WO1988008747A1 - Microencapsulation - Google Patents

Abstract

Microcapsules of a polymeric material comprising gelatin and the polysaccharide XM6 obtainable from the bacterium NCIB 11870 can be prepared by complex coacervation of these polymers over a wide range of pH and concentration.

Description

POLYMORPHS OF XANTHOSINE AND METHODS OF MAKING AND USING THEM

This application is a continuation-in-part of application Serial No. , for "Polymorphs Of

Xanthosine And Methods Of Making And Using Them," filed May 15, 1987. Attention is also drawn to Applicant's co-pending application Serial No. , for

"Polymorphs of Inosine 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 xanthosine are known. One of these crystal polymorphs is the dihydrate form of xanthosine. The final crystallization procedure used in the preparation of this form of xanthosine in¬ volves the heating of a two percent suspension of xanthosine in water until solution of the inosine occurs at about 100 C. This is followed by precipitation and harvesting of the xanthosine. The resultant crystals are long prisms which decomposes on heating and have no well-defined melting range. "Standard xanthosine" is defined herein to be xanthosine dihydrate which is pre¬ pared using this crystallization procedure as the final step.

In addition to the dihydrate form of xanthosine, a second xanthosine crystal polymorph is known which is the anhydrous form. This form of xanthosine crystallizes from alcohol to form felted clusters (warts). It also decomposes upon heating and has no distinct melting range.

Polymorphism in the solution state has long been believed to be non-permissible for energetic reasons. It has generally been assumed that the mole¬ cules 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 po¬ tential energy necessary to create the solution state. The basic solution event may even be followed by aggrega¬ tion of the solute, but it has been assumed that there is an absence of energy barriers between solute aggre¬ gate forms. Indeed, the tendency of nucleosides such as xanthosine to aggregate in aqueous solution has been known for many years. This association has been re¬ ported 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 solu¬ tion.

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 hydrogen bond between solute and solvent, and no form of solute-solute association other than the base stack¬ ing discussed above and hydrogen-bonding has been anti¬ cipated 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 readings, they speculate that the differences do reflect different conformations of the solute forms of the two crystal polymorphs and that these differences in conformation may give rise to differences in the biological accessibility (rate of passage into bio¬ logical fluids of an animal) and the 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-diethylbar- bituric 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 principle 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 be obtained using water as the solvent. Further, it is merely speculated in Leonidov and Agafonov that polymorphs of the same compound might have different biological activities. No data were presented to sup¬ port this theory.

Although they do not deal with polymorphism, U.S. Patents Nos. 3,646,007, 3,728,450, 3,857,940 and 4,512,981 do disclose complexes of inosine and lower alkyl aminoalcohols of the formula:

^N<^Cn^H2n>^0H

wherein R- and R₂ are lower alkyl and n is an integer of 2 to 4. The complexes are prepared by mixing the inosine and alkyl aminoalcohol in a ratio of from 1:1 to 10:1 at room temperature. Complexes can also be prepared by mixing, in these same ratios and at room temperature, inosine and a salt of the alkyl aminoalcohol which has been formed by reacting the alkyl aminoalcohol with a pharmacologicallyacceptable acid. No heating is used, and no neutralization or other chemical reaction takes place during complex formation or is needed for complex formation. The complexes have pharmacological activity, including antiviral, anti-inflammatory and immunoregulatory activities and the ability to restore deteriorated learning and memory behavior.

BRIEF DESCRIPTION OF THE INVENTION

According to one aspect of the invention, there is provided a method of preparing a crystal polymorph of a xanthosine salt comprising: providing a solvent; adding to the solvent a quantity of xanthosine; adding to the xanthosine an equimolar amount of a strong base; heating the solvent, base and xanthosine at a predetermined rate to a temperature sufficient to cause the xanthosine to go into solution and to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration; precipitating the xanthosine salt; cooling the salt at a predetermined rate for a predetermined period of time; and completely lyophilizing the salt to yield the crystal polymorph of the xanthosine salt. The invention also comprises a method of syn¬ thesizing a crystal polymorph of xanthosine identical to the method just described, except that no extraneous heat is applied, and the heat generated by the neutrali¬ zation reaction alone causes the temperature to rise to a temperature sufficient to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration. Under some conditions of synthesis in both of these processes, the xanthosine salts precipitate spontaneously, while under other con¬ ditions, additional steps must be taken to precipitate them. Especially preferred bases are the alkali metal hydroxides and alkylamino alcohols such as choline and dimethyl minoisopropanol.

Also part of the invention are the crystal polymorphs produced by these processes.

According to another aspect of the invention, there are provided solute polymorphs of the xanthosine salts. These polymorphs exhibit different physical- chemical properties, compared one to the other and to the solute form of standard xanthosinate, which reflect their different configurations. The solute polymorphs of the invention also exhibit dramatically different biological activities compared to standard xanthosinate.

There is also provided a method of preparing a solute polymorph of a xanthosine salt comprising: providing a solvent; adding to the solvent a quantity of xanthosine; adding to the xanthosine an equimolar amount of a strong base; heating the solvent, base and xanthosine at a predetermined rate to a temperature sufficient to cause the xanthosine to go into solution and to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration; and cooling the solution at a predetermined rate for a predetermined period of time.

The invention also comprises a method of pre¬ paring a solute polymorph of xanthosine identical to the method just described, except that no extraneous heat is applied, and the heat generated by the neutrali¬ zation reaction causes the temperature to rise to a temperature sufficient to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration. Solute polymorphs of xanthosine salts may further be prepared by dissolving the xanthosinate crystal polymorphs of the invention, described above, in a solvent.

According to another aspect of the invention, there are provided crystal and solute polymorph of xanthosine.

According to yet another aspect of the invention, there are provided novel salts of xanthosine having the formula:

(Xs)^" (CB)⁺

wherein Xs is the xanthosinate ion and CB is the cation of a strong base.

Finally, there are provided: (1) a method of modulating the immune response of an animal comprising administering to the animal a composition comprising a pharmaceutically-acceptable solvent or carrier and an effective amount of a solute or crystal polymorph of xanthosine or of a solute or crystal polymorph of a xanthosine salt; and (2) compositions for use in this method.

DEFINITIONS As used herein, the term "conformation" refers to differences in the structure of the xanthosine or xanthosine salt monomeric 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 different patterns of association between the xanthosine or xanthosine salt monomeric units.

"Xanthosine" will be used herein to refer to all forms of xanthosine, including the known crystal polymorphs of xanthosine and the novel crystal and solute polymorphs of the invention. However, Applicant does not intend the known crystal polymorphs of xanthosine to be a part of the invention, and they are specifically disclaimed.

"Xanthosinate" or "xanthosine salt" will be used herein to refer to all salts of xanthosine formed by neutralizing xanthosine with a strong base.

"Standard xanthosinate" is defined herein to mean standard xanthosine which has been neutralized in aqueous solution at low concentration (1 mg/ml) by the addition of a molar equivalent of sodium hydroxide over a period of about three hours, with stirring, but without heating and without generating heat.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a bar graph showing the increase in pH for-several salts when the concentration of the salts is changed from 2.7 x 10 -4M to 0.4M.

FIGURE 2 is a graph of concentration versus relative extinction coefficient for standard xanthosinate and for three solute xanthosinate polymorphs which shows the deviation from Beer's law produced by these compounds.

FIGURE 3 is the x-ray powder diffraction pattern for standard xanthosine.

FIGURE 4 is the x-ray powder diffraction pattern for crystal xanthosinate polymorph NaXs-I. FIGURE 5 is the x-ray powder diffraction pattern for the crystal xanthosinate polymorph NaXs-II.

FIGURE 6 is the x-ray powder diffraction pattern for the crystal xanthosinate polymorph ChXs-I.

FIGURE 7 is the x-ray powder diffraction pattern for the crystal xanthosinate polymorph ChXs-II.

FIGURE 8 shows the optical rotary dispersion spectra of aqueous solutions of xanthosinate polymorphs ChXs-I, ChXs-II, NaXs-I and DMAIPXs.

FIGURE 9 is a graph of mortality versus time after lethal irradiation for mice treated with several xanthosinate solute polymorphs.

FIGURE 10 shows the suppression of trypsin footpad edema by xanthosinate solute polymorphs ChXs-I and ChXs-II and a mixture of choline chloride and standard xanthosine at various treatment doses.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The invention comprises novel xanthosine solute and crystal polymorphs and novel xanthosinate solute and crystal polymorphs. Each polymorph has distinct physical, chemical and biological properties. As regards the individual xanthosine monomers of which the polymorphs are composed, the changes produced in them by the methods of the invention can only be stereo- chemical, i.e., changes in relative position-in-space of functional and other chemical groups that contribute to the isomeric, tautomeric, and rotomeric systems of xanthosine.

The interrelation between these stereochemical conformations and the energetics of their interconversion have been studied by Ludemann and co-workers in liquid ammonia (Eur. J. Biochem, 49, 143 (1974) ). Xanthosine is sufficiently soluble in this solvent to effect a 0.6 M solution without engaging in complexation as would be the case in water. In ammonia, the energy barrier between the various stereochemical forms of xanthosine is very low, in the neighborhood of the energy of a single hydrogen bond. Thus the time taken to achieve equilibrium is much less than one second.

If the different preparative methods of the invention led only to a different distribution between possible forms-in-space of the xanthosine monomers, then when such monomers were placed in solution, all differences in distribution would be erased in less than a second regardless of the distribution at the start. However, this is not the case. The xanthosine and xanthosinate polymorphs of the invention retain their physical and chemical identity in solution for several days. The persistence of differences in poly¬ morphic configuration between solutions of polymorphs can only be explained by interaction between monomers (i.e., aggregation). This would increase the energy barrier between monomer forms by the energy of an aggregation term.

Synthesis of Xanthosine Salt Polymorphs

The following variables are important in the synthesis of the novel xanthosine salt polymorphs (both crystal and solute polymorphs) of the invention:

1. Solvent. Xanthosine is virtually in¬ soluble in nonpolar solvents. Thus, polar solvents must be used. Suitable polar solvents include water, dimethylformamide, ammonia and lower alkyl alcohols such as ethanol, methanol, propanol and butanol.

The kinds of bonds that the solvent forms with the xanthosine prior to formation of the crystal and solute xanthosinate polymorphs of the invention is an important factor in polymorph formation. The use of one polar solvent versus another leads to the formation of different polymorphic configurations of xanthosine salts 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 xanthosine salt polymorphs. The temperature must be elevated suffi¬ ciently high so that the xanthosine goes into solution and so that the energy barriers which prevent conver¬ sion of one polymorphic configuration of xanthosine to another are overcome. The heat generated by the neutralization of xanthosine with the strong base is sufficient to overcome these energy barriers. However, heating the xanthosine and strong base to a higher tem¬ perature by applying extraneous heat further enhances polymorph formation and influences the character of the final polymorph. It is not known yet what types of forces create these energy barriers, but their exist¬ ence is shown by the fact that the use of elevated tem¬ peratures is necessary to obtain formation of the crystal and solute polymorphs of the invention. In addition, the variation of temperature with time in both the heating and cooling cycles affects the development of the xanthosine salt polymorphs and the nature of the final polymorph which is formed.

3. Concentration. The concentration of the xanthosine used is also critical to the formation of the novel xanthosine salt polymorphs of the invention. Further, as the xanthosine salts form, higher concen¬ trations of xanthosine go into solution since the resultant salts are highly soluble in the polar solvents, thereby enhancing polymorph formation.

Concentration is important because contact between solute molecules is necessary for the aggrega¬ tion of molecules. As a first principle, as concen¬ tration increases, contact and aggregration become more likely. This is not necessarily true, however, if the molecules in question possess domains that attract each other and possess fixed charges that naturally repel. The anionic functional group of xanthosinate after neutralization puts xanthosinate in this class of mole¬ cules. However, aggregrates do form between ions of like sign. The capacity for the interplay between attraction and repulsion, especially in the presence of counter ions (e.g. , choline , Na ) makes the associative ' outcome between xanthosinate ions unpredictable.

4. Lvophi1ization. Although lyophilization is not necessary for polymorph formation, the use of lyophilization instead of other methods of crystal iso¬ lation, such as suction filtration and air drying, con¬ tributes to the nature of the final crystal polymorph and of the solute polymorph which forms when this crys¬ tal polymorph is dissolved in a solvent.

5. Base. The strong base used to neutralize the xanthosine also affects the final nature of the polymorph formed.

Example 1 To a 600 ml VirTis lyophilization flask were added 0.4 mole of standard xanthosine (xanthosine dihy¬ drate from Pharma Waldhof) and 0.4 mole of choline in methanol (110 ml a solution containing 45% choline weight-to-volume) . The flask and its contents were then heated with stirring so that the temperature rose from room temperature to 80°C in 20 minutes. The xanthosine dissolved during the course of the heating, but when the temperature was between 75° and 80°C, an abrupt precipitation occurred. The precipitated material became custard-like and adhered to itself and the glass walls of the flask to such a degree that the flask could be turned upside down without causing spillage. The flask was next removed from the heater- stirrer and was allowed to cool at room temperature for 30 minutes. After cooling, the contents of the flask were slurried using a glass stirring rod. Then the flask was placed on a VirTis lyophilizer (model Freeze- mobile 12), and the material was lyophilized for 48 hours, at which time lyophilization was complete. The flask was removed from the lyophilizer, and the resultant lyophilized material was harvested and pul¬ verized to a fine powder. The powder obtained was a slightly off-white powder. It proved not to have the hygroscopicity of the common choline salts, such as choline chloride. The powder should still, however, be routinely stored in a desiccator under vacuum. This xanthosine salt will be referred to hereinafter as ChXs-I.

Example 2

To a 600 ml VirTis lyophilization flask were added 0.4 moles of standard xanthosine (xanthosine dihydrate from Pharma Waldhof) and 0.4 moles of choline in methanol (110 ml of a solution of choline 45% weight- to-volume). The contents of the flask were actively stirred for 45 minutes at room temperature without any heating.

A thin precipitate appeared after about 40 minutes of stirring, but the reaction mixture remained fluid and pourable. After 45 minutes, stirring was discontinued and the reaction mixture was allowed to sit at room temperature for another 20 to 30 minutes.

The flask was next placed on a VirTis lyophi¬ lizer for 48 hours, at which time lyophilization was complete. The resultant lyophilized material was harvested and pulverized to a fine powder which was slightly off-white in color and which, like the material produced in Example 1, lacked the hygroscopicity of the common choline salts. This xanthosine salt will be referred to herein as ChXs-II.

Example 3 To a 600 ml VirTis lypohilization flask were added 0.4 moles of standard xanthosine (xanthosine dihydrate, Pharma Waldhof), 0.4 moles of redistilled dimethylamino isopropanol and 51 ml of methanol. The contents of the flask were stirred and progressively heated from room temperature to 80°C over a 20-minute period. Significant events in regard to solubilization, precipitation and gross appearance of the precipitate were the same as those set forth in Example 1. The remainder, of he processing was also- as set forth in Example 1, and the resultant powder was off-white and show a lack of hygroscopicity. This xanthosine salt will be referred to hereinafter as DMAIPXs.

Example 4

To a 600 ml VirTis lyophilization flask were added 0.4 mole of standard xanthosine (xanthosine dihy¬ drate, Pharma Waldhof), 0.4 mole of sodium hydroxide in pellet form and 160 ml of water. The contents of the flask were stirred and heated progressively from room temperature to 80°C over a period of 30 minutes. No precipitate developed.

Next, 200 ml of methanol was added to the flask while heating and stirring continued. Heavy pre¬ cipitation then developed similar in nature to that observed in Example 1. The flask was then removed from the heat and allowed to sit at room temperature for another 45 minutes. The material within the flask was then lyophilized for 48 hours, at which time lyophili¬ zation was complete. The resultant lyophilized material was a non-hygroscopic white powder. This xanthosine salt will hereinafter be referred to as NaXs I.

Example 5 Another xanthosine salt was prepared using the procedure described in Example 4, except that the ingredients were not heated. They were simply stirred for 30 minutes before the addition of the methanol. The resultant lyophilized material was a white, non- hygroscopic powder. This xanthosine salt will be referred to hereinafter as NaXs-II.

Example 6

Another xanthosine salt was prepared according to the procedure of Example 4, except that 160 ml, rather than 200 ml, of methanol was used. As a consequence of using a smaller amount of methanol, a thready precipitate was formed, but this precipitate subsequently re-dissolved. Thus, the flask placed on the lyophilizer contained a solution rather than a solid, and the solid emerged during the lyophilization step.

The solution in the flask was lyophilized for 48 hours, at which time lyophilization was complete. Then, the flask was removed from the lyophilizer and the lyophilized material was pulverized to a powder. The resultant white powder was non-hygroscopic. This xanthosine salt will hereinafter be referred to as NaXs-III.

Example 7 Another xanthosine salt was made according to the procedure of Example 5, except that only 160 ml of methanol were added. As a consequence, a precipitate developed, but this precipitate did not persist. The material put on the lyophilizer was a solution, and the precipitate developed during lyophilization. The product obtained following lyophilization was a white non-hydroscopic powder. This xanthosine salt will be referred to hereinafter as NaXs-IV.

Example 8 Another xanthosine salt was made according to the procedure of Example 6, except that prior to the addition of 160 ml of methanol, the reaction mixture^' was cooled to 20°C by placing it in an ice bath. Thus, the period of heating was followed by cooling before addition of the alcohol. As in Example 6, a precipitate formed upon addition of the methanol. The precipitate subsequently re-dissolved, and a solution was put on the lyophilizer. The final product obtained after lyophilization was a white non-hygroscopic powder, and this material will be referred to herein as NaXs-V.

Example 9 Another xanthosine salt was made according to the procedure set forth in Example 4, except that only 100 ml of water was added. Subsequent stirring and heating produced an active and permanent precipitation. This occurred approximately 20 minutes following the initiating of heating when the temperature of the reac¬ tion mixture was at about 70°C. This precipitate was lyophilized. The product obtained following lyophili¬ zation was a white non-hygroscopic powder. This xanthosine salt will be herein referred to as NaXs-VI.

Synthesis of Xanthosine Polymorphs The same variables as described above for the formation of the xanthosinate polymorphs are also impor¬ tant in the synthesis of the novel xanthosine crystal and solute polymorphs of the invention. With respect to concentration, xanthosine is not ionized so the factor of repulsive forces between ions of like charge does not come into play.

Physical and Chemical Properties

The existence of polymorphic configurations of the xanthosine salt polymorphs is demonstrated by the fact that these polymorphs have:

(1) different degrees of hydrolysis at high concentration, and thus different pHs of concentrated aqueous solutions;

(2) different solubilities in butanol, a relatively hydrophobic solvent, and in heavy water, a solvent of high hydrogen bond strength;

(3) unique departures from Beer's law;

(4) different x-ray powder diffraction patterns; and

(5) different optical rotary dispersion (ORD) spectra.

1. Hydrolysis The nucleoside xanthosine is an organic acid, weak in the formal sense, whose pK Cl (half-ionization pH) is identified in the literature variously, as either 5.5. or 5.7. Xanthosine salts can be formed by reacting xanthosine with strong bases. This neutraliza¬ tion of xanthosine proceeds as follows: (xanthosine) + OH~ +

(strong-base cation) (xanthosinate)^" + H,0 + (strong- base cation) . The xanthosinate salt and water subse¬ quently undergo hydrolysis which elevates the pH of the solution. This interaction is typical of salts formed from weak acids and strong bases and occurs because the anion of these salts has a far stronger tendency to associate with hydrogen ions in water than the cation has to associate with hydroxide ions. The hydrogen and hydroxide ions, of course, are provided by water it¬ self, according to its dissociation constant: H,0 = H⁺ + OH^", K ^*= 10 . Hydrolysis proceeds as fol-

— + 5 6 lows: (xanthosinate) + H = (xanthosine), K = 10 , and is governed in its intensity by the "weakness", or pK ex of xanthosine, and by the concentration of the xanthosinate anion.

The hydrolysis of a salt of a weak acid and a strong base renders the solution alkaline. This alkaline

• _ change reflects the extent of the hydrolysis reaction and depends on solute anion pK and on solute anion concentration. Across the same concentration range, disparate salts of the weak base-strong acid type, in the absence of association or aggregation, will exhibit nearly identical relative pH increases due to hydrolysis.

As shown in Eigure 1, when the concentration of dimethylamino isopropanol hydrochloride, dibasic sodium phosphate, inosine which had been neutralized at room temperature with sodium hydroxide or standard xanthosinate was increased from 0.0027 M to 0.4 M, the increase in pH due to hydrolysis of these salts was between 1.50 and 1.75 pH units. On the other hand, the analogous increase in pH for the sodium xanthosinates was between 2.3 and 3.40 pH units (see Figure 1). The xanthosine salts of the invention all exhibit greater hydrolysis than the other weak acid-strong base salts, including standard xanthosinate. Given that pH is a log scale, the average difference in terms of relative change in hydroxide ion concentration is 10-100 times as great. For the xanthosine salts of the invention, pH differences are largely absent at low concentration.

The elevation in pH produced during hydrolysis is a function of the dissociation constant of water, the solute anion concentration and the solute anion pK . The relationship between these variable is ex¬ pressed in the equation of Glasstone: pH = 1/2 pK + 1/2 pK + 1/2 log (concentration) . Since the dissociation constant of water is a constant and the concentration is the same for all of the xanthosine salts, the only remaining variable is the pK ex, the 50% ionization pH of the acidic group.

The pKa's of the xanthosinates of the invention become uniquely alkaline with increasing concentration. In general, the pK cL of an acidic compound reflects the anionic charge density of the ionized group, and

• _ the higher (more alkaline) the pK ex, the more electro- negative would be its polarization. Thus the ionizing groups on the claimed xanthosine salt polymorphs change in character, each uniquely, as xanthosinate concentra¬ tion increases. This change must reflect aggregration between xanthosinates as their concentration increases. Since the number of ionizable sites is constant, the difference in the pKa's of the xanthosine salts of the invention must reflect a different organization of those sites, and the increase in pKx must reflect differences in the configuration of the xanthosine salts of the invention.

The use of the strong base choline radically changes the physical and chemical properties of the xanthosinate salts formed compared to the alkali metal xanthosinates. Choline xanthosinate exhibited a very small degree of hydrolysis in aqueous solution at the concentrations under consideration. Also two separate lines of investigation have established that, in aqueous solution, choline remains bound to xanthosinate to a considerable degree. Since this ion pairing eliminates the anionic feature of xanthosinate, it could explain the reduced degree of hydrolysis of the choline xantho¬ sinates. As a consequence, the pH of concentrated solu¬ tion of choline (and related alkylamino alcohols) salts of xanthosine do not rise into the alkaline range (8.5 - 10.5) but remain nearly neutral (7.5). This is an important characteristic of alkylamino alcohol salts of xanthosine since it permits them to be used jLn vivo in high concentration without producing injury.

2. Solubility

Choline and alkylamino alcohol salts of xanthosine exhibit other unique physical-chemical features. Thus while the alkali metal salts have solubilities in n-butanol that are very much less than unionized xanthosine, being reduced by from 95.0 to 99.5% according to the species, choline salts have solubilities in n-butanol that are very close to that of unionized xanthosine. These results are presented in Table 1.

Table 1

Compound Relative Solubility in n-butanol compared to standard xanthosine

Standard Xanthosine 1.00

ChXs-I 0.93

ChXs-II 0.67

DMAIP-Xs 0.20

NaXs-I 0.047

NaXs-II 0.,013

NaXs-III 0..004

NaXs-IV 0.,015

NaXs-V 0.,045

NaXs-VI 0,.005

The high solubility of the alkylamino alcohol salts of xanthosine in aqueous solution is anomalous as well. While alkali metal salts of xanthosine dissolve in water up to about 0.45 M, alkylamino alcohol salts begin to precipitate at about 0.015 M. However, unlike the alkali metal salts and most solutes, the solubility of alkylamino alcohol salts cannot be described by a simple solubility relationship. Normally, when the same molecular species locates in the solid form and in solution, the solute dissolves until its saturation concentration is ex¬ ceeded; adding more solute does not increase the amount that goes into solution. There is an equilibrium be¬ tween the solid molecules and the molecules in solution.

However, although the alkylamino alcohol xanthosinates begin to precipitate at the low con¬ centration of 0.015 M, or about 6 mg/ml, adding more solute does increase the amount that goes into solu¬ tion. With further addition, one can actually make a 250 mg/ml solution, although 20-25% of all solute above 6 mg/ml will not dissolve.

For this to occur, the laws of solubility require that multiple solute species exist in equili¬ brium with each other and that one of these solute species be identical with and in equilibrium with the same species in the solid state. This state of affairs would be facilitated by the tendency for the alkylamino alcohol cations to engage in ion pair formation with the xanthosinate anions.

3. Beer's Law

Ultraviolet spectra make very important contri¬ butions to the identification of polymorphic configura¬ tions at low solute concentration. Shifts in absorbance peaks and shoulders reveal novel electronic states.

The ultraviolet absorbance of solutions also can reveal departures from Beer's law as interactions between solute molecules develop. Beer's law states that the absorbance of monochromatic light per molecule of solute is a constant in solution. Doubling the con¬ centration of a solute in aqueous solution in the absence of solute-solute interaction doubles the amount of irradiant energy absorbed, etc. However, as a result of interaction between solute molecules in solution, such as by aggregration, the relationship of light absorbance to solute concentra¬ tion can depart from Beer's law. Thus, the absorbance per molecule can decrease as concentration increases. This is an example of hypochromicity. Hypochromicity is characteristic of the absorbance of highly organized DNA and RNA which reflects a complexation between adjacent nucleotides.

With other compounds the absorbance of light per molecule increases as concentration increases, an example of hyperchro icity. Although this can occur when associations are breaking down, i.e., hypochromicity is being reversed, it can also occur as a result of an increased interaction of a compound with water. That is to say, if with increase in concentration a component of the solute molecule whose electrons are responsible for the absorbance of relevant protons becomes more accessible to and surrounded by water, the absorbance of monochromatic light energy per molecule can increase. These events can occur as a consequence of a shift in structure in space initiated by aggregration. Also, hyperchromicity can occur as a consequence of aggrega¬ tion that is followed by charge transfer. This is a unique mode of sharing a mobile electron that can develop with an aggregate.

Using a Hitachi double beam recording spectrophotometer, the ultraviolet spectrum of alkylamino alcohol and xanthosinate alkali metal salts were examined over a range of concentrations and under Ξ conditions that generated both the unionized and v;^*n.iz . states. Unique ultraviolet spectral shoulders and peaks were generated by the ionization process. Further, deviations from Beer s Law th.a^*c are unique for ^•■r:. . salt were found. All of the salts became more hypochromic than standard xanthosinate as concentration - -

increased, a result that is reflective of a greater degree of aggregration.

The relative extinction coefficients of stan¬ dard xanthosinate, NaXs-I, NaXs-II and ChXs-I are pre¬ sented in Table 2. Also, Figure 2 is a graph of con¬ centration versus relative extinction coefficient for these compounds. Table 2 and Figure 2 show that the xanthosinate polymorphs of the invention exhibit deviations from Beer's law. Also, the polymorphs give different deviations from Beer's law compared one to other and to standard xanthosinate, showing that the polymorphs of the invention have different patterns of aggregation, i.e. , have different polymorphic configurations, compared to each other and to standard xanthosinate.

Table 2

Relative Extinction Coefficients

Beer's Standard

Concentration Law Xanthosinate NaXs-I NaXs-II ChXs-

3.0 X 10^"6M 1.00 1.00 1.00 1.00 1.00

6.0 x 10^"6M 1.00 1.09 0.87 1.06 1.04

12.0 x 10^"6M 1.00 1.17 0.79 0.97 0.82

30.0 x 10^"6M 1.00 1.23 0.76 0.96 1.06

60.0 x 10^"6M 1.00 1.26 0.75 0.95 0.83

4. X-ray Powder Diffract:ion

X-ray powder diffraction patterns were de¬ termined for several crystal xanthosinate polymorphs using an Enraf-Nonius CAD4 single crystal diffracto- meter in association with a VAX 11/730 computer. The results are shown in Figures 3-7. As can be seen there, the spectra of standard xanthosine, NaXs-I and NaXs-II are largely amorphous (Figures 3, 4 and 5). The choline xanthosinates, however, reveal a degree of crystallinity not seen with standard xanthosine, NaXs-I or NaXs-II (Figures 6 and 7). Further, the spectrum of ChXs-I is different than that of ChXs-II (compare Figure 6 with Figure 7).

5. ORD Spectra

The ORD spectra of aqueous solutions of ChXs-I, ChXs-II, NaXs-II and DMAIPXs were determined using a Cary Spectropolarimeter. The results are shown in Figure 8. These tracings represent the average of triplicate runs, and the standard error in the tracings is virtually zero. As can be seen from Figure 8, each polymorph gives a different ORD spectrum which is evidence of different conformations which is in turn evidence of solute polymorphism.

Biological Assays Another piece of evidence that shows that the xanthosinates of the invention are polymorphic is the fact that they exhibit unexpected and remarkably dif¬ ferential biological activities. Xanthosine is not known to have any biological activity, but the xanthosine salt polymorphs of the invention are immunomodulators. In a standard mouse contact hypersensitivity system they can modify the intensity of the cellular immune response whether given solely during the time of immuni¬ zation or following antigen challenge. Further, the xanthosine salt polymorphs can function to enhance the immune response when the response is spontaneously depressed and can down-regulate the response when it is very active. This capacity to buffer the immune response is not, however, shared by all xanthosine salt polymorphs as is shown in the following examples. Exa ple 10

Adjuvant arthritis was induced in 120 gram male Sprague-Dawley rats, purchased from Harlan, Sprague Dawley, Indiana, by injection of complete Freund's adju¬ vant into the footpads of the anesthetized rats. These animals were also injected with 1.25 X 10 heat-killed H. pertusis organisms in the footpads to further stimu¬ late the inflammatory response. Primary'inflammation in the injected paws developed by the fourth day after the injections and persisted through the thirty-fifth day after the injections. Secondary (autoimmune) in¬ flammation appeared in other extremities on the tenth day after the injections, and persisted through the thirty-fifth day after the injections. The rats exhi¬ bited weight loss, joint swelling and joint immobiliza¬ tion.

Three groups of nine rats each, treated as just described, were injected interperitoneally daily with either saline, 0.079 mg/kg NaXs-I or 0.079 mg/kg NaXs-II beginning on the tenth day after the footpad injections. Secondary joint swelling in the hind foot was quantified daily by the standard scoring system described in Pearson and Wood, Arthritis and Rheumatism, 2_., 440 (1959), which is incorporated herein by reference. The results are shown in Table 3.

Table 3

Secondary Treatment Inflammation Index

Control (Saline) 4.00 + 0.28

NaXs-I 2.06 ± 0.70

NaXs-II 4.17 ± 0.39

As can be seen from Table 3, NaXs-I signifi¬ cantly reduced the secondary inflammation index (P less than 0.02). The secondary inflammation index for animals treated with NaXs-I was also significantly less than that for animals treated with NaXs-II (P less than 0.01). The results show that NaXs-I is anti-inflammatory while NaXs-II is not in this disease model. Indeed, NaXs-II enhanced secondary inflammation slightly. These results are significant because the inflammatory disease model described in this example is one that is predictive of anti-arthritic activity in the humans.

Example 11 Myocarditis was produced in the Balb/c mice (males, 4-5 weeks old) by injection of an unadapted human coxsackie-virus (B₃ Nancy strain) . Dilutions of between 1:50 and 1:400 of a stock containing g

1.75 X 10 plaque units/ml were used. Virus stock was maintained frozen at -70°C, and re-growths and assay titrations to desired stock strength were carried out in a Hep-2 cell system.

The disease produced in mice by this infec¬ tion results in death beginning seven days after they are injected, at which time the virus is no longer demonstrable in the heart, although cardiac inflam¬ mation progresses. This inflammation has been identi¬ fied as autoimmune in character.

Treatment with ChXs-I was begun seven days after the mice were injected with the virus. The ChXs-I was added to the drinking water of the mice at either 0.2 mg/ml or 0.4 mg/ml.

Twenty-eight days after the mice were injected with the virus, representative animals from control and treatment groups were sacrificed for histopathology. The following indices of disease were evaluated: the death rate; the intensity of myocardial inflammatory cellular exudate; and the intensity of myocardial necrosis, determined by microscopic examination by two -27-

independent assessments, each being done blind and the cellular exudate and the necrosis parameters being scored separately on a +1 to +4 basis. Hearts of animals dead within 24 hours were also processed for histopathology. Table 4 shows the results of this study.

Table 4

Mean

Relative

Death Rate Cardiac Relative through^ Cellular Cardiac

Day 28 Infiltrate Necrosis

Control (water 13/21 1.40 ± 0.13 2.0 ± 0.2 only)

*** **

ChXs-I, 0.2 mg/ml 0/21 1.21 ± 0.11 0.8 ± 0.1

***

ChXs-I, 0.4 mg/ml 0/2l^' 1.21 ± 0.13 0.9 ± 0.2

* All deaths occurred between days 8 and 27.

** Significant by student's t test, P less than 0.01,

*** Significant by Chi square analysis, P less than 0.001.

Example 12

Exposure to phagocytic stimuli causes neutro¬ phils to induce cell injury, death and lysis, events that are initiated by means of the action of released superoxide, peroxide and hydroxyl free radical. In inflammatory sites, such as arthritic joint spaces, such events are presumed to contribute to the short working lifetime of neutrophils, to exacerbate tissue destruction via the release of enzymes from disinte¬ grating neutrophils and to perpetuate the inflammatory responses by generation and build up of chemotactic peroxidized and otherwise modified cellular debris. Peripheral blood leukocytes (containing 70-90% neutrophils) were harvested from human volunteers and

7 were incubated for 22 hours with 3.5 X 10 Eschericia coli, according to the method of Salin & McCord, "Free radicals in leukocyte metabolism and inflammation," in

Superoxide and Superoxide Dismutases 257-270 (Michelson and Fridovick, eds. 1977) which is incorporated herein by reference. E. Coli is a stimulator of macrophages.

An aliquot of the leukocytes was exposed to trypan blue dye and examined microscopically in the hemocytometer counting chamber at zero time for total cells (generally to 5 X 10 neutrophiIs/ml) and via¬ bility. Viable cells exclude trypan blue. The leukocyte samples were found to be 90% viable.

The leukocytes were incubated in Hank's Balanced Salt Solution (HBSS) (1 ml total incubation volume) with added homologus serum, with the E^_ coli and with varying amounts of ChXs-I, standard xanthosinate, choline chloride (ChCl) or superoxide dismutase (SOD) .

Twenty-two hours after incubation began, a quantitative assessment of living, total dead, and lysed (absent) cells was made. The data were then converted so that all treatment groups were expressed as percent of control. The average of these converted data for three neutrophi1 studies and results for one lymphocyte study are presented in Table 5. A suppression of cell death and lysis by ChXs-I and by the active oxygen scavenger SOD was observed, but suppression by standard xanthosinate or ChCl alone was not observed. - -

Table 5

Neutrophils Lymphocytes

Treatment Total Dead Lysed Total Dead

Control

ChXs-I, 25 ug/ml 106 95 76

2.5 ug/ml 82 69 51

0.25 ug/ml 57 43 76

0.025 ug/ml 55 55 63

0.0025 ug/ml 81 81 78

Standard xantho¬ sinate, 0.20 ug/ml 110 102 106

ChCl, 0.07 ug/ml 100 98 104

SOD, 100 ug/ml 59 64 ND

Example 13

A drop of 0.5% dinitrofluorobenzene (DNFB) in acetone-olive oil was spread on the shaven abdomens of male CD-I mice to immunize them. The mice were challenged four or five days later on both ears with 0.2% DNFB under ether anesthesia. Immediately after the ear chal¬ lenge and on the day following ear challenge, the mice were given 0.079 mg/kg NaXs-I and NaXs-IV inter- peritoneally.

Measurement of ear thickness with a micrometer caliper under ether anesthesia was made before challenge and 24 hours after challenge. This measurement reflects the intensity of the local cellular immune response.

Of the 16 animals in each group, the 8 with the lowest responses were selected for analysis so that weak immune responses could be studied. The data in Table 6 shows that NaXs-I increases this minimal cellular immune response, while NaXs-IV does not. This increase is statistically significant.

Table 6 Treatment Ear Thickness (Units of 0.1mm)

Control 2.30 ± 0.45

NaXs-I 5.03 + 0.39

NaXs-IV - 1.40 ± 0.31

Example 14 A comparison was made between the effects of concanavalin A, Isoprinosine, ChXs-I and ChXs-II on mouse suppressor cells in the mixed lymphocyte reaction (MLR). Female BALB/c(H-2 ) mice were used as the source of the responder and suppressor populations. Male C57BL/6

^• -

(CH-2 ) mice were used as as source of the stimulator populations. The culture medium used throughout was RPM1-1460 Hepes supplemented with 5% heat inactivated fetal calf serum (FCS), penicillin (100 U/ml), strepto¬ mycin (100 ug/ml) and fungisome.

In vitro activation of suppressor cells was

₇ obtained by culturing 1 X 10 Balb/c splenocytes for three days in the presence of sterile filtered solutions of Con A (3.0 ug/ml, Sigma), Isoprinosine (1.0 ug/ml), ChXs-I or ChXs-II at various concentrations. After 3 days of culture, cells were harvested with a pasteur pipet. Con A treated cells were then washed in 0.3 M sterile methylmannoside (Sigma) solution, treated with 50 ug/ml mitomycin C (Sigma) for 30 minutes at 37°C, washed two additional times with methylmannoside and one time in culture medium. Other suppressor cell popu¬ lations were washed after harvest with sterile HBSS, mitomycin C treated as described above, washed two additional times with HBSS and one time with culture medium. The mixed lymphocyte reactions were performed g using 1 X 10 responder BALB/c splenocytes, 1 X

10 mitomycin C-treated C57BL/6 stimulator splenocytes and 2 X 10 of either Con A, Isoprinosine, ChXs-I or ChXs-II treated cells. The^' volume of each culture was 0.2 ml. The MLR were pulsed with 1 uCi tritiated thymidine (specific activity of 6.7 Ci/mmole) 56 hours after culture began and were harvested 72 hours after culture began. Each experiment was performed in quintuplicate.

Table 7 depicts the data from this experiment expressed as the mean counts per minute (cpm) ± standard error of the mean (S.E.M.). The addition of syngeneic-Con A-activated and mitomycin-C-treated cells resulted in a 69% suppression of tritiated thymidine up-take in the MLR system when compared with the sup¬ pressor cell activity spontaneously occurring in BALB/c lymphocytes cultured for three days in the absence of any mitogen. ChXs-I at the optimum dose is as effective as Con A in inducing suppressor cell activity above that spontaneously evoked in BALB/c lymphocytes cultured for three days in the absence of mitogen. ChXs-I enhance¬ ment of suppressor cell activity increased progressively with dose throughout the dose range studied. Isoprinosine enhancement of suppressor cell activity was also evident, although it was significantly smaller than the effect of ChXs-I at the one dose that allows comparison.

Table 7

3 Percen

Re- Stimu¬ Supres- H-thymidineδ⁸ Sup¬ sponder lator sor incorporation presso

Cell Cell Cell (c.p.m.±S.E.M. ) Effec

BALB B6m BALBm 67994 ± 2517 BALB B6m BALBm ^"Con A+ 20765 ± 14 -69 BALB B6m BALBm Iso 1.0++ 49546 ± 1775 -27 BALB B6m BALBm ChXs-I 0.01Δ 59577 ± 2991 -12 BALB B6m BALBm ChXs-I 0.03 28552 ± 2245 -58 BALB B6.m BALBm ChXs-I 1.00 25900 ± 906 -62 BALB B6m. BALBm ChXs-I 3.00 19915 ± 784 -71

BALB = BALB/c mice; B6=C57BL/6 mice.

* Subscript m designates mitomycin C-treated cultures

+ Con A, 3 ug/ml, added to spleen lymphocyte cultures

++ Isoprinosine, 1.0 ug/ml, added to spleen lymphocyte cultures

Δ ChXs-I added at either 0.01, 0.03, 1.00 or

3.00 ug/ml to l.OxlO⁷ spleen lymphocyte cultures as indicated

§ c.p.m. ± standard error of the mean for 5 cultures per group harvested at 72 hours after a 16-hour labeling period

V Suppressor effects highly significant, suppressor effects relative to control P less than 0.01 - -

Table 8 compares Isoprinosine, ChXs-I and ChXs-II for their effect on the induction of splenic suppressor lymphocytes. Both ChXs-I and ChXs-II are more potent than Isoprinosine at inducing suppressor lymphocytes. The dose response curve of ChXs-I is linear while that of ChXs-II is bell shaped. Thus, ChXs-I and ChXs-II both stimulate lymphocyte suppressor cell activity in the mouse.

Table 8

₃ H-thymidine §

Dose incorporation

Compound ug/ml (c.p.m.±S.E.M. ) % Suppression

None 0.00 67,994 + 2,517

ChXs-I 0.01 59,577 ± 2,911 -12

ChXs-I 0.03 28,552 + 2,245 -58

ChXs-I 1.00 25,900 ± 906 ^■62

ChXs-I 3.00 19,915 ± 784 ^■71

ChXs-II 0.01 45,110 ± 1,495 ■33

ChXs-II 0.03 52,246 ± 2,551 ■23

ChXs-II 1.00 12,382 ± 321 ^■82

ChXs-II 3.00 43,796 ± 4,195 •35

Isoprinosine 1.00 49,546 ± 1,775 -24

Counts per minute +_ standard error of the mean for 3 - 5 cultures per group harvested at 72 hours after a 16-hour labelling period.

Suppressor effects are highly significant relative to the control and to each other when comparison is made of equivalent dose, P less than 0.02 Example 15

In the previous example, it was shown that ChXs-I can strikingly increase mouse lymphocyte sup¬ pressor cell activity in mouse MLR. In this example, the effects of ChXs-I and DMAIPXs were examined for their capacity to induce suppressor cells.

The methods used were the same as those em¬ ployed in Example 14. Compounds examined for effects on suppressor cell populations were Con A (3 ug/ml), Isoprinosine (ISO) (1.0 ug/ml), ChXs-I (0.01 - 1.00 ug/ml), and DMAIPXs (0.01 - 1.00 ug/ml).

The results are shown in Table 9. Paradoxi¬ cally, Con A exerted a facilitator effect, rather than inducing suppressor cells, in this experiment. Isoprino¬ sine at 1.0 ug/ml caused a slight but not significant increase in suppressor cell activity. As in Example 14, ChXs-I increased suppressor cell activity at all doses used. The effect was statistically significant at the 0.03 and 1.0 ug/ml doses. DMAIPXs exerted a significant facilitator effect at 0.1 ug/ml, but did not signi¬ ficantly enhance suppressor cell activity at the other doses tested.

Thus, ChXs-I stimulated suppressor cell activity in mouse spleen cells in an experiment in which Con A exerted a facilitator rather than a suppressor-enhancing effect. The DMAIPXs did not significantly stimulate suppression but also acted as a facilitator.

-35-

Table 9

lender ftatimour- Ssuoprres- iHnc-tohr .p^*ymoirdai L^"nni^".ceo.n % Cell Cell Cell (c.p.m. ± S.E.M. ) Chan

BALB B6 17873 ± 3342

BALB B6_ ± m BALBm„, 9505 1317 - BALB B6m_ BALBmCon A 39725 ± 4989 +31 BALB B6m BALBmISO 1.0 8116 + 663 -15

BALB B6m BALBm ChXs-I 0.01 7712 + 1250 -19 BALB B6m. BALBm ChXs-I 0.03 4081 + 290 -57 BALB B6m_ BALBm ChXs-I 1.00 5243 ± 652 -45

BALB B6m_ BALBm DMAIPXs 0.01 7034 ± 1770 - BALB B6_m BALBm DMAIPXs 0.03 11654 ± 1714 + BALB B6_m BALBm_m DMAIPXs 0.10 13475 + 1896 +4 BALB B6m. BALB„ DMAIPXs 1.00 7875 ± 682

Example 16

In patients, whole-body irradiation may be administered in certain disseminated cancers as well as prior to bone marrow transplantation. A morbidity often results from this procedure that is associated with defects in the blood forming organs, generating the red cells, phagocytes and lymphocytes. This results in jJancytopenia. Similar side-effects result from treat¬ ment with cytotoxic agents.

As shown in the above examples, ChXs-I enhances depressed cellular immune responses and pro¬ motes phagocytosis. Since multiple defects in host defense systems occur following lethal irradiation, it was decided to determine whether ChXs-I could affect the course >pf the post radiation period.

Twenty-one male rats of the Fisher strain (180-200 gm) were given 750R of irradiation from a cobalt source. This irradiation dose normally produces 100% mortality in this strain of rats by five weeks.

All animals were injected with 2 mg/kg of the antibiotic Gentamicin daily throughout the study, starting one day after irradiation. The animals also received two antibiotics in their drinking water. These were neomycin at 0.56 ug/ml, bactrim at 44 ug/ml trimethoprim component, and 222 ug/ml sulfa component.

Beginning one day after irradiation, eight of the twenty-one irradiated animals were injected inter- peritoneally with 28 mg/kg of ChXs-I once daily for fourteen days. On the fifteenth day, the dose of ChXs-I was reduced to 0.1 mg/kg. Treatment and observations were continued through day 35.

Blood samples were taken for assessment of hematocrit and white blood cell count (WBC) on days 4, 7, 10, 14 and 21. WBC and hematocrit values were assigned to dead animals, and these numbers were - -

included in the calculations to provide a total morbidity index. The values assigned in this situation were 7.0% for the hematocrit and 1.0 x 10 3/mm3 for the WBC. These values are a small increment lower than the lowest value typically found in survivors. Normal WBC for rats are between 7.0 and 11.0 x 10 3/mm3. Normal hematocrits for rats of this strain, are between 48 and 55%.

Table 10

Treatment WBC's Counts Following Irradiation

Day 4 Day 7 Day 10 Day 14 Day 21

Control Avg. 0.90 0.88 1.16 2.38 3.12 SE 0.06 0.06 0.15 0.22 0.93

ChXs-I Avg. 0.71 1.04 1.80 3.36* 10.33*' SE 0.08 0.10 0.58 0.38 2.11

Significantly different from the irradiated control (P less than 0.05).

** Significantly different from the irradiated control (P less than 0.02).

Table 11

Treatment Hematocrits Following Irradiation

Day 4 Day 7 Day 10 Day 14 Day 21

Control Avg. 58.60 51.26 36.24 21.60 18.86 SE 0.58 1.87 2.41 2.29 3.67 ChXs-I Avg. 56.90 47.83 27.21 17.66 29.71* SE 0.65 1.82 2.24 1.63 2.58

Significantly different from the irradiated control (P less than 0.05). The final mortality on day thirty-five follow¬ ing an irradiation dose known to produce a 100% mortal¬ ity was:

Treatment Deaths

Control: 5/5 dead ChXs-II 5/8 dead

Thus, post-irradiation treatment with ChXs-I tends to suppress mortality and significantly promotes recovery of the white and red blood cell series.

Example 17

A second irradiation study was performed as described in Example 16, except that the rats only received 650R of irradiation. Also, a group of rats that received no irradiation was included for comparison purposes.

The results are shown in Tables 12, 13 and 14. Once again ChXs-I suppressed mortality and promoted recovery of white and red blood cells following irradiation.

Table 12

White Counts Following Irradiation

Treatment: Day 7 Day 14 Day 21 Day 28 Day 35 Day 42

Normal Avg. 6.44 9.16 7.74 8.00 9.56 12.32 SE 0.74 0.65 0.81 0.68 0.40 1.14

Control Avg. 2.27 2.15 2.39 1.83 4.34 6.83 SE 0.15 0.16 0.31 0.45 1.55 2.68

ChXs-I Avg. 2.04 3.72 5.33 * 4.13 21.45** 15.31* SE 0.26 0.26 0.45 1.47 6.03 3.00

* Significantly different from control (P less than 0. ** Significantly different from control (P less than 0. *** Significantly different from control (P less than 0. -39-

Table 13

Hematocrits Following Irradiation

Treatment: Day 7 Day 14 Day 21 Day 28 Day 35 Day 42

Normal Avg. 56.00 53.40 51.00 50.20 52.40 52.80 SE 2.74 0.51 0.45 0.97 0.87 0.58

Control Avg. 51.70 40.30 38.60 16.20 16.10 23.^*60 SE 1.45 1.17 2.18 4.33 4.70 7.26

ChXs-I Avg. 50.67 41.60 43.80* 32.60** 32.30* 42.80* SE 1.67 1.53 0.92 4.07 5.37 5.28

* Significantly different from control (P less than 0. ** Significantly different from control (P less than 0.

Table 14 Treatment Mortality on Day 42 Control 6/10 dead

ChXs-I 1/10 dead**

** Significantly different from control (P less than 0.0

Example 18 A third irradiation study was performed as described in Example 16, except that C57BL/6 mice were used instead of rats. The substances tested were: ChXs-I, DMAIPXs and a mixture of choline chloride with an equimolar amount of standard xanthosine. The mice received 0.1 mg/kg of these substances. Standard xanthosinate alone was also tested (at 0.07 mg/kg). The results are shown in Figure 8. As can be seen there, ChXs-I gave the lowest mortality of all the compounds tested at all times post irradiation. Also, all of the compounds provided some protection from irradiation effects as compared to control. Example 19 Ten groups of eight 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 Normal saline.

Groups 2-4 A solution of a mixture of equimolar amounts of choline chloride and xanthosine in normal saline. Three separate solutions were used at concentrations chosen so that Group 2 was given 25 mg/kg, Group 3 was given 1 mg/kg and Group 4 was given 0.25 mg/kg in the 0.1 ml.

Groups 5-7 A solution of ChXs-I in normal saline. Three separate solutions were used at concentrations chosen so that Group 5 was given 25 mg/kg, Group 6 was given 1 mg/kg and Group 4 was given 0.25 mg/kg in the 0.1 ml.

Groups 8-10 A solution of ChXs-II in normal saline. Three separate solutions were used at concentrations chosen so that Group 8 was given 25 mg/kg. Group 9 was given 1 mg/kg and Group 10 was given 0.25 mg/kg in the 0.1 ml.

One hour after the oral gavage treatment, 6.6 ug of trypsin in 0.05 ml of 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 to the nearest 0.1 mm employing a Schnelltaster caliper. Also, the increase in dorsoventral footpad diameter produced by injection of the solvent alone was determined by measuring the dorsoventral footpad diameters of a group of mice before and at *2 hours after injection of the solvent. This nonspecific swelling (the passive solvent effect) was subtracted from the mean increase in swelling evoked by trypsin injection to provide a measure of the specific swelling developed as part of the inflammatory response to trypsin itself.

The results of this experiment are presented in Figure 10. As shown _t there, ChXs-I gave significant suppression of specific trypsin-induced footpad swelling at 2 hours at 0.25 mg/kg and 1 mg/kg. ChXs-II and the equimolar mixture of choline chloride and xanthosine actually enhanced trypsin edema at higher doses.

Claims

I CLAIM:

1. A method of preparing a crystal polymorph of a xanthosine salt comprising: providing a solvent; adding to the solvent a quantity of xanthosine; adding to the xanthosine an equimolar amount of a strong base; heating the solvent, base and xanthosine at a predetermined rate to a temperature sufficient to cause the xanthosine to go into solution and to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration; precipitating the xanthosine salt; cooling the salt at a predetermined rate for a predetermined period of time; and completely lyophilizing the salt to yield the crystal polymorph of the xanthosine salt.

2. The method of Claim 1 wherein the solvent is water.

3. The method of Claim 1 wherein the solvent is methanol.

4. The method of Claim 1 wherein the xanthosine is standard xanthosine.

.

5. The method of Claim 1 wherein the xanthosine salt is precipitated by adding methanol.

6. The method of Claim 1 wherein the precipitation occurs spontaneously.

7. The method of Claim 1 wherein the base is selected from the group consisting of alkylamino alcohols and alkali metal hydroxides.

8. The method of Claim 7 wherein the base is choline.

9. The method of Claim 7 wherein ^"the base is dimethylaminoisopropanol.

10. The method of Claim 7 wherein the base is

_ sodium hydroxide.

11. A method of preparing a crystal polymorph of a xanthosine salt comprising: providing a solvent; adding to the solvent a quantity of xanthosine; adding to the xanthosine an equimolar amount of a strong base to neutralize the xanthosine, the neutralization reaction generating enough heat so that the temperature rises to a temperature sufficient to cause the xanthosine to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration; precipitating the xanthosine salt; cooling the salt at a predetermined rate for a predetermined period of time; and completely lyophilizing the salt to yield the crystal polymorph of the xanthosine salt.

12. The method of Claim 11 wherein the solvent is water.

13. The method of Claim 11 wherein the solvent is methanol.

14. The method of Claim 11 wherein the xanthosine is standard xanthosine.

15. The method of Claim 11 wherein the xanthosine salt is precipitated byadding methanol.

16. The method of Claim 11 wherein the precipitation occurs spontaneously.

17. The method of Claim 11 wherein the base is selected from the group consisting of alkylamino alcohols and alkali metal hydroxides.

18. The method of Claim 17 wherein the base is choline.

19. The method of Claim 17 wherein the base is dimethylaminoisopropanol.

20. The method of Claim 17 wherein the base is sodium hydroxide.

21. The xanthosinate crystal polymorph produced by the process of any of Claims 1-20.

22. A solute polymorph of a xanthosinate.

23. A solute polymorph of choline xanthosinate.

24. A solute polymorph of sodium xanthosinate.

25. A solute polymorph of a dimethyla ino- isopropanol xanthosinate.

26. A method of preparing a solute polymorph of a xanthosine salt comprising dissolving the crystal poly¬ morph of the xanthosine salt of Claim 21 in a solvent.

27. A method of preparing a solute polymorph of a xanthosine salt comprising: providing a solvent; adding to the solvent a quantity of xanthosine; adding to the xanthosine an equimolar amount of a strong base; heating the solvent, base and xanthosine at a predetermined rate to a temperature sufficient to cause the xanthosine to go into solution and to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration; and cooling the solution at a predetermined rate for a predetermined period of time.

28. A method of preparing a solute polymorph of a xanthosine salt comprising: providing a solvent; adding to the solvent a quantity of xanthosine: adding to the xanthosine an equimolar amount of a strong base to neutralize the xanthosine, the neutralization reaction generating enough heat so that the temperature rises to a temperature sufficient to cause the xanthosine to overcome the energy barriers which prevent the conversion of the xanthosine to another polymorphic configuration; and cooling the solution at a predetermined rate for a predetermined period of time.

29. The method of either Claim 27 or 28 wherein the xanthosine is standard xanthosine.

30. The method of either Claim 27 or 28 wherein the solvent is water and the base is sodium hydroxide.

31. A crystal polymorph of xanthosine.

32. A solute polymorph of xanthosine.

33. A salt of xanthosine having the formula:

(Xs)^" (CB)⁺ wherein Xs is the xanthosinate ion and CB is the cation of a strong base.

34. The salt of Claim 33 wherein the base is selected from the group consisting of alkylamino alcohols and alkali metal hydroxides.

35. Choline xanthosinate.

36. Sodium xanthosinate.

37. Dimethylaminoisopropanol xanthosinate.

38. A composition for modulating the immune response of an animal comprising: a pharmaceutically-acceptable solvent; and an amount of a solute polymorph of xanthosine effective to modulate the immune response of the animal.

39. A method for modulating the immune response of an animal comprising administering to the animal a composition comprising: a pharmaceutically-acceptable solvent; and an effective amount of a solute polymorph of xanthosine.

40. A composition for modulating the immune response of an animal comprising: a pharmaceutically-acceptable carrier; and an amount of a crystal polymorph of xanthosine effective to modulate the immune response of the animal.

41. A method for modulating the immune response of an animal comprising administering to the animal a composition comprising: a pharmaceutically-acceptable carrier; and an effective amount of a crystal polymorph of xanthosine.

42. A composition for modulating the immune response of an animal comprising: a pharmaceutically-acceptable carrier; and an amount of the xanthosinate crystal polymorph of Claim 21 effective to modulate the immune response of the animal.

43. A method for modulating the immune response of an animal comprising administering to the animal a composition comprising: a pharmaceutically-acceptable carrier; and an effective amount of the xanthosinate crystal polymorph of Claim 21.

44. A composition for modulating the immune response of an animal comprising: a pharmaceutically-acceptable solvent; and an amount of a xanthosinate solute polymorph effective to modulate the immune response of the animal.

45. A method for modulating the immune response of an animal comprising administering to the animal a composition comprising: a pharmaceutically-acceptable solvent; and an effective amount of a xanthosinate solute polymorph.