, in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavor imparting agents, absorption enhancers, complexing agents, solubilizing agents, wetting agents and surfactants.
In one embodiment, the pharmaceutical composition comprises a compound of cardiogenin major isomer or a pharmaceutically acceptable salt, solvates, tautomers, polymorphs, hydrates, structural derivative or prodrug thereof, and a pharmaceutically acceptable carrier.
In another embodiment, the pharmaceutical composition comprises an aglycone of cardiogenin major isomer or a pharmaceutically acceptable salt, solvates, tautomers, polymorphs, hydrates, structural derivative or prodrug thereof, and a pharmaceutically acceptable carrier.
The inventive compositions can be administered orally, parenterally, by inhalation or spray, percutaneously, intravaginally, or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intrathecal, intraventricular, peritoneal, intracardiac injection or infusion techniques as well as via direct injection into any of numerous additional tissues or organs.
Inventive compositions suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the inventive compounds contain one or more agents selected from the group consisting of sweetening agents, solubilizers, dispersing agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations of the isomer.
For tablet compositions, the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, carboxymethylcellulose, hydroxypropyl methylcellulose, mannitol, polyvinylpyrolidone, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For instance, a time-delay material such as glyceryl monostearate or glyceryl distearate may be employed. Additional tablet formulations that afford slow leaching of the active ingredient can be used to provide sustained release, including the use of hydrogels, osmotic pump tablets, and wax matrices.
Formulations for oral use may also be presented as hard or soft gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, lactose, mannitol, methylcellulose or derivatives thereof, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
For aqueous suspensions the inventive compound is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.
Oral suspensions can also contain dispersing or wetting agents, such as lecithin, or the condensation product of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or the product of ethylene oxide with long chain aliphatic alcohols, such as, heptadecaethyleneoxycetanol, or compounds such as polyoxyethylene sorbitol monooleate, or polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, e.g., ethyl or n-propyl p-hydroxybenzoate, as well as one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, also may be present.
Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil, sesame, peanut or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents include without limitation, naturally-occurring gums, for example gum acacia or gum tragacanth, other naturally-occurring compounds, for example, soy bean, lecithin, Tweens, and esters or partial esters derived from fatty acids and hexitol, anhydrides, sorbitan monoleate and polyoxyethylene sorbitan monoleate. The emulsions also may contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The collected material was dried and percolated with methanol at room temperature three times, for 6 days each time. The EGJ methanol extract was dried under reduced pressure using a spray drying procedure to yield a powder residue. 500 g EGJ extract was stirred in 2.5 L methanol at Ti=41° C. for 1 h. After slow cooling to room temperature, the suspension was filtered off and rinsed with 200 ml methanol. The filter cake was resuspended in 1.5 L methanol at Ti=41° C. for 1 h and stirred for an additional 20 hours at room temperature. After filtration, the filter cake was rinsed with 750 ml methanol. The combined methanol filtrates were concentrated to yield 280 g crude #1.
Crude #1 was stirred in 1 L of TBME plus 1.2 L of water, resulting in a thick emulsion. The thick emulsion was diluted with 0.5 L of methanol and filtered, the filter cake was rinsed intensively with 0.5 L methanol and the combined filtrates (3.2 L) were concentrated to a final volume of 2 L. At this point a fine aqueous methanolic suspension had formed which could be filtered over a paper filter without pressure to furnish a clear dark, homogenous solution. The aqueous methanolic solution was extracted 3 times with 0.3 L of dichloromethane. The aqueous methanolic solution was then extracted 5 times with 0.3 L TBME portions. The remaining aqueous methanolic layer was diluted with 0.9 L n-butanol, followed by 0.8 L of water, to result in good phase separation.1 The aqueous layer then was washed three times with 0.3 L of n-butanol. The combined n-butanol layers (approximately 3.5 L) were washed with 0.5 L brine furnishing 1.4 L of aqueous layer and 2.5 L of n-butanol layer. The n-butanol was concentrated at the rotary evaporator removing approximately 0.5 L of solvents. The remaining n-butanol layer (approximately 2 L) was extracted twice with 0.3 L of brine. Complete concentration of the n-butanol layer eventually furnished 36.4 g of crude #2. 1 Alternatively, the aqueous methanolic suspension can be filtered with the aid of Celite with cake washed. The aqueous methanolic phase is next extracted 3 times with 0.3-0.6 L of dichloromethane and 5 times with 0.3-0.6 L TBME portions. The aqueous methanolic phase is rendered into a sodium chloride saturated solution and finally extracted with n-butanol. This process reduces emulsion formation during the DCM extraction and avoids need to concentrate aqueous methanolic phase in an attempt to remove methanol, avoiding severe foaming.
An amount of 545 g dry HP-20 resin (approximately 15 mass equivalents dry resin per crude #2) was slurried in methanol, transferred to the column (7.5 cm×21 cm=927 ml column volume), and exchange for 20% methanol/water. Crude #2 was resuspended in approximately 1-1.5 vol. of 20% methanol/water and applied on the column. It was eluted with increasing concentration of methanol in water (10% step). For the 20-30% methanol/water steps, 2 fractions of 2 L were taken; for the 40-70% methanol/water steps, four 1 L fractions were taken.
The fractions were analyzed by HPLC. Most cardiogenin content are in fraction 9-17. Fractions 9 and 10 were pooled together as pool-1; fractions 11-17 were pooled as pool-2; and fractions 1-8 and 18-20 were discarded. After concentration to dryness, pool-1 yielded 3.17 g solids containing approximately 370 mg of cardiogenin, while pool-2 yielded 5.48 g solids containing approximately 1.2 g of cardiogenin.
Pool-2 solids (5.4 g) were slurried in 30 ml starting eluent DCM/methanol 90:10 to prepare the feed. Separation was performed on 100 g silica gel (approximately 20 mass equivalents) equilibrated with DCM/methanol 90:10 (v:v). After allowing for 150 ml forerun, fractions were taken in 50 ml aliquots until fraction 32, then the eluent was changed for DC/methanol 85:15 and fractions of 100 ml size were taken. At fraction 42 the eluent was changed for DCM/methanol 80:20.
Fractions 38 and 39 were pooled together as pool-11; fractions 40-44 were pooled as pool-22; and fractions 45-51 were pooled as pool-33. After concentration to dryness, pool-11 yielded 0.11 g solids containing approximately 21 mg of cardiogenin (18.9% w/w); pool-22 yielded 0.67 g solids containing approximately 391 mg of cardiogenin (58.4% w/w); and pool-33 yielded 1.36 g solids containing approximately 775 mg of cardiogenin (57.0% w/w).
To increase the overall purity of cardiogenin, reserve phase separation was performed using the following parameter:
The isolation procedure consisted of evaporating the ACN at the rotary evaporator at 40° C. and reduced pressure and subsequently lyophilizing the remaining solution. The isolated foam was re-dissolved in ACN/water approximately 1:2 (v:v) and lyophilized again to remove residual traces of NH4OAc. Pool-22 and pool-33 were combined and separated in 60 runs and yielded 1.29 g of white foam. As shown in
To separate the two isomers in the 1.29 g mixture, chiral phase separation was performed using the following parameter:
Fractions 3 and 4 comprises primarily the major isomer of cardiogenin, while fraction 1 and 2 comprise primarily the minor isomer of cardiogenin. Fractions 2 and 3 were evaporated to dryness and re-processed with the same method to ensure a maximum yield. Fractions 1 and 2 and fractions 3 and 4 are combined, respectively, and worked up. The workup consisted again of evaporation to dryness followed by a lyophilization step after dissolution in water:ACN=75:25 (v:v). The workup gave 900 mg of white powder for the major isomer (96.49% a/a), and 180 mg of white powder for minor isomer.
Stress tests were performed to determine whether the two cardiogenin isomers separated from each other are stable and not converting into each other. In particular, the two cardiogenin isomers were stressed by storage in MTBE/IPA=50:50 or as solids at 40° C. (the major isomer in solution and solid; the minor isomer only in solution) for 24 hours.
All single impurity peaks of the cardiogenin major isomer and also the ratio of the two isomers were identical within the accuracy of the measurement. The same picture was obtained for the cardiogenin minor isomer. No changes were found in the composition within the accuracy of the method. It can be concluded that the two cardiogenin isomers are stable compounds, which resist thermal stress well.
Isolated cardiogenin major isomer as white powder (900 mg) was stirred in 10 volumes of methanol at To=40° C. until a clear solution was obtained. Water (40 volumes) was added slowly, within 50 minutes, at To=40° C. After about 6 drops of water, crystallization started. After complete addition the thick white suspension was cooled to room temperature and filtered, and residual solids were flushed from the flask with small amounts of mother liquor. The filter cake was washed with methanol/water (1:4) and dried at the rotary evaporator to furnish 756 mg cardiogenin as white solid crystal.
The purity of the isolated cardiogenin major isomer after crystallization is 98.97% a/a, as shown by HPLC at 210 nm (see
The isolated cardiogenin major isomer was subjected to saponification in MeOH:H2O 1:1, using excess NaOH, as shown in
Tested for activity in inducing cardiogenic differentiation of MSCs were five compounds: the isolated cardiogenin major isomer (HUYA-1), the isolated cardiogenin minor isomer (HUYA-2), the aglycone of the cardiogenin minor isomer (HUYA-3), the aglycone of the cardiogenin major isomer (HUYA-4), and the cardiogenin composition prepared according to the method of Cheng et al., 2009 (Car). In
Testing was performed according to the procedures described in the following: The tibias/femur bones of rats were removed and the BM was flushed out of the bones with alpha IMDM culture medium. The BM was mixed well and centrifuged at 1,500 rpm for 5 minutes. The cell pellet was suspended with 3 ml culture medium, and the forming cell suspension was carefully put on 4 ml Ficoll solution, to minimize disturbance, and then was centrifuged at 200 rpm for 30 minutes. The second layer was transferred into a tube and washed twice with PBS to remove Ficoll (1,200 rpm for 5 minutes). The resulting cell pellet was resuspended in IMDM culture medium containing 10% heat-inactivated FBS (GIBCO) and 1% penicillin/streptomycin antibiotic mixture, and this was used for the tests. Non-adherent cells were discarded after 24 hours culturing. The adherent cells were cultured by changing medium every 3 days. The cells became nearly confluent after 14 days culture. To activate the cardiogenic morphology transition, the MSCs were cultured for 7 days in the presence of the HUYA-1, HUYA-2, HUYA-3, HUYA-4 or Car (10 μg/ml IMDM culture medium), respectively. The treating period-dependent morphological transition was evaluated, on a time-lapse basis, with a phase contrast microscope.
After 3- or 7-day treatment of the MSCs in culture with HUYA-1, HUYA-2, HUYA-3, HUYA-4 or Car (10 μg/ml IMDM culture medium), respectively, fluorescent immunocytochemistry was performed, using antibodies specific to early cardiogenic differentiation factor 2 (MEF2a) at 3 days post-treatment and the contractile protein myosin heavy chain beta (MHC beta) at 7 days post-treatment, in order to demonstrate the cardiogenic differentiation of the treated MSCs in vitro. The method for the fluorescent-immunostaining is summarize briefly: The cultured cells were fixed with 4% paraformaldehyde in PBS for 15 minutes and permeabilized with 0.5% Triton X-100 for 15 minutes. Dilution of antibodies was as follows: rabbit polyclonal antibodies specific to rat MEF2a (1:500) and mouse monoclonal antibodies specific to MHC (1:500) (both antibodies from Abcam®). Secondary antibodies were goat anti-mouse and rabbit anti-IgG antibodies conjugated with fluorophore (FITC 495/528 and Cy5 650/667, products of Abcam®), respectively. The nuclei were stained with DAPI. Examined by fluorescent microscopy were the cardiogenic differentiation-associated morphological transition and specific marker protein expression of the cultured MSCs.
Bone marrow GFP-MSCs were isolated from the tibias/femur bones of GFP-transgenic mice. The GFP-MSCs were co-cultured with the cardiac myocytes isolated from neonatal SD rats in the presence of HUYA-1 (10 μg/ml IMDM culture medium) to mimic the cardiac micro-environment. The cultures were investigated daily, under a fluorescent microscope, to identify beating GFP-positive cells and to gauge the morphological transition of the cultured GFP-MSCs.
As shown in
This application claims priority to U.S. provisional application No. 61/426,929, filed Dec. 23, 2010, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/066469 | 12/21/2011 | WO | 00 | 9/11/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61426929 | Dec 2010 | US |
