Attention is drawn to Applicant's co-pending application, Serial No. 054,376 ^~ _or "Polymorphs of Xanthosine And Methods Of Making And Using Them, " filed on May 26, 1987.

BACKGROUND OF THE INVENTION 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.

Two crystal polymorphs of inosine are known. One of these crystal polymorphs is the commercially- available anhydrous form of inosine. The final crystallization procedure used in the preparation of this form of inosine involves the heating of a three to seven percent suspension of crude inosine in 80% ethanol:20% water until solution of the inosine occurs at about 80° C. This is followed by filtration of the inosine solution. The filtrate is next cooled to room temperature and then stored at refrigerator temperatures for 72 hours to induce and maximize precipitation. The precipitate is then harvested by suction filtration and dried. The resultant crystals

are anhydrous needles which decompose at 218 C. All commercially-available inosine is prepared using this same final crystallization procedure (presumably chosen because of its low cost), and "standard inosine" is defined herein to be inosine which is prepared using this crystallization procedure as the final step.

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.

Polymorphism in the solution state has long been believed to be non-permissible for energetic reasons. It has generally been assumed that the molecules in a solvent will be unaware of their origin and that all crystal polymorphs of a compound will give rise to solutions having identical properties. The underlying assumption on which this conclusion is based is that all the intermolecular solid state bonds of the crystal will initially be given up to release the potential energy necessary to create the solution state. The basic solution event may even be followed by aggregation of the solute, but it has been assumed that there is an absence of energy barriers between solute aggregate forms. Indeed, the tendency of nucleosides such as inosine to aggregate in aqueous solution has been known for many years. This associa¬ tion has been reported to occur essentially exclusively by vertical base stacking. However, it has been assumed that energy barriers do not exist between the aggregated forms of nucleoside molecules in solution and that monomers are the form of nucleoside most frequently found in solution.

Further, although the monomeric purine nucleo¬ sides can potentially assume 20 to 26 different con¬ formations in space as a result of spontaneous bond

rotation, it has been assumed that in aqueous solution these forms are instantly interconvertible. The energy barrier between next-neighbor conformations is held to be trivial since it is assumed to derive from one hydro¬ gen bond between solute and solvent, and no form of solute-solute association other than the base stacking discussed above and hydrogen-bonding has been antici¬ pated for purine nucleosides.

Agafonov, Leonidov and Kobzareva, Zhurnal Obshchei Khimii. 50, 166 (1980) (hereinafter 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. Although the authors state that the differences in the ORD spectra of the two polymorphs cannot serve as a criterion in conformational analysis because of high background read¬ ings, they speculate that these differences do reflect different conformations of the solute forms of the two crystal polymorphs and that these differences in con¬ formation may give rise to differences in the biological accessibility (rate of passage into biological fluids of an animal) and activity of the two polymorphic forms of prednisolone.

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.

However, all of the changes in optical properties reported in Leonidov and Agafonov were observed only in organic solvents. Neither of these two publications reports any changes in optical properties of polymorphs in aqueous solutions. Water, the biologically significant solvent, has properties fundamentally different than those of other liquids. It is totally unpredictable from the results reported in Leonidov and Agafonov whether solute polymorphism would occur in water. Indeed, the compounds tested in the Leonidov and Agafonov publications are all virtually insoluble in water. Further, it is merely speculated in Leonidov and Agafonov that polymorphs of the same compound might have different biological activities. No data are presented to support this theory.

BRIEF DESCRIPTION OF THE INVENTION According to one .aspect of the invention, there is provided 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.

Also part of the invention are the crystal polymorphs produced by this process. 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.

According to another aspect of the invention, there are provided solute polymorphs of inosine. The solute polymorphs of the invention 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.

According to another aspect of the invention, there is provided 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.

Again 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.

Finally» there are provided: (1) a method of reducing an inflammatory response in an animal comprising administering to the animal a composition comprising a pharmaceutically-acceptable solvent or carrier and an effective amount of an inosine solute or

crystal polymorph of the invention; and (2) the anti- inflammatory compositions for use in this method.

DEFINITIONS

As used herein, 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. The term "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.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE PRESENTLY PREEERRED EMBODIMENTS

Synthesis of Inosine Polymorphs The following variables are important in the synthesis of the novel inosine crystal and solute polymorphs of the invention:

1. 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.

2. Temperature. Elevation of the tempera¬ ture is necessary for the formation of inosine crystal

and solute polymorphs. The temperature must be elevated sufficiently high so that the inosine goes into solution and so that the energy barriers which prevent conversion of one polymorphic configuration of inosine to another are overcome. It is not yet known what types of forces create these energy barriers, but their existence is shown by the fact that the use of elevated temperatures is necessary to obtain formation of the crystal and solute polymorphs of the invention. Further, the variation of temperature with time in both the heating and cooling cycles affects the development of the inosine polymorphs and the nature of the final polymorph which is formed.

3. Concentration. 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. Treatment of a solution of standard inosine according to the methods of the invention at con¬ centrations above about 3% results in the formation of an inosine polymorph, but when the concentration of the treatment solution exceeds 10-15%, the resulting poly¬ morphs do not exhibit the remarkable differences in biological activity, as compared to standard inosine, that polymorphs made using concentrations of inosine from about 3 to 10-15% exhibit.

4. ^' Freezing and Lyophilization. The choice of lyophilization over conventional techniques of crystal precipitation and harvesting, such as suction filtration and air drying, and the length of time the solution is frozen prior to lyophilization also

contribute to the character of the final inosine crystal polymorph. Lyophilization and freezing are not necessary for the formation of the solute or crystal polymorphs of the invention, but these factors contribute to the final nature of the inosine crystal polymorph and to the solute polymorphs which are formed when these crystal polymorphs are dissolved. Freezing creates pressure and solute proximity, and it is believed that these two results of freezing lead to the production of the crystal seeds. Lyophilization completes the crystallization and gives the crystal its final form.

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. Next, 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- ^ς C thirty-two minutes after the flask was removed from the heater-stirrer.

During the heating cycle, all of the inosine went into solution 14 minutes after heating began when the flask temperature was 47°C. The inosine did not ^■ reprecipitate during subsequent cooling.

A complete record of how the temperature varied wit -time for this synthesis is presented below:

Time After Heat- Time After Heat- ing Began (min. ) Temp. (°C) ing Began (min. ) Temp.

0 25 35 89

5 29 38 90

10 36 45 73

14 47 50 61

15 50 55 57

20 63 60 52

25 77 65 49

30 85 70 46

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.

During the heating cycle, all of the inosine went into solution 15 minutes after heating began when the flask temperature was 49°C. The inosine did not reprecipitate during subsequent cooling.

A complete record of how the temperature varied with time for this synthesis is presented below:

Time After Heat- Time After Heat- ing Began (min. ) Temp. (° C) ing Began (min. ) Temp. (° C)

0 26 45 84

5 28 50 79

10 37 55 75

15 49 60 73

20 66 65 61

25 78 70 57

30 86 75 51

35 91 80 48

40 89 85 45

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. Next, 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.

During this heating cycle the inosine dissolved completely between 42 and 60°C and between the 10th and 15th minute of heating. Inosine did not reprecipitate during cooling. -»

A completed record of how the temperature varied with time for this synthesis is presented below:

Time After Heat- Time After Heat- ing Began (min. ) Temp. ( ° C) ing began (min. ) Temp. ( ° C)

0 23 27 90

5 28 35 73

10 42 40 60

15 60 45 54

20 78 50 48

25 89 55 44

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. Then, the heater setting was increased to number 4.7, and the temperature was kept between 73 and 77°C for 15 minutes. Next, 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.

During this heating cycle, all of the inosine went into solution 13 minutes after heating began when the flask temperature was 53°C. The inosine did not reprecipitate during subsequent cooling.

A complete record of how the temperature varied with time for this synthesis is presented below:

Time After Heat- Time After Hea - ing Began (min. ) Temp. f° C) ing began ( in. ) Temp. (° C)

0 24 40 79

5 29 45 73

10 .43 50 73

13 53 55 75

15 62 60 77

20 80 65 64

25 90 70 55

30 89 75 50

35. 84 80 45

The cooled solution was shell frozen in an acetone and dry ice mixture, placed in a -40°C freezer overnight and then lyophilized as described in Example 3. The resulting dry white crystalline powder was ground with mortar and pestle and stored at 25° C. This inosine crystal polymorph will be referred to hereinafter as GR-SP98-15.

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.

Example 6

Two grams of standard inosine (Sigma, Lot 13F-0738) and two grams of GR-SP98-15 were added to separate 250 ml beakers. Then, 95 ml of a solution of 80% ethanol in water and a magnetic stirring bar were added to each beaker. The beakers were placed on Fisher Thermix heater-stirrers, and the beakers and their contents were heated uncovered, with stirring, with the heater control set at number 4.5. When the inosine or GR-SP98-15 was completely dissolved, the beaker containing each compound was removed from its heater, allowed to cool to room temperature and then placed in a refrigerator at 4 C for 72 hours.

The standard inosine dissolved completely after 20 minutes of heating, and the beaker containing it was removed from the heater-stirrer at that time. The GR-SP98-15 dissolved completely after 15 minutes of heating, and the beaker containing it was removed from the heater-stirrer at that time. In each case, complete dissolution occurred when the temperature was between 75 to 84°C.

During refrigeration, recrystallization was active, and at 72 hours, crystals were harvested by suction filtration. The products were then air-dried for 15 minutes at room temperature and for 4 hours at 105°C in a drying-oven. The resultant materials will be referred to hereinafter as Inosine-R and GR-SP98-15-R.

Example 7

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 91°C thirty-three minutes after heating 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 17 minutes. Then, the heater setting was increased to number 4.7. The temperature, however, 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. At this 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 temperature of the flask reached 46°C twenty minutes after the flask was removed from the heater-stirrer.

During this heating cycle, all of the inosine went into solution 14 minutes after heating began when the flask temperature was 48°C. The inosine did not reprecipitate during subsequent cooling.

A complete record of how the temperature varied with time for this synthesis is presented below:

Time After Heat- Time After Heat- ing Began (min. ) Temp. ( ° C) ing Began (min. ) Temp. ( ° C)

0 23 40 85

5 25 45 78

10 36 50 75

14 48 55 70

15 50 60 75

20 66 65 63

25 78 70 56

30 87 75 __

33 91 80 46

35 91

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 ground with mortar and pestle and stored at 25 C. Hereinafter this inosine crystal polymorph will be referred to as GR-SP98-15.2.

Example 8

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 stirred, uncovered, without heating, for 80 minutes. All of the inosine did not go into solution.

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.

Example 9

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 91°C thirty minutes after heating began. The heater setting was then reduced to number 2, and the heater was kept at this setting so that the temperature dropped to 73°C over the next 20 minutes. Then, the heater setting was increased to number 4.7 and then to number 6. Next, 55 minutes after heating initially began, 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.

During this heating cycle, all of the inosine went into solution 13 minutes after heating began when the flask temperature was 53°C. The inosine did not reprecipitate during subsequent cooling.

A complete record of how the temperature varied with time is presented below:

Time After Heat- Time After Heat- ing Began (min.) Temp.(° C) ing Began (min. ) Temp. ( ⁰ C)

0 24 70 73

5 28 75 73

10 40 80 73

13 53 90 74

15 59 100 74

20 78 110 73

25 87 120 73

30 91 130 73

35 87 140 72

40 82 150 12

45 77 160 12

50 73 170 73

55 73 175 59

60 75 185 48

65 73 190 40

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 ground with mortar and pestle and stored at 25°C. This inosine crystal polymorph will be hereinafter referred to as GR-SP98-18.

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

Heating began Heater

(minutes) Temperature (°C) Setting Comments

0 23 6

5 27 4-3

10 50

11 53 4 inosine dissolved

15 54

20 58 5.5-6

25 68 8

30 81 10

35 90 0

40 89 2

45 79

50 75 4-5-6

55 73 6

60 77 5-4

65 76 3.5

70 72 4-4.2

75 70 4.5-4.7

80 72 4.7

90 73

100 73

110 73

120 73

130 72

140 72

150 72

160 72

170 72 ^•

180 72

190 71 5.5

200 73

210 73

220 73

230 73 flask removed from heater

235 60

245 49

255 46 no reprecipitation

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.

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. Then, the heater setting was increased to number 5 and the temperature was kept at about 75°C for 120 minutes. Next, 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. Then, the

heater setting was increased to number 5, so that the temperature was maintained at about 75°C for the next 60 minutes. At this 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 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. The glass dish was placed in an exhaust fume hood at 25°C for 2 to 3 hours to dry the precipitate. The resulting dry white crystalline powder was ground with a mortar and pestle and stored at 25°C. Hereinafter 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. Next, 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.

Example 16

Twenty-one grams of standard 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 twenty-five minutes after heating began. The heater setting was reduced to 2 so that the temperature dropped to 75°C over a period of twenty minutes. The heater setting was then changed to 5.5 so that the temperature was maintained at about 75°C for the next 10 minutes. Next, 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.

Physical and Chemical Properties 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. As can be seen, 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-ΞP98-26 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.

Further, significant differences in the details of the x-ray diffraction patterns of one crystal polymorph of the invention as compared to another and to standard inosine have been observed (compare e.g. , Figures 1, 2 and 3 with each other). These differences vary with the conditions of synthesis of the particular polymorph. In particular, the position and height of the principal peak varies.

Another piece of evidence that establishes crystal polymorphism is the fact that the limiting eguili- brium solubility of the inosine crystal polymorphs of the invention in ordinary water is increased by about 60 to 80%, in heavy wter by about 20-30% and in methanol by about 100% compared to standard inosine. This change in solubility is not due to change in crystal size.

Example 19

The solubility of standard inosine (Sigma,

Lot 13F-0738), GR-SP98-15, Inosine-R and GR-SP98-15-R in methanol (reagent grade, Fisher), ethanol (200 proof,

Commercial Solvents Corp. ) and heavy water (deuterium oxide, 99.8% D, Sigma, Lot 33F-0705) was determined at room temperature (23°C) . To do so, 33.5 mg, or 0.125 x

_3 10 moles, of each type of inosine were added to a 50 ml conical centrifuge tube with a ground-glass stopper, and solvent was added in steps with vigorous shaking between each step. When all but the last visible trace of compound was dissolved, the sample was held to be in solution.

To assess solubility in methanol and ethanol, solvent was added in 1 ml increments. Heavy water was added in 1 ml increments up to a volume of 4 ml and then was added in 0.5 ml increments.

The results are shown in Table 1. Standard inosine has different solubility properties than GR-SP98-15. Also, Inosine-R and GR-SP98-15-R have similar solubility properties, but their solubilities in heavy water are increased over that of standard inosine and GR-SP98-15.

TABLE 1

LIMITING SOLUBILITY (mg/ml)

COMPOUND METHANOL ETHANOL HEAVY WATER

Inosine 0.66 0.66 .6.09

GR-SP98-15 1.24 0.66 7.44

Inosine-R 0.66 N.D. 11.17

GR-SP98-15-R 0.86 N.D. 11.17

Example 20

The solubilities of standard inosine, GR-SP98-15, Inosine-R and GR-SP98-15-R in deionized ordinary water were determined. At room temperature (23°C), 67 mg, or 0.25 x 10 moles, of each type of inosine was added to a 16 x 125 mm pyrex test tube and water added in steps, with vigorous buzz-stirring between each addition. When all but the last visible trace of compound was dissolved, the sample was held to be in solution.

The results are shown in Table 2. Standard inosine has a solubility of about 16 mg/ml in ordinary water at room temperature (20-23°C). W.H. Elliott (1959) PURINES, PYRIMIDINES AND NUCLEOSIDES, in Data for Biochemical Research, R.M.C. Dawson, D.C. Elliott, W.H. Elliott, K.M. Jones (eds.), p. 70. P. Gordon, INOSINE DERIVATIVES, U.S. Patent, 3,857,940, Dec. 31, 1974. 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.

TABLE 2 COMPOUND - LIMITING SOLUBILITY IN WATER AT 23°C

Inosine 16.3 mg/ml

GR-SP98-15 26.8 mg/ml

Inosine-R 17.2 mg/ml

GR-SP98-15-R 20.9 mg/ml

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. Further, if solvent addition of standard inosine is stopped when the more soluble polymorph becomes completely dissolved, additional shaking for another twenty-four hours does not cause further dissolution of the standard inosine, as determined by eye and also by ultraviolet absorbance measurements made on the supernatant.

Elemental analysis revealed that the crystal polymorphs of the invention were inosine. Those crystal

polymorphs of inosine made using standard inosine and water as the preparative solvent were anhydrous like standard inosine, and the decomposition point of these polymorphs was not changed.

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. First, 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 ORD spectra of solutions of standard ino¬ sine and inosine polymorph GR-SP98-15 in water are shown in Figure 5. These spectra are the result of triplicate runs, and the standard error in the tracings is virtually zero. Differences between the ORD spectra of the solute polymorphs of the invention compared to that of standard inosine have also been observed for other solute poly¬ morphs of the invention.

It has been observed, however, that there are no differences between the infrared spectra of standard inosine and GR-SP98-15.2 in water. This establishes that keto-enol tautomerism does not play a role in inosine solute polymorphism.

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.

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

—6 —8 progressively reduced from 1 x 10~ to 4 x 10 ^~ M. The instrument employed was a Gilford Response Recording Spectrophotometer. From the digital data generated, the extinction coefficients for each dilution at 248.5 nm and at 225 n and the ratio of extinction coeffi¬ cients at the two wave lengths for each dilution, ₅ were calculated.

The calculated data is shown in Figure 4. These data reveal that the extinction coefficients for each compound at each wave length increased in value below 1 x 10~ , but not above this dilution. For both standard inosine and GR-SP98-15, the extinction coeffi¬ cient for the lower wave length increased more rapidly than did the extinction coefficient for the higher wave length with progressive dilution. This differential increase of low wave length molar absorbance is known as a "blue shift". An increase in extinction coeffir cient and a shift in absorbance from higher to lower wave lengths are hallmarks of dissociation. Therefore, the data establish that both standard inosine and the inosine polymorph GR-SP98-15 dissociated as dilution is increased. However, 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).

In the carrageenan assay, 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. These results are significant because the carrageenan footpad edema test is routinely used by pharmaceutical companies to predict the anti-arthritic activity of new drugs in man.

Both trypsin and chymotrypsin are released during inflammatory responses. For instance, 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.

Finally, 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:

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.

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.

Two hours after the oral gavage treatment, 0.05 ml of a 2 mg/ml solution of carrageenan in normal saline was injected into the dorsoventral footpads of each of the mice of each group. To prepare the injec¬ tion solution, type IV lambda-carrageenan, purchased from Sigma Chemical Company, was mixed with normal saline, and the mixture was stirred overnight. Injections were carried out under ether anesthesia.

Just prior to and at 2 and 3 hours after the carrageenan injection, 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.

The results of this experiment are presented in Table 3. As shown there, GR-SP98-15 produced a sig¬ nificant (95%) suppression of carrageenan-induced footpad swelling at 2 hours, whereas standard inosine produced insignificant (less than 10%) suppression. This study establishes that a single oral dose of 10 mg/kg GR-SP98-15 exerts an anti-inflammatory effect in the mouse carrageenan edema model, while standard inosine does not.

TABLE 3

MEASURED SPECIFIC FOOTPAD SWELLING _# CARRAGEENAN SWELLING^

GROUP (in units of 0.10mm) (in units of 0.10mm)

2 hours 3 hours 2 hours 3 hours

1 6, ,86 ± 0.86 6.29 ± 0.70 2.86 2.29

2 6. .57 ± 1.08 6.14 ± 3.74 2.57 2.14

3 4. ,07 + 0.61 4.86 ± 0.63 0.07 0.86

* Analysis using the student's t test shows that, at two hours, the footpad swelling of Group 3 (GR-SP98-15) is significantly smaller than that of Group 1 (distilled water) (P less than 0.02).

** Mean measured footpad swelling minus the nonspecific swelling caused by solvent alone (4.00 in units in 0.10 mm) .

Example 23 Three groups of eight CD-I mice, 8 weeks old, average weight 33.6 grams, purchased from Charles River, Wellington, Massachusetts were given 0.1 ml of the following substances by oral gavage:

Treatment

Group 1 Distilled water

Group 2 Solution of standard inosine (Sigma, Lot 13F-0738) in distilled water

Group 3 Solution of GR-SP98-15 in distilled water

The solutions of standard inosine and of GR-SP98- 15 were at concentrations chosen so that each mouse received 50 mg/kg of each substance in the 0.1 ml.

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.

The results of this experiment are presented in Table 4 and show that, at 50 mg/kg, both GR-SP98-15 and standard inosine significantly suppressed carrageenan footpad edema in mice.

TABLE 4

MEASURED SPECIFIC FOOTPAD SWELLING _ft CARRAGEENAN SWELLING**

GROUP fin units of 0.10mm) (in units of 0.10mm) .

2 hours 3 hours 2 hours 3 hours

1 13. ,63 ± 0.97 14.88 ± 0.83 ^• 9.63 10_88

2 8, .78 ± 0.73 10.94 + 0.72 4.78 6.94

3 7 .56 ± 1.56 9.69 ± 0.99 3.56 5.69

* Analysis using the student's t test shows that, at 2 and 3 hours, the footpad swelling of both Groups 2 and

3 is significantly smaller than that of Group 1 (P less than 0.002) .

** Mean measured footpad swelling minus the nonspecific swelling caused by the solvent alone (4.00 in units of 0.10 mm) .

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. Four groups of six CD-I mice, 7 weeks old, average weight 30 grams, purchased from Charles River, were given 0.1 ml of the following substances by oral gavage: Group 1 Distilled water Group 2 A solution of standard inosine

(Sigma Lot 13F-0738) in distilled water Group 3 A solution of Indomethacin in distilled water Group 4 A solution of GR-SP98-11 in distilled water The solutions of Indomethacin, standard inosine and GR-SP98-11 were at concentrations chosen so that each mouse received 50 mg/kg of each inosine compound and 5 mg/kg of Indomethacin in the 0.1 ml.

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 results of this experiment are shown in Tables 5 and 6. These results establish that 50 mg/kg standard inosine or GR-SP98-11 exert anti-inflammatory effects on carrageenan footpad edema that are at least as powerful as the anti-inflammatory effects of 5 mg/kg

Indomethacin. Also, the effect of GR-SP98-11 is sig¬ nificantly greater than that of standard inosine.

TABLE 5

MEASURED SPECIFIC FOOTPAD SWELLING * CARRAGEENAN SWELLING*

GROUP (in units of 0.10mm) (in units of 0.10mm)

2 hours 3 hours 2 hours 3 hours

1 13. .17 ± 0.88 11.42 ± 0.85 9.17 7.42

2 10, .58 ± 0.79 7.00 ± 0.35 6.58 3.00

3 8. .50 ± 0.72 9.17 ± 0.75 4.50 5.17

4 6, .50 ± 0.47 6.83 ± 0.52 2.50 2.83

* Analysis using the student's t test shows that the carrageenan-induced footpad swelling at two hours of Groups 3 and 4 is significantly less than that of Group 1, (P less than 0.005). Also, the carrageenan-induced footpad swelling of Group 4 at two hours is significantly less than that of Group 2 (P less than 0.01), and sig¬ nificantly less than that of Group 3 (P less than 0.05) .

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¬ duced 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) .

** Mean measured footpad swelling minus the nonspecific swelling caused by the solvent (4.00 in units of 0.1 mm).

TABLE 6

Effect on Carrageenan Foot¬ pad Edema (% decrease in Treatment specific carrageenan swelling)

2 Hours 3 Hours

Indomethocin -51% -30%

Standard inosine (Sigma) -28% -39%

GR-SP98-11 -73%

Example 25 Five groups of six CD-I mice each, 6 week old males, average weight 27 grams, purchased from Charles River, 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 Group 4 A solution of inosine-R in distilled water Group 5 A solution of GR-SP98-15-R in distilled water

The solutions of standard inosine, GR-SP98-15, Inosine-R and GR-SP98-15-R were at concentrations chosen so that each mouse received 10 mg/kg of each substance in the 0.1 ml.

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.

The results of this experiment are shown in Tables 7-9 and demonstrate that, at 10 mg/kg, GR-SP98-15 exerts the most powerful anti-inflammatory effect of all the tested compounds at 2 hours, the time of peak inflammation. The results also show that this activity is largely destroyed by recycling GR-SP98-15 "through hot 80% ethanol. In contrast the recycling of standard inosine through hot 80% ethanol does not modify its

anti-inflammatory activity. This data .shows that the changes which took place in the formation of GR-SP98-15 are reversible. The reversibility of the changes sup¬ ports the propositions that the changes are in the con¬ figuration of inosine and that the formation of new covalent bonds is not involved. Further, this data supports the general hypothesis that inosine is changed in both physical-chemical and biological properties when it is exposed to different conditions in which the solvent, temperature, concentration and other parameters discussed above are varied.

TABLE 7

MEASURED SPECIFIC FOOTPAD SWELLING * CARRAGEENAN SWELLING**

GROUP (in units of 0.10mm) (in units of 0.10mm)

2 hours 3 hours 2 hours 3 hours

1 10.50 ± 0.81 9.67 ± 1.08 6.50 5.67

2 7.50 ± 0.58 5.50 ± 0.56 3.50 1.50

3 5.67 ± 0.74 6.17 ± 0.55 1.67 2.17

4 7.33 ± 0.45 5.25 ± 0.48 3.33 1.25

5 8.33 ± 0.71 6.92 ± 0.76 4.33 2.92

* Analysis using the student's t test shows that, at two hours, the footpad swelling of Groups 2, 3 and 4 is significantly less than that of Group 1 (P less than 0_01, 0.001 and 0.05, respectively). Also at two hours, the footpad swelling of Group 3 is significantly less than that of Group 5 (P less than 0.02). At three hours, the footpad swelling of Groups 2, 3, 4 and 5 are sig¬ nificantly less than that of Group 1 (P less than 0.005, 0.01, 0.002 and 0.05, respectively).

** Mean measured footpad swelling minus the nonspecific swelling caused by the solvent alone (4.00 in units of 0.10 mm)

TABLE 8

MEASURED ACTIVE

FOOTPAD SWELLING CARRAGEENAN SWELLING GROUP (% increase of diameter) (% increase in diameter)

2 hours 3 hours 2 hours 3 hours

1 29.08 ± 2.35 26.83 ± 3.06 17.90 15.60

2 20.75 ± 1.69 15.08 ± 1.61 9.60 4.10

3 15.08 + 1.99 16.67 ± 1.56 4.60 6.00

4 19.17 ± 1.49 14.17 ± 1.38 9.20 3.40

5 22.50 ± 1.96 18.58 ± 2.03 11.90 8.00 TABLE 9

DRUG EFFECT ON INFLAMMATION AS % OF CONTROL

2 Hours 3 Hours

Inosine -46 -74

GR-SP98 -15 -74 -62

Inosine -R -51 -78

GR-SP98 -15-R -33 -49

Example 26

Five groups of six CD-I mice each, 6 weeks old, average weight 27.5 grams, purchased from Charles River, were given 0.1 ml of the following substances by oral gavage:

Treatment Dose given mouse

Group 1 Distilled water

Group 2 A solution of standard inosine

(Sigma Lot 13F-0738) 1 mg/kg

Treatment (cont'd)

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

The solutions of standard inosine and GR-SP98-15 were at concentrations chosen so that each mouse received the dose designated in the third column in the 0.1 ml.

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.

The results are shown in Tables 10-12 and establish that the optimum anti-inflammatory oral dose of GR-SP98-15 is about 1 mg/kg.

TABLE 10

MEASURED SPECIFIC FOOTPAD SWELLING ** CARRAGEENAN SWELLING*

GROUP (in units of 0.10mm) (in units of 0.10mm)

2 hours 3 hours 2 hours 3 hours

1 6.67 ± 0.47 6.17 ± 0.65 2.67 2.17

2 7.83 ± 0.55 5.25 ± 0.73 3.83 1.25

3 5.17 ± 1.15 4.42 ± 0.80 1.17 0.42

4 3.58 ± 0.62 3.83 ± 0.47 0 0

5 5.42 ± 0.36 6.83 ± 0.24 1.42 2.83

* Mean measured footpad swelling minus the nonspecific swelling caused by the solvent alone (4.00 in units of 0.10 mm) .

** Analysis of the data using the student's t test shows that, at two hours, the mean footpad swelling of Groups 4 and 5 are significantly less than that of Groups 1 and 2 (P less than 0.001, 0.05 respectively). Also the swelling of Group 5 is significantly less than that of Group 2 (P less than 0.05). At three hours the footpad swelling of Group 4 is significantly less than that of Group 1 (P less than 0.01).

TABLE 11

MEASURED SPECIFIC FOOTPAD SWELLING CARRAGEENAN SWELLING

GROUP (% increase of diameter) (% increase in diameter)

2 hours 3 hours 2 hours 3 hours

1 18.17 ± 1.29 16.83 ± 1.93 9.39 7.43 2 20.42 ± 1.46 13.92 ± 1.95 11.02 4.52 3 14.00 ± 3.20 12.08 ± 2.29 4.60 2.68

4 9.67 ± 1.65 10.33 ± 1.31 0.21 0.93 5 14.17 + 0.90 17.92 ± 0.68 4.77 8.52

TABLE 12

EFFECT ON INFLAMMATION AS 5 If. OF CONTROL

-

- 2 Hours 3 Hours

Inosine +43 -42 (1 mg/kg)

GR-SP98-15 -56 -80 (10 mg/kg)

GR-SP98-15 -100 -100 (1 mg/kg)

GR-SP98-15 -47 +30 (0.1 mg/kg)

Example 27

Six groups of six HA/ICR mice, 7 weeks old, average weight 25.6 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 Group 4 A solution of GR-SP98-15.2 in distilled water Group 5 A solution of GR-SP98-17 in distilled water Group 6 A solution of GR-SP98-18 in distilled water Group 7 A solution of GR-SP98-19 in distilled water

The solutions of standard inosine and of the other substances listed above were at concentrations chosen so that each mouse received 1 mg/kg of each sub¬ stance in the 0.1 ml volume.

Two hours after the oral gavage treatment, the 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.

The results are given in Tables 13-15. These results establish that mouse carrageenan footpad edema is not inhibited by 1 mg/kg standard inosine or 1 mg/kg of GR-SP98-17. In contrast 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).

GR-SP98-17 was prepared without heating the standard inosine (see Example 8). Thus, these results show that an energy barrier separates the different polymorphic configurations of inosine.

TABLE 13

MEASURED SPECIFIC FOOTPAD SWELLING * CARRAGEENAN SWELLING**

GROUP J'i units of 0.10mm) (in units of 0.10mm)

2 hours 3 hours 2 hours 3 hours

1 7.33 ± 0.71 7.17 ± 0.53 3.33 3.17

2 7.58 ± 0.58 7.17 ± 0.66 3.58 3.17

3 4.50 ± 0.56 5.25 ± 0.60 0.50 1.25

4 6.08 ± 0.62 7.17 ± 0.58 2.08 3.17

5 7.30 ± 0.68 6.00 ± 0.70 3.30 2.00

6 4.08 ± 0.72 5.58 ± 0.91 0.08 1.58

7 4.58 ± 0.82 4.75 ± 0.70 0.58 0.75

* Analysis of the data using the student's t test shows that, at two hours, the mean footpad swelling of Groups 3, 6 and 7 is significantly less than that of Group 1 (P less than 0.005, 0.005 and 0.02, respectively). Also, the mean footpad swelling of Groups 3, 6 and 7 is significantly less than that of Group 2 (P less than 0.001, 0.002, 0.01, respectively). At three hours Groups 3 and 7 remain significantly less than Group 1 (P less than 0.05, 0.02 respectively). Also, the mean footpad swelling of Groups 3 and 7 remains significantly less than that of Group 2 (P less than 0.05, 0.02 respectively)

** Mean measured footpad swelling minus the nonspecific swelling caused by the solvent alone.

TABLE 14

MEASURED SPECIFIC

FOOTPAD SWELLING CARRAGEENAN SWELLING

GROUP (% increase of diameter) (% increase in diameter)

2 hours 3 hours 2 hours 3 hours

1 19.75 ± 1.85 19.25 ± 1.46 10.35 10.84

2 29.50 ± 1.66 19.50 ± 1.91 11.10 10.10

3 11.70 ± 1.44 13.58 ± 1.50 2.30 4.18

TABLE 14 ( cont ' d)

MEASURED SPECIFIC

FOOTPAD SWELLING CARRAGEENAN SWELLING GROUP (% increase of diameter) (% increase in diameter)

2 hours 3 hours 2 hours 3 hours

4 15.41 ± 1.56 18.30 ± 1.46 6.01 8.90

5 17.00 ± 2.08 15.70 ± 1.93 7.60 6.30

6 10.90 ± 2.15 14.90 ± 2.76 1.50 5.50

7 17.67 ± 2.02 12.70 ± 1.96 3.30

TABLE 15

EFFECT ON INFLAMMATION AS % OF CONTROL 2 Hours 3 Hours

Standard Inosine +7.5 0

GR-SP98-15 -85 61

GR-SP98-15. 2 -38 0

GR-SP98-17 -1 -37

GR-SP98-18 -98 -50

GR-SP98-19 -83 -76

Example 28

Three groups of six Sprague Dawley rats, 7 weeks old, average weight 208 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

The solutions of standard inosine and GR-SP98-15 were at concentrations chosen so the rats each received 50 mg/kg of these substances in. the 0.1 ml volume.

Two hours after the oral gavage treatment, 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.

The results are shown in Tables 16-18. These results demonstrate that the anti-inflammatory activity of GR-SP98-15 can be distinguished from that of stan¬ dard inosine in rats as well as in mice. However, in this study, the anti-flanimatory effect of GR-SP98-15 was seen only in the rats exhibiting milder inflamma¬ tion (the lowest 1/3 or 1/2 of all paws)..

TABLE 16

MEASURED FOOTPAD SWELLING. GROUP (in units of 0.10mm)

1 29.00 ± 2.04

2 32.25 ± 1.85

3 28.17 ± 3 .36

* Analysis using the student's t test shows that the mean footpad swelling for all animals of the three groups are not significantly different from each other.

TABLE 17

MEASURED FOOTPAD SWELLING

GROUP (in units of 0.10mm)

Lowest 1/3 Upper 2/3 Of All Paws Of All Paws

1 21.25 ± 2.39 32.88 ± 1.47 2 26.00 ± 1.00 35.38 ± 1.91 3 14.50 ± 2.25 35.00 ± 2.35

* Analysis using the student's t test shows that the mean footpad swelling of the lowest 1/3 of paws in rats treated with GR-SP98-15 is significantly less than the swelling of the lowest 1/3 of paws in rats treated with standard inosine (P less than 0.001). The mean footpad swelling of the upper 2/3 of paws is not significantly different for the three treatment groups.

TABLE 18

MEASURED FOOTPAD SWELLING

GROUP (in units of 0.10mm)

Lowest 1/2 Upper 1/2 Of All Paws Of All Paws

1 23 . 67 ± 2 . 16 34. 33 ± 1 . 52 2 27 . 00 ± 0. 93 37 . 50 ± 1 . 77

18. 67 ± 3 . 04 37 . 67 ± 2 . 09

* Analysis using the student's t test shows that the mean footpad swelling of the lowest 1/2 of paws in rats treated with GR-SP98-15 is significantly less than the swelling of the lowest 1/2 of paws in rats treated with standard inosine (P less than 0.005). The mean footpad swelling of the upper 1/2 of paws is not significantly different for the three treatment groups.

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

The solutions of standard inosine and GR-SP98-15 were at concentrations chosen so that the rats each received 10 mg/kg of these substances in the 0.1 ml volume.

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.

The results are shown in Table 19. These results demonstrate that the anti-inflammatory activity of GR-SP98-15 is far more powerful than that of standard inosine in older rats.

TABLE 19

MEASURED FOOTPAD SWELLING GROUP (in units of 0.10mm)

1 31.50 ± 2.17

2 27.75 ± 2.51

3 22.41 ± 1.71

* Analysis using the student's t test shows that the mean footpad swelling of Group 3 is significantly less

than that of Group 1 (P less than 0.005). The mean footpad swelling of Group 2 is not significantly dif¬ ferent from that of Group 1 or 3.

Example 30

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.

Three groups of eight Sprague Dawley rats, retired female breeders, average weight 270 grams, pur¬ chased 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

The solutions of standard inosine and GR-SP98-15 were at concentrations chosen so that each rat received 10 mg/kg of these substances in the 0.1 ml volume.

Two hours after the oral gavage treatment, 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.

The results are shown in Table 20. In con¬ trast to results in aged male rats, neither standard inosine nor GR-SP98-15 affected carrageenan footpad edema in retired female breeders.

TABLE 20

MEASURED FOOTPAD SWELLING GROUP (in units of 0.10mm)

1 30.00 ± 2. 15

2 30.50 ± 1.99

3 32.25 ± 1.94

* Analysis using the student's t test shows that the mean footpad swelling of each of these groups is not significantly different from the others.

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:

Treatment Group 1 Saline

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.

After the subcutaneous injections, 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 .

Prior to the zymosan injection and 2 hours ^" after the zymosan injection, dorsoventral footpad diameters were measured as described in Example 22.

The results are presented in Tables 21-23. These results show that, at 20 mg/kg, both standard inosine and GR-SP98-7 significantly inhibited zymosan- induced inflammation in the mouse paw.

TABLE 21

MEASURED SPECIFIC ZYMOSAN- FOOTPAD SWELLING * INDUCED SWELLING **

GROUP (in units of 0.10mm) (in units of 0.10mm)

1 16.70 ± 0.56 12.70

2 • 12.00 ± 0.84 8.00

3 11.70 ± 1.08 ^• 7.70

* The mean footpad swellings of Groups 2 and 3 are both significantly different from that of Group 1 (P less than 0.001).

** Mean measured footpad swelling minus the nonspecific swelling caused by the solvent alone.

TABLE 22

MEASURED ACTIVE ZYMOSAN-

F00TPAD SWELLING INDUCED SWELLING GROUP (%increase of diameter) (% increase in diameter)

1 70.30 ± 4.00 54.24

2 49.50 ± 4.66 33 .44

3 47.10 ± 5.68 31.04

TABLE 23

EFFECT ON INFLAMMATION AS % OF CONTROL

Inosine -37

GR-SP98-7 -39

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.

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.

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.

One hour after the oral gavage treatment, 6.6 ug chymotrypsin in 0.05 ml saline was injected into each of the dorsoventral footpads of each of the mice of each group. Injections were carried out under ether anesthesia.

Just prior to and at 2 hours after the chymotrypsin injection, dorsoventral footpad diameters of each mouse in each group were measured as described in Example 22.

The results of this experiment are presented in Figure 6. As shown there, GR-SP98-15 produced a significant suppression of chymotypsin-induced footpad swelling at 2 hours compared to control, whereas standard inosine did not. Also, GR-SP98-15 gave significantly greater suppression of footpad swelling than did standard inosine.

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

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. Group 4 A solution of GR-SP98-22 in distilled water. Group 5 A solution of GR-SP98-26 in distilled water.

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.

One hour after the oral gavage treatment, 10 ug of chymotrypsin in 0.05 ml saline was injected into each of the dorsoventral footpads of each of the mice of each group. Injections were carried out under ether anesthesia.

Just prior to and at 2 hours after the chymotrypsin injection, dorsoventral footpad diameters of each mouse in each group were measured as described in Example 22.

The results of this experiment are presented in Figure 7. As shown there, standard inosine and all of the inosine polymorphs significantly decreased chymotrypsin-induced footpad swelling compared to the control. GR-SP98-26 also produced a significant sup¬ pression of chymotrypsin-induced footpad swelling at 2 hours compared to standard inosine.

Example 34 Seven 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. Group 2 A solution of standard inosine

(Sigma, Lot 13F-0738) in distilled wate . 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.

One hour after the oral gavage treatment, 10 ug of chymotrypsin in 0.05 ml saline was injected into each of the dorsoventral footpads of each of the ^' mice of each group. Injections were carried out under ether anesthesia.

Just prior to and at 2 hours after the chymotrypsin injection, dorsoventral footpad diameters of each mouse in each group were measured 'as described in Example 22.

The results of this experiment are presented in Figure 8. As shown there, GR-SP98-26, -28, -38, -39

and -66 produced significant suppression of chymotrypsin- induced footpad swelling at 2 hours compared to control and to standard inosine.

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:

Treatment Group 1 Saline Group 2 A solution of standard inosine

(Sigma, Lot 13F-0738) in saline. 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.

One hour after the oral gavage treatment, 10 ug of chymotrypsin in 0.05 ml saline was injected into each of the dorsoventral footpads of each of the mice of each group. Injections were carried out under ether anesthesia.

Just prior to and at 2 hours after the chymotrypsin injection, dorsoventral footpad diameters of each mouse in each group were measured as described in Example 22.

The results of this experiment are presented in Figure 9. As shown there, GR-SP98-15 and 26 produced significant suppression of chymotrypsin-induced footpad swelling at 2 hours compared to the control. The two standard inosines also produced significant suppression compared to control.

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:

Treatment Group 1 Distilled water. Groups 2 and 3 A solution of standard inosine

(Sigma, Lot 13F-0738) in distilled water. Groups 4 and 5 A solution of GR-SP98-15 in distilled water.

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.

One hour after the oral gavage treatment, 6.6 ug of trypsin in 0.05 ml saline was injected into each of the dorsoventral footpads of each of the mice of each group. Injections were carried out under ether anesthesia.

Just prior to and at 2 hours after the trypsin injection, dorsoventral footpad diameters of each mouse in each group were measured as described in Example 22.

The results of this experiment are presented in Figure 10. As shown there, GR-SP98-15 produced a significant suppression of trypsin-induced footpad swelling at 2 hours compared to control and to standard inosine at the two doses tested.