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WO1988009335A1 - Formes polymorphes de l'inosine, procedes de preparation et d'utilisation - Google Patents

Formes polymorphes de l'inosine, procedes de preparation et d'utilisation Download PDF

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Publication number
WO1988009335A1
WO1988009335A1 PCT/US1988/001468 US8801468W WO8809335A1 WO 1988009335 A1 WO1988009335 A1 WO 1988009335A1 US 8801468 W US8801468 W US 8801468W WO 8809335 A1 WO8809335 A1 WO 8809335A1
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Prior art keywords
inosine
solution
period
standard
water
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PCT/US1988/001468
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English (en)
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Paul Gordon
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Paul Gordon
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals

Definitions

  • Crystal polymorphism The occurrence of several crystalline forms of the same compound is called crystal polymorphism. Crystal polymorphs are chemically identical but differ in their crystalline structure and physical-chemical properties.
  • inosine crystal polymorph In addition to the anhydrous form of inosine, a second inosine crystal polymorph is known which is the dihydrate form. This form of inosine crystallizes slowly from water in long rectangular plates. The dihydrate form has a melting point of 90 C.
  • Agafonov teaches that two known forms of-prednisolone which are produced by recrystallization from two different organic solvents are crystal polymorphs which have different physical-chemical properties, including different rates of dissolution and different solubilities. Agafonov further teaches that solutions of these two crystal polymorphs of prednisolone in ethanol exhibit different optical rotary dispersion (ORD) spectra.
  • ORD optical rotary dispersion
  • Leonidov Russian Journal of Physical Chemistry, 59, 760 (1985) (hereinafter Leonidov) teaches that crystal polymorphs of certain organic compounds (5,5-diethylbarbi- turic acid, p-aminobenzenesulphanilamide,. prednisolone, caffeine and L-camphor) exhibit different indices of refraction and volumes of optical indicatrix when they are put into solution in certain organic solvents (chloro- form, ethanol and dimethyl formamide) .
  • the differences in- these two optical properties reported in Leonidov are extremely small (about 0.01-0.07% variation for the index of refraction and 0.04-0.21% variation for the volume of indicatrix) .
  • Leonidov suggests that the prin ⁇ ciple that polymorphic modifications of a compound differ in crystal structure but are identical when they are put into solution may not be applicable afterall to organic substances and that the reported differences in biological activity of some pharmaceuticals could be explained by the persistence of the different crystal polymorphic modifications of the compounds in the solution state.
  • a method of preparing a crystal poly ⁇ morph of inosine comprising: providing a solvent; adding to the solvent at least about three grams of inosine per about 100 milliliters of solvent; heating the solvent and the inosine at a predetermined rate to a temperature sufficient to cause the inosine to go into solution and to overcome the energy barriers which prevent the conversion of the inosine to another polymorphic configuration; cooling the solution at a predetermined rate for a predetermined period of time; and precipitating the crystal polymorph of inosine.
  • the cooling procedure of the method may be accomplished in a single step or may comprise several steps such as: cooling the solution at a predetermined rate to a second predetermined temperature; maintaining the temperature of the solution at this second tempera ⁇ ture for a predetermined period of time; and further cooling the solution at a predetermined rate for a pre ⁇ determined period of time.
  • the precipitation may be accomplished by conventional techniques.
  • Crystal forma ⁇ tion may also be accomplished by freezing the cooled solution and completely lyophilizing the frozen solu ⁇ tion. Water is the preferred solvent for use in this process.
  • crystal polymorphs produced by this process are also part of the invention.
  • Particularly pre ⁇ ferred are the crystal polymorphs of inosine which are formed when water is the solvent used in the process.
  • These crystal polymorphs are characterized in that they are anhydrous and have x-ray diffraction patterns and solubilities in water and methanol that are different than those of standard inosine.
  • solute polymorphs of inosine exhibit different physical-chemical properties, compared one to the other and to the solute form of standard inosine, which reflect their different configurations.
  • the solute polymorphs of the invention also exhibit dramatically different biological activities compared to standard inosine.
  • a method of preparing a solute poly ⁇ morph of inosine comprising: providing a solvent; adding to the solvent at least about three grams of inosine per about 100 milliliters of solvent; heating the solvent and the inosine at a predetermined rate to a temperature sufficient to cause the inosine to go into solution and to overcome the energy barriers which prevent the conversion of the inosine to another polymorphic configuration; and cooling the solution at a predetermined rate for a predetermined period of time.
  • the cooling procedure of the method may be accomplished in a single step or may comprise several steps such as: cooling the solution at a predetermined rate to a second predetermined temperature; maintaining the solution at this second temperature for a predeter ⁇ mined period of time; and further cooling the solution at a predetermined rate for a predetermined period of tim .
  • a solute polymorph of inosine may also be prepared by dissolving the crystal polymorphs of the invention in a solvent.
  • Particularly preferred are pharmaceutically-acceptable solvents.
  • the term “conformation” refers to differences in the structure-in-space of monomeric inosine units, such as the conversion of the ribose ring from the chair to the boat form.
  • configuration will be used herein ' to mean differences in conformation combined with differences in patterns of association between the monomeric inosine units.
  • Inosine will be used herein to refer to all forms of inosine, including the known crystal polymorphs of inosine and the novel crystal and solute polymorphs of the invention. However, Applicant does not intend the known crystal polymorphs of inosine to be a part of the invention, and they are specifically disclaimed.
  • Figure 1 shows the x-ray diffraction pattern of standard inosine.
  • Figure 2 shows the x-ray diffraction pattern of inosine crystal polymorph GR-SP98-15.
  • Figure 3 shows the x-ray diffraction pattern of inosine crystal polymorph GR-SP98-26.
  • Figure 4 shows the high-dilution-induced "blue shift" of the ultraviolet absorbance of standard inosine and inosine polymorph GR-SP98-15.
  • Figure 5 shows the ORD spectra of standard inosine and inosine solute polymorph GR-SP98-15.
  • Figure 6 is a graph showing the suppression of chy otrypsin footpad edema caused by standard inosine and inosine polymorph GR-SP98-15.
  • Figure 7 is a graph showing the suppression of chymotrypsin footpad edema caused by standard inosine and inosine polymorphs GR-SP98-15, GR-SP98-22 and GR-SP98-26.
  • Figure 8 is a graph showing the suppression of chymotrypsin footpad edema caused by standard inosine and inosine polymorphs GR-SP98-26, GR-SP98-28, GR-SP98-38, GR-SP98-39 and GR-SP98-66.
  • Figure 9 is a graph showing the suppression by chymotrypsin footpad edema caused by standard inosine and inosine polymorphs GR-SP98-15 and GR-SP98-26.
  • Figure 10 is a graph showing the suppression of trypsin footpad edema caused by standard inosine and inosine polymorph GR-SP98-15.
  • Solvent The kind of bonds that the solvent forms with the inosine prior to formation of the polymorphs according to the methods of the invention is an Important factor in inosine solute and crystal polymorph formation.
  • the use of polar solvents versus aliphatic solvents in practicing the invention leads to the formation of different polymorphic configurations of inosine. Further, the use of one polar solvent versus another leads to the formation of different polymorphic configurations of inosine depending on the intensity of proton donation and acceptance of the various polar solvents.
  • the concentration of the inosine used is also critical to the formation of the novel inosine solute and crystal polymorphs of the in ⁇ vention. For instance, when a 1% solution of standard inosine and a 3% solution of standard inosine are treated identically, the treatment of the 1% solution does not result in the formation of an inosine having a polymorphic configuration different than that of standard inosine, whereas treatment of the 3% solution does.
  • Example 1 Ten grams of standard inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask suit ⁇ able for use on a VirTis lyophilizer. Next, 300 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. " The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the- flask reached 90°C thirty-eight minutes after heating began.
  • the flask was removed from the Fisher Thermix heater- stirrer and was cooled, at room temperature, with stir ⁇ ring,, so that the temperature of the flask reached 45- ⁇ C thirty-two minutes after the flask was removed from the heater-stirrer.
  • the cooled solution was block frozen in a -40°C freezer overnight (8-16 hours) and lyophilized on a VirTis lyophilizer (model Freezemobile 12) for 120 hours, at which time the frozen reaction mixture was completely lyophilized.
  • the resultant dry white crystal ⁇ line powder was harvested, ground with mortar and pestle and stored at 25°C. This inosine crystal polymorph will hereinafter referred to as GR-SP98-11.
  • Example 2 Ten grams of standard inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask. Next, 300 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the flask reached 91°C thirty-five minutes after heat ⁇ ing began. The heater setting was then reduced to number 2, and the heater was maintained at this setting so that the temperature of the flask dropped to 75°C by 55 minutes after heating initially began. Next, the heater setting was increased to number 4.3.
  • the heater setting was maintained at number 4.3 for five minutes after which time the heater was turned off, and the flask was removed from the heater-stirrer and was cooled at room temperature, with stirring, so that the temper- ature of the flask reached 45°C forty-five minutes after the flask was removed from the heater-stirrer.
  • Example 2 The cooled solution was block frozen in a -40°C freezer overnight and was then lyophilized as described in Example 1.
  • the resultant dry white crystalline powder was ground with mortar and pestle and stored at 25° C. This inosine crystal polymorph will hereinafter be referred to as GR-SP98-12.
  • Example 3 Seven grams of standard inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask. Next, 210 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the flask reached 90°C twenty-seven minutes after heat ⁇ ing began.
  • the flask was removed from the heater- stirrer and was cooled at room temperature, with stirring, so that the temperature of the flask reached 45°C twenty-eight minutes after the flask was removed from the Fisher Thermix heater-stirrer.
  • the cooled solution was shell frozen in an acetone and dry ice mixture, placed in a -40°C freezer overnight and then lyophilized on a VirTis lyophilizer (Model Freezemobile 12) for 24 hours, at which time the frozen solution was completely lyophilized.
  • the result ⁇ ing dry white crystalline powder was ground with mortar and pestle and stored at 25° C. This inosine crystal polymorph will hereinafter be referred to as GR-SP98-13.
  • Example 4 Seven grams of standard inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask. Then, 210 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the flask reached 90°C twenty-five minutes after heat ⁇ ing began. The heater setting was then reduced to number 2, and the heater was maintained at this setting so that the temperature dropped to 73°C over the next 20 minutes.
  • the heater setting was increased to number 4.7, and the temperature was kept between 73 and 77°C for 15 minutes.
  • the heater was turned off, and the flask was removed from the heater-stirrer and was cooled at room temperature with stirring so that the temperature of the flask reached 45°C twenty minutes after the flask was removed from the heater-stirrer.
  • Example 5 Another inosine crystal polymorph was prepared as described in Example 4, except that the solution was maintained between 73° C and 77° C for 60 minutes. This inosine crystal polymorph will be referred to hereinafter as GR-SP98-16.
  • the temperature fell to 70°C over the next 5 minutes, and the setting was increased to number 7.0.
  • the chemical hood air-flow had been inadvertently left on, slightly retarding heating.
  • the air-flow was turned off, and the flask temperature rose to 75°C over the next 5 minutes.
  • the heater was turned off, and the flask was removed from the heater-stirrer and was cooled at room temperature, with stirring, so that the temperature of the flask reached 46°C twenty minutes after the flask was removed from the heater-stirrer.
  • the resultant mixture was shell frozen in an acetone and dry ice mixture and placed in a -40°C freezer overnight.
  • the flask was then attached to a VirTis lyophilizer (Freezemobile 12), and the mixture was lyophilized for 24 hours, at which time the frozen suspension was completely lyophilized.
  • the resultant dry white crystalline powder was ground with mortar and pestle and stored at 25 C. This material will hereinafter be referred to as GR-SP98-17.
  • the heater setting was changed to number 5.
  • the heater was kept at this setting for 115 minutes, during which time the tempera ⁇ ture remained between 72 and 75°C. Then, the heater was turned off, and the flask was removed from the heater-stirrer and was cooled at room temperature, with stirring, so that the temperature of the flask reached 40°C twenty minutes aft-er the flask was removed from the heater-stirrer.
  • the cooled solution was shell frozen in an acetone and dry ice mixture, placed in a -40°C freezer overnight, and then completely lyophilized as described in Example 3.
  • Example 10 Seven grams of standard inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask. Then, 210 ml of distilled water and a magnetic stirring bar were added, and the flask was placed on a Thermolyne Nuova II heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the settings set forth below: Time After
  • the heater settings were chosen to achieve the same rates of temperature increase and decrease as those developed with the Fisher Thermix heater-stirrer.
  • the flask was removed from the heater-stirrer 230 minutes after heating initially began and was cooled at room temperature, with stirring, so that the temperature of the flask reached 46°C twenty-five minutes after the flask was removed from the heater- stirrer.
  • the inosine did not reprecipitate during cooling.
  • Example 3 The cooled solution was shell frozen in an acetone and dry ice mixture, placed in a -40°C freezer overnight, and then completely lyophilized as described in Example 3. The resulting dry white crystalline powder was harvested, ground with mortar and pestle and stored. This inosine crystal polymorph will be referred to hereinafter as GR-SP98-19.
  • Example 11 Another material was synthesized as described in Example 9 except that only 3.2 grams of standard inosine were used as the starting material. The resultant material of this synthesis will hereinafter be referred to as GR-SP98-20.
  • Example 12 Another material was synthesized as described in Example 9 except that 21 grams of standard inosine were used as the starting material. The resultant material of this synthesis will hereinafter be referred to as GR-SP98-21.
  • Example 13 Seven grams of standard inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask. Then, 210 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the flask reached 90°C twenty-five minutes after heating began. The heater setting was then reduced to number 5 for five minutes and then to number 2. The heater was maintained at this setting so that the temperature dropped to 75°C over the next fifteen minutes.
  • the heater setting was increased to number 5 and the temperature was kept at about 75°C for 120 minutes.
  • the heater was turned off, and the flask was removed from the heater-stirrer and was cooled at room temperature with stirring so that the temperature of the flask reached 38°C thirty minutes after the flask was removed from the heater-stirrer.
  • the inosine crystal polymorph was precipitated from the cooled solution by placing the solution in a refrigerator at 4°C overnight. The precipitate was harvested by suction filtration. The resultant filtrate was washed with approximately 100 ml of cold acetone, after which it was removed from the filter paper and placed on a glass dish. The glass dish was placed in an exhaust fume hood to dry for 2 hours at 25 C. Finally, the resultant dry white crystalline powder was ground with a mortar and pestle and stored at 25°C.
  • This inosine crystal polymorph will be referred to hereinafter as GR-SP98-22.
  • Example 14 Seven grams of inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask. Next, 210 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the flask reached 90°C twenty-five minutes after heating began. The heater setting was then reduced to number 7. Five minutes later, the heater setting was reduced to number 2, and the heater was maintained at this setting so that the temperature dropped to 75°C over the next twenty to twenty- ive minutes.
  • the heater setting was increased to number 5, so that the temperature was maintained at about 75°C for the next 60 minutes.
  • the heater was turned off, and the flask was removed from the heater-stirrer and was cooled at room temperature, with stirring, so that the temperature of the flask reached 43°C twenty minutes after the flask was removed from the heater-stirrer.
  • the inosine crystal polymorph was precipi ⁇ tated from the cooled solution by placing the solution in a refrigerator at 4 C overnight. The precipitate was harvested by suction filtration, and the filtrate was washed with 200 ml cold acetone. The filtrate was then removed from the filter paper and placed on a glaps dish.
  • GR-SP98-26 This inosine crystal polymorph will be referred to as GR-SP98-26.
  • Example 15 Seven grams of inosine (Sigma, Lot 13F-0738) were placed in a 600 ml VirTis flask suitable for use on a VirTis lyophilizer. Next, 210 ml of distilled water and a magnetic stirring bar were added to the flask, and the flask was placed on a Fisher Thermix heater-stirrer. The flask and its contents were heated uncovered, with stirring, at the maximum setting (number 10) so that the temperature of the flask reached 90°C thirty-five to forty minutes after heating began.
  • the flask was removed from the Fisher Thermix heater-stirrer and was cooled, at room tempera ⁇ ture, with stirring, so that the temperature of the flask reached 58°C fifteen minutes after the flask was removed from the heater-stirrer.
  • the inosine crystal polymorph was precipi ⁇ tated and harvested as described in Example 14.
  • the resultant dry white crystalline powder was stored at 25°C. This inosine crystal polymorph will hereinafter be referred to as GR-SP98-28.
  • the heater setting was then changed to 5.5 so that the temperature was maintained at about 75°C for the next 10 minutes.
  • the flask was removed from the Fisher Thermix heater-stirrer and was cooled, at room temperature, with stirring, so that the temperature of the flask reached 46°C twenty minutes after the flask was removed from the heater-stirrer.
  • the inosine crystal polymorph was precipi ⁇ tated and harvested as described in Example 14.
  • the resultant dry white crystalline powder was stored at 25°C.
  • This inosine crystal polymorph will hereinafter be referred to as GR-SP98-38.
  • Example 17 Another inosine crystal polymorph was made as described in Example 16, except that 31.5 grams of standard inosine were used.
  • the resultant inosine crystal polymorph will be referred to herein as GR-SP98-39.
  • Example 18 Another inosine crystal polymorph was made as described in Example 17, except that the temperature was maintained at 75°C for 60 minutes. The resulting dry white crystalline powder was ground with mortar and pestle and stored at'25°C. This inosine crystal polymorph will hereinafter be referred to as GR-SP98-66.
  • the inosine crystal polymorphs of the inven ⁇ tion exhibit major differences in their x-ray powder diffraction spectra as compared to that of standard inosine.
  • the x-ray powder diffraction spectra of stan ⁇ dard inosine, GR-SP98-15 and GR-SP98-26 are shown in Figures 1, 2 and 3 respectively.
  • there is a striking increase in the crystal "d" spacing of the x-ray powder spectra of GR-SP98-15 and GR-SP98-26 as compared to that of standard inosine.
  • There is also a difference in the "d" spacing of GR-SP98-15 as com ⁇ pared to GR-SP98-26.
  • the "d" spacing is the degree number at which the first peak on the right side of the tracing appears.
  • the observed increase in "d” spacing shows that the size of the organizational unit (the repeating subunit of the crystal) of GR-SP98-15 and GR-SP98-26 is increased compared to that of standard inosine and that size of the organizational unit of GR- ⁇ P98-26 is increased compared to that of GR-SP98-15.
  • the increased size of the organizational unit must be the result of major changes in the conformation and patterns of association (the configuration) of the monomeric units of inosine in standard inosine, GR-SP98-15 and GR-SP98-26 compared one to the other. Changes in the "d" spacing relative to standard inosine have also been obtained for other inosine crystal poly ⁇ morphs of the invention.
  • Standard inosine has a solubility of about 16 mg/ml in ordinary water at room temperature (20-23°C).
  • the previously reported value for the solubility in water of standard inosine corresponds to the value shown in Table 2.
  • the three modified compounds exhibited a solubility greater than standard inosine. This increase in water solubility of GR-SP98-15 is substantially reversed by one recrystallization from hot 80% ethanol (GR-SP98-15-R) as shown in Table 2.
  • the method of determining solubilities employed in Examples 19 and 20 differs from the standard approach to solubility assessment in that an excess of solute is not shaken with a standard volume of solvent for 24-48 hours to achieve equilibrium. Rather, the test used in Examples 19 and 20 determines how much solvent is re ⁇ quired to dissolve the same fraction of a millimole of standard inosine and of inosine polymorph. The results are obtained in about one hour, but the results approximate the equilibrium condition as evidenced by the fact that the solubility of inosine in ordinary water determined using this test corresponds to the previously reported equilibrium solubility of inosine.
  • the inosine crystal polymorphs of the invention When the inosine crystal polymorphs of the invention are dissolved in a solvent, they unexpectedly exhibit solute polymorphism. The following behavior establishes that these materials are solute polymorphs.
  • the ORD spectra of aqueous solutions of the inosine polymorphs of the invention were substantially different than that of an aqueous solution of standard inosine. This change in the ORD spectra is evidence of differ ⁇ ences in the conformation of the solute polymorphs of the invention as compared to standard inosine.
  • the second piece of evidence that establishes solute polymorphism is the fact that the inosine polymorphs of the invention exhibit different patterns of ultraviolet (UV) absorbance as compared to that of standard inosine when samples of the polymorph and of standard inosine are diluted to dilutions much lower than those routinely studied by other workers.
  • UV ultraviolet
  • Example 21 Solutions of standard inosine and solute poly ⁇ morph GR-SP98-15 in water were examined by scanning UV spectroscopy in quartz-windowed cells of both one and ten centimeter pathlength (the pathlength used being dependent on the concentration) as concentration was
  • inosine polymorph GR-SP98-15 gives up its association less readily with further dilution than does standard inosine.
  • These data are evidence of solute polymorphism and of the persistence of differences in the configurations of standard inosine and the inosine solute polymorphs of the invention at high dilutions that are close to the drug levels found in biological systems.
  • Biological Activity The final piece of evidence that establishes that the novel inosine polymorphs of the invention exhibit solute polymorphism is the fact that the inosine polymorphs surprisingly exhibit unanticipated and important differences in biological activity as compared to standard inosine.
  • the solute polymorphs exhibit dramatically increased efficacy in reducing inflammatory responses in animals as shown by results in the carrageenan, zymosan, trypsin and chymotrypsin footpad edema tests.
  • the anti-inflammatory activity of the inosine solute polymorphs of the invention is substantially lost along with their unique physical- chemical properties when they are recycled through the crystallization procedure which is used to produce standard inosine (see Example 6).
  • the inosine solute polymorphs of the invention exhibit as much as a fifty- fold increase in anti-inflammatory activity compared to standard inosine. Also, the solute polymorphs of the invention exhibit profound anti-inflammatory activity in this test at very low doses at which standard inosine shows no anti-inflammatory effect at all.
  • trypsin and chymotrypsin are released during inflammatory responses.
  • chymo ⁇ trypsin is released by neutrophils and other cells during joint inflammation and degeneration and is known to participate in arthritis.
  • Trypsin is known to be released into the pancreas during pancreatitis due to blockage of the pancreatic duct and consequent " spilling of the trypsin-rich duct fluid into the pancreas.
  • the cells of the pancreas are severely damaged by trypsin directly and by the ensuing inflammatory response.
  • Cystic fibrosis is also a disease of the pancreas in which the cellular damage is caused by trypsin.
  • zymosan edema is also a widely-used model of arthritis.
  • Zymosan stimulates phagocytes which release oxygen-free radicals and lysosomal enzymes.
  • Example 22 Three groups of seven HA/ICR mice, 19 weeks old, average weight 40.4 grams, purchased from Harlan, Sprague-Dawley, Indianapolis, were given 0.1 ml of the following substances by oral gavage:
  • the concentrations of the standard inosine and GR-SP98-15 solutions were such that the mice received 10 mg/kg of each substance in the 0.1 ml.
  • dorsoventral footpad diameters of each mouse in each group were measured to the nearest 0.1 mm employing a Schnelltaster caliper. Also, the increase in dorsoventral footpad diameter produced by injection of the carrageenan solvent alone was determined by measuring the dorsoventral footpad diameters of a group of mice before and at 2 and 3 hours after injection of the solvent. This nonspecific swelling (the passive solvent effect) was subtracted from the mean increase in swelling evoked by carra ⁇ geenan injection to provide a measure of the specific swelling developed as part of the inflammatory response to carrageenan itself.
  • mice Two hours after the oral gavage treatment, the mice were injected with 0.5 ml of a 2 mg/ml solu ⁇ tion of carrageenan, prepared and injected as described in Example 22. Also, dorsoventral footpad diameters were measured as described in Example 22.
  • Example 24 The anti-inflammatory effects of standard inosine and of inosine solute polymorph GR-SP98-11 were compared with those of indomethacin, a potent anti- inflammatory drug currently in clinical use.
  • mice Two hours after the oral gavage treatment, the mice were injected with 0.05 ml of a 2 mg/ml solution of carrageenan, prepared and injected as de ⁇ scribed in Example 22. Also, dorsoventral footpad diameter measurements were performed as described in Example 22.
  • the carrageenan-induced footpad swelling of Groups 2 and 4 at 3 hours is significantly less than that of Group 1 (P -less than 0.005), and the carrageenan-in ⁇ quizd swelling of Group 4 at 3 hours is not significantly less than that of Group 2 ; but the swelling of both is significantly less than that of Group 3 (P less than 0.05) .
  • mice Two hours after the oral gavage treatment, the mice were injected with carrageenan, prepared and injected as described in Example 22. Dorsoventral footpad diameter measurements were also performed as described in Example 22.
  • Group 3 A solution of GR-SP98-15 10 mg/kg Group 4 A solution of GR-SP98-15 1 mg/kg Group 5 A solution of GR-SP98-15 0.1 mg/kg
  • mice Two hours after the oral gavage treatment, the mice were injected with 0.05 ml of a 2 mg/ml carrageenan solution, prepared and injected as described in Example 22. Dorsoventral footpad diameter measure ⁇ ments were also performed as described in Example 22.
  • mice in each group were injected with 0.05 ml of a 2 mg/ml solution of carrageenan in each dorsoventral footpad, prepared and injected as described in Example 22.
  • Dorsoventral footpad diameter measurements were also performed as described in Example 22.
  • mice carrageenan footpad edema is not inhibited by 1 mg/kg standard inosine or 1 mg/kg of GR-SP98-17.
  • mouse carrageenan footpad edema is strikingly inhibited by the inosine solute polymorphs GR-SP98-15, GR-SP98-15.2, GR-SP98-18 and GR-SP98-19. This inhibition is as high as 98% (treat ⁇ ment with GR-SP98-18).
  • each rat in each group was injected with 0.10 ml of a 10 mg/ml solution of carrageenan in each dorsoventral footpad.
  • the carrageenan was prepared and injected as described in Example 22.
  • Dorsoventral footpad dia ⁇ meters were also measured as described in Example 22 just prior to and 3 hours after carrageenan injection.
  • Example 29 The anti-inflammatory activity of GR-SP98-15 was compared with that of inosine in male rats of about 12 months of age. These rats had lived approximately of their life span. Three groups of eight Sprague Dawley rats, retired male breeders, average weight 370 grams, purchased from Harlan, were given 0.1 ml of the following substances by oral gavage:
  • Treatment Group 1 Distilled water Group 2 A solution of standard Inosine (Sigma, Lot 13F-0738) in distilled water Group 3 A solution of GR-SP98-15 in distilled water
  • Example 22 Two hours after the oral gavage treatment, the rats were injected with 0.10 ml of a 10 mg/ml solution of carrageenan, prepared and injected as described in Example 22. Also, dorsoventral footpad diameter measurements were performed as described in Example 22.
  • GR-SP98-15 The anti-inflammatory activity of GR-SP98-15 was compared with that of standard inosine in female rats of about 12 months of age. These rats had lived approximately 60% of their life span.
  • the rats were injected with 0.10 ml of a 10 mg/ml solu ⁇ tion of carrageenan, prepared and injected as described in Example 22.
  • Dorsoventral footpad diameter measure ⁇ ments were also performed as described in Example 22.
  • Example 31 Three groups of five male CD-I mice, 8 weeks old, average weight 30.5 grams, purchased from Harlan, were injected subcutaneously with 0.5 ml of the following substances:
  • Group 2 A solution of standard inosine (Sigma, Lot 13F-0738) in distilled water
  • Group 3 A solution of GR-SP98-7 in distilled water
  • the solutions of standard inosine and GR-SP98-7 were at concentrations chosen so that each mouse received 20 mg/kg of each substance in the 0.5 ml volume.
  • each mouse in each group was injected in each dorsoventral footpad with 0.05 ml of a 2 mg/ml suspension of zymosan A in saline.
  • the zymosan A was obtained from Sigma Chemical Compan .
  • Example 32 Three groups of seven CD-I mice, 5-6 weeks old, purchased from Charles River, were given 0.1 ml of the following substances by oral gavage: Treatment Group 1 ' " Distilled water.
  • the concentrations of the standard inosine and GR-SP98-15 solutions were such that the mice received 30 mg/kg of each substance in the 0.1 ml.
  • dorsoventral footpad diameters of each mouse in each group were measured as described in Example 22.
  • Example 33 Five groups of six CD-I mice, 5-6 weeks old, purchased from Charles River, were given 0.1 ml. of the following substances by oral gavage: Treatment
  • the concentrations of the standard inosine and the various inosine polymorphs were such that the mice received 30 mg/kg of each substance in the 0.1 ml.
  • dorsoventral footpad diameters of each mouse in each group were measured as described in Example 22.
  • Group 3 A solution of GR-SP98-26 in distilled water.
  • Group 4 A solution of GR-SP98-28 in distilled water.
  • Group 5 A solution of GR-SP98-38 in distilled water.
  • Group 6 A solution of GR-SP98-39 in distilled water.
  • Group 7 A solution of GR-SP98-66 in distilled water.
  • the concentrations of the standard inosine and the inosine polymorph solutions were such that the mice received 30 mg/kg of each substance in the 0.1 ml.
  • dorsoventral footpad diameters of each mouse in each group were measured 'as described in Example 22.
  • Example 35 Five groups of six CD-I mice, 5-6 weeks old, purchased from Charles River, were given 0.1 ml of the following substances interperitoneally:
  • Group 3 A solution of standard inosine in saline (Pharma Waldhof) .
  • Group 4 A solution of GR-SP98-15 in saline.
  • Group 5 A solution of GR-SP98-26 in saline.
  • the concentrations of the standard inosine and of the inosine polymorph solutions were such that the mice received 15 mg/kg of each substance in the 0.1 ml.
  • Example 36 Five groups of six CD-I mice, each, 5-6 weeks old, purchased from Charles River, were given 0.1 ml of the following substances by oral gavage:
  • the concentrations of the standard inosine and GR-SP98-15 solutions were such that the mice in Groups 2 and 4 received a dose of 3 mg/kg and the mice in Groups 3 and 5 received a dose of 30 mg/kg of the substances indicated in the 0.1 ml.

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Abstract

Nouvelles formes polymorphes de l'inosine, solutés et cristaux. Procédé de préparation d'un cristal polymorphe de l'inosine consistant à: utiliser un solvant; à ajouter au solvant au moins 3 grammes d'inosine pour 100 ml de solvant environ; à chauffer le solvant et l'inosine, selon une vitesse prédéterminée, pour atteindre une température suffisante pour provoquer la mise en solution de l'inosine et pour vaincre les barrières énergétiques qui empêchent la conversion de l'inosine en une autre configuration polymorphe; à refroidir la solution selon une vitesse prédéterminée pendant une période prédéterminée, et à précipiter le cristal d'inosine polymorphe. Le refroidissement peut s'effectuer en une ou plusieurs étapes. On peut remplacer la précipitation par la lyophilisation pour produire le cristal polymorphe. L'invention se rapporte également à une procédé de prépartion de solutés polymorphes de l'inosine consistant à dissoudre les cristaux polymorphes d'inosine dans un solvant. On peut égalent préparer des solutés polymorphes selon l'invention en utilisant le procédé décrit précédemment pour la préparation de cristaux polymorphes, seules les étapes de précipitation et de lyophilisation étant omises. Compositions anti-inflammatoires comprenant une quantité d'un soluté ou d'un cristal polymorphe d'inosine efficace pour atténuer la réponse inflammatoire d'un animal, et procédés de réduction de la réponse inflammatoire d'un animal consistant à administrer ces compositions à l'animal.
PCT/US1988/001468 1987-05-26 1988-05-10 Formes polymorphes de l'inosine, procedes de preparation et d'utilisation WO1988009335A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7338666B2 (en) 1997-09-02 2008-03-04 Children's Medical Center Corporation Methods for modulating the axonal outgrowth of central nervous system neurons
US8912144B2 (en) 2003-12-16 2014-12-16 Children's Medical Center Corporation Method for treating stroke via administration of NEP1-40 and inosine
CN110627854A (zh) * 2019-10-18 2019-12-31 海南顿斯医药科技有限公司 一种1/10水肌苷化合物

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Bulletin of the Chemical Society of Japan, (Tokyo, JP), Y. Suzuki et al.: "Polymorphism of inosine. III. The equilibrium for the inosine-dimethyl sulfoxide-water system", pages 2551-2552 *
Bulletin of the Chemical Society of Japan, volume 43, no. 5, May 1970, (Tokyo, JP), Y. Suzuki et al.: "Polymorphism of inosine", page 1600 *
Bulletin of the Chemical Society of Japan, volume 47, no. 10, October 1974, (Tokyo, JP), Y. Suzuki: "The polymorphism of inosine. II. The measurement of solubilities in water", pages 2549-2550 *
Chemical Abstracts, volume 82, 1975, (Columbus, Ohio, US), see page 483 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7338666B2 (en) 1997-09-02 2008-03-04 Children's Medical Center Corporation Methods for modulating the axonal outgrowth of central nervous system neurons
US7935680B2 (en) 1997-09-02 2011-05-03 Children's Medical Center Corporation Methods for modulating the axonal growth of central nervous system neurons
US8912144B2 (en) 2003-12-16 2014-12-16 Children's Medical Center Corporation Method for treating stroke via administration of NEP1-40 and inosine
CN110627854A (zh) * 2019-10-18 2019-12-31 海南顿斯医药科技有限公司 一种1/10水肌苷化合物

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