Scope of the Invention This invention relates to the dihydrate of a compound which is useful for treating asthma, and other diseases caused by or related to leukotrienes, particularly LTD4. The entity is (S)-β-[(2-carboxyethyl)thio]- 2-(8-phenyloctyl)benzenepropanoic acid disodium salt dihydrate.

Area of the Invention This invention has application in the area of "Slow Reacting Substance of Anaphylaxis," or SRS-A as it is also known. SRS-A has been shown to be a highly potent broncho- constricting substance which is released primarily from mast cells and basophils on antigenic challenge. SRS-A has been proposed as a primary mediator in human asthma. SRS-A, in addition to its pronounced effects on lung tissue, also produces permeability changes in skin and may be involved in acute cutaneous allergic reactions. Further, SRS-A has been shown to effect depression of ventricular contraction and potentiation of the cardiovascular effects of histamine.

The discovery of the naturally occurring leukotrienes and their relationship to SRS-A has reinforced interest in SRS-A and other arachidonate metabolites. SRS-A derived from mouse, rat, guinea pig and man have all been characterized as mixtures of leukotriene-C4 (LTC4), leukotriene-D4 (LTD4) and leukotriene-E4 (LTE4), the structural formulae of which are represented below.

γ Glu

LTC4 R" = Cys-Gly TD4 R" = Cys-Gly

LTE4 R" = Cys Leukotrienes are a group of eicosanoids formed from arachidonic acid metabolism via the lipoxygenase pathway. These lipid derivatives originate from LTA4 and are of two types: (1) those containing a sulfidopeptide side chain (LTC4, LTD4, and LTE4), and (2) those that are nonpeptidic (LTB4). Leukotrienes comprise a group of naturally occurring substances that have the potential to contribute significantly to the

pathogenesis of a variety of inflammatory and ischemic disorders. The pathophysiological role of leukotrienes has been the focus of recent intensive studies.

Leukotrienes have been directly or indirectly implicated in a variety of pulmonary and non-pulmonary diseases in the ocular, dermatologic, cardiovascular, renal, trauma, inflammatory, carcinogenic and other areas. By antagonizing the effects of LTC4, LTD4 and LTE4 or other pharmacologically active mediators at the end organ, for example airway smooth muscle, the compound administered via the compositions of the instant invention are valuable in the treatment of diseases in subjects, including human or animals, in which leukotrienes are a key factor.

Summary of the Invention This invention relates to the dihydrate form of (S)-β-[(2- carboxyethyl)thio]-2-(8-phenyloctyl)benzenepropanoic acid disodium. Brief Description of the Drawings

Fig. la shows a TGA moisture curve for samples of the acid salt exposed to approximately 24% relative humidity (RH) for 4 weeks (Lotr 1).

Fig. lb shows a TGA moisture curve for samples of the acid salt exposed to approximately 24% relative humidity (RH) for 4 weeks (Lot 2). Fig. 2a shows a TGA moisture curve for samples of the acid salt exposed to approximately 84% relative humidity (RH) for 4 weeks (Lot 1).

Fig. 2b shows a TGA moisture curve for samples of the acid salt exposed to approximately 84% relative humidity (RH) for 4 weeks (Lot 2).

Fig. 3a shows a TGA moisture curve for samples of the acid salt exposed to approximately 24% relative humidity (RH) for 1 weeks (Lot 2).

Fig. 3b shows a TGA moisture curve for samples of the acid salt exposed to approximately 24% relative humidity (RH) for 1 weeks (Lot 2).

Fig. 4a shows an initial DSC thermogram for the disodium salt (Lot 1).

Fig. 4b shows an initial DSC thermogram for the disodium salt (Lot 2). Fig. 5a shows a DSC thermogram for the disodium salt compound stored at

24% relative humidity for 4 weeks (Lot 1).

Fig. 5b shows a DSC? thermogram for the disodium salt compound stored at

24% relative humidity for 4 weeks (Lot 2).

Fig. 6a shows a DSC thermogram for the disodium salt compound stored at 84% relative humidity for 4 weeks (Lot 1).

Fig. 6b shows a DSC thermogram for the disodium salt compound stored at

84% relative humidity for 4 weeks (Lot 2).

Fig. 7a shows a DSC thermogram for the disodium salt compound stored at

84% relative humid y for 1 weeks (Lot 1).

Fig. 7b shows a DSC thermogram for the disodium salt compound stored at

84% relative humidity for 1 week (Lot 2).

Fig. 8a shows an initial X-ray diffraction pattern for the disodium salt (Lot 1).

Fig. 8b shows an initial X-ray diffraction pattern for the disodium salt compound (Lot 2).

Fig. 9 shows a powder X-ray diffraction pattern for the disodium salt stored at 24% relative humidity for 4 weeks. Fig. 10a shows a powder X-ray diffraction pattern for the disodium salt stored at 84% relative humidity for 1 week.

Fig 10b shows a powder X-ray diffraction pattern for the disodium salt stored at 84% relative humidity for 4 weeks.

Fig. 11a shows a powder X-ray diffraction pattern for the disodium salt stored at 84% relative humidity for 1 week.

Fig. lib shows a powder X-ray diffraction pattern for the disodium salt stored at 84% relative humidity for 2 weeks.

Fig. 12 shows the representative FT-IR tracings of a control sample (top) versus one exposed to 84% humidity for 4 weeks (bottom tracing). Fig. 13a shows the representative FT-IR tracings of a control sample for the range of 2000-700 cm" ¹ .

Fig. 13b shows the representative FT-IR tracings of a sample exposed to

84% humidity for 4 weeks for the range of 2000-700 cm" ¹ .

Specific Embodiments The benzenepropanoic acid involved in this invention is a known compound. It has been described in the patent literature, specifically in

U.S. patent 4,874,792 issued 17 October 1989. This U.S. patent is incorporated herein by reference. It discloses not only how to make this acid, it also shows how the (S) form may be isolated. Methods for making pharmaceutically acceptable salts, including the disodium form are set out in this patent. Likewise a utility statement is given there along with certain methods for demonstrating utility.

The propanoic acid disodium salt comprising part of this invention has a significant non-crystalline component and exists as a mixture of different (though closely related) polymorphic forms. The most stable form exhibits a melting endotherm at approximately 247°C. Under polarized light, this material exhibited bifringent areas suggesting some crystalline character but also some areas of dark non-crystalline character.

Theoretically a dihydrate would have 6.9% total water content.

Thermogravimetric analysis (TGA) and Karl Fischer data indicated a total water content, based upon the weight loss curves, of approximately 7% after 3 days which remained constant through 4 weeks. The TGA weight loss curves showed this water loss occurred over the temperature range from 40°C to 165°C and the first derivative indicates a distinct change in the rate of water loss. Initial water loss rate occurred in two steps at about 40°C and at about 126°C, whereas the dihydrate form dehydrates at 75°C. Differential scanning calorimetry (DSC) scans were consistent with the water loss curves obtained thermogravimetrically for samples stored at 84% relative humidity. Two solvent transitions were observed at about 63°C and at about 75°C as were observed with the TGA weight-loss curves. Initial DSC scans showed two endotherms at about 243° and 245°C. After 1 week at 84% relative humidity, the lower melting endotherm became the predominant transition. TGA and DSC data support the formation of a dihydrate of (S)-β-[(2-carboxyethyl)thio]-2-(8-phenyloctyl)benzenepropan oic acid possessing unique thermal properties.

Experimental data for 4 weeks, analyzed by elemental analysis, agreed with the theoretical value for the dihydrate form. The stability of the dihydrate was investigated by placing a sample in a low humidity chamber (20%±4%) for 2 weeks. There were no physical changes observed using DSC and photomicroscopy. TGA curves showed similar water content and rate of water loss.

No chemical degradation was observed in samples of the dihydrate after exposure to 84% relative humidity over 4 weeks.

After essentially anhydrous samples of the disodium salt were exposed to moisture in a chamber having a relative humidity of about 84% (±4%), an increase in moisture was observed which was stoichmetrically equivalent to formation of a dihydrate (ca 7%). DSC scans exhibited conversion of two closely related endotherms (ca. 243° C and ca. 247° C) to the lower melting endoderm at 243°C. This dihydrate was stable in the solid state using HPLC. Photomicrographs showed a significant increase in birefringence although some dark areas remained. Scanning electron microscope photographs showed a considerable change in drug particle morphology which appeared highly structured with plate-like surfaces containing cracks and fissures. X-ray diffraction patterns showed a considerable increase in peak positions and intensities indicative of higher crystallinity.

Example 1 Hygroscopicity experiments were carried out by placing samples pans of the anhydrous disodium salt of (S)-β-[(2-carboxyethyl)thio]-2-(8- phenyloctyDbenzenepropanoic acid in desiccators at room temperature. The desiccators contained saturated salt solutions of potassium acetate and sodium chloride to obtain relative humidities of 20%±4% and 84%±4% respectively. The samples were randomly mixed with a spatula to avoid stickiness resulting from surface water absorption. Samples were analyzed at 0 days, 3 days, 1 week, 3 weeks, and 4 weeks by TGA, DSC, SEM, powder X-ray diffraction and photomicroscopy. Samples exposed to high humidity were also analyzed by HPLC (Waters Assoc. phenyl, 30cm, 10 micron particle size; solvent 50% acetonitrile/50% water/0.1% trifluoroacetic acid; flow rate: 2ml/min.; detector: 215nm). Water content was corroborated using Karl Fischer titration.

Example 2 Thermogravimetric Analysis (TGA):

The moisture content of two lots prepared as per Example 1 was determined thermogravimetrically by heating the samples from 40°C to 165°C at a scanning rate of 20°C/min under a N2 atmosphere, as well as, by Karl Fischer titration. Initial moisture content was low ( <2%) and occured in two distinct step. This weight loss obtained by TGA was assumed to be water (Karl Fischer titration data supports this assumption). Figures la and lb contains the TGA moisture curves for samples exposed to approximately 24% relative humidity (RH) for 4 weeks. The first derivatives of these curves (b) were similar to the initial samples with moisture being lost in two-distinct steps. Samples from both lots did not have increased water content under low humidity conditions. At ca. 84% RH, both lots exhibited an increase in water content after 3 days which remained constant during the duration of the experiments (4 weeks). TGA curves indicated a total water content of about 7%. TGA curves for two lots are shown in Figures 2a/2b and 3a/3b, respectively. The first derivative of the water loss curves (b) shows a marked change in the rate of water loss for the samples exposed to high humidity. A sample of one lot was stored for two weeks at high humidity. This sample was then placed into the low humidity chamber for 2 weeks. The moisture content of this sample did not change from the original value (i.e. 7%).

Example 3 Differential Scanning Calorimetry (DSC):

DSC scans were performed on samples from two different lots which had been prepared as per Example 1 by heating samples from 25 °C to

265°C at a scan rate of 10°C/min. Initial scans of both lots, shown in Figure 4a and 4b, exhibited two melting endotherms, the first at ca. 243°C and the second ca. 247°C, suggesting a mixture of different polymorphic forms. Both lots also showed two broad endotherms at ca. 126°C and ca. 202°C. DSC thermograms of samples exposed to ca. 24% RH for 4 weeks are shown in Figure 5a and 5b. These data are similar to the initial DSC scans, however, the endothermic transitions occurred at ca. 240°C and 245°C.

After 3 days at ca. 84% RH, the two lots exhibited a distinct conversion to the lower melting polymorphic form at ca. 243 °C with a small endotherm at 247°C. A sharp endotherm was observed at ca. 63°C, and a broad transition at ca. 75°C. After cooling and re-heating, these transitions did not occur suggesting solvent loss. DSC thermograms for two lots stored at ca. 84% RH for 1 week and 4 weeks are found in Figures 6a/6b and 7a/7b, respectively. This data suggests that both lots converted to a stable crystalline form possessing different thermal properties after exposure to high humidity.

Example 4 Power X-Ray Diffraction:

Power X-ray diffraction patterns are contained in Figure 8a and 8b for two different lots at initial conditions. Both lots were similar, possessing low crystallinity. The X-ray diffraction pattern of lot of one stored for 4 weeks at ca. 24% RH, shown in Figure 9, is similar to the initial sample with respect to peak intensities and degree of crystallinity. Powder X-ray diffraction patterns obtained at 1 week and 4 weeks for samples stored at ca. 84% RH showed more peaks indicative of crystalline materials and greater intensity suggesting much higher crystallinity. The XRD patterns for 1 week and 4 weeks at ca. 84% RH are similar, as shown in Figures lOa/lOb and lla/llb. X-ray data was not obtained at 3 days.

Example 5 Scanning Electron Microscopy (SEM):

SEM photographs revealed a significant change in morphology for samples stored for 3 days at 84% RH. Initially, the samples appeared to be porous globular particles. Exposure to high humidity for 3 days caused a significant change in morphology evidenced by cracks and fissures on the particle surface and the morphology was observed to be more structured and ordered after 1 week. Samples stored for 4 weeks at high humidity conditions appeared more structured than 3 day samples, however, this is a qualitative observation and was not supported by X-ray diffraction data. The data did not suggest any significant change in morphology for samples stored at ca. 20% RH for 4 weeks.

Example 6 Microscopy:

Photomicrographs of initial samples and low humidity samples exhibited low order birefringence. Under polarized light, both lots exhibited some birefringent areas suggesting some crystalline character, but also possessed some areas of dark non-crystalline character.

Samples stored at ca. 84% RH exhibited much greater birefringence under polarized light suggesting increased crystalline character beginning at 3 days and continuing for the duration of these experiments (4 weeks). The particles appeared more plate-like with less conglomeration than the initial samples.

Example 7 Elemental Analysis:

Samples of two lots exposed to ca. 84% RH for 4 weeks were submitted to Elemental Analysis. The resulting data suggests the formation of the dihydrate based upon the percentage of elements found in the sample. TGA and Karl Fischer data suggested the increase in mass of these samples is attributed to water absorption in the amount stochiometrically necessary to form a dihydrate.

Example 8 Transmittance and Reflectance (FT-IR) Microscopy Several different samples of the diacid salt were analyzed using microscopic FT-IR spectroscopy. Two sets of samples were from control lots which had not been exposed to water or a humid atmosphere. The other two sets were from lots exposed to 80% RH for four weeks prior to analysis.

All samples were run as is using a Nicolet 800 RT-IR spectrometer equipped with a NicPlan microscope and a 16X objective.

Figure 12 gives a representative FT-IR tracings of a control sample (top) versus one exposed to 84% humidity for 4 weeks (bottom tracing). The samples were visually different in appearance under the microscope.

Control samples were amorphous while those exposed to 84% RH appeared crystalline and were easily visualized using cross polars. One distinguishing IR spectral changes occurs in the O-H stretching region (3600-3100cm ^-1 ) of the infrared spectrum. Less dramatic but noticeable changes in spectra were observed from 2000-700 cm" ¹ . Figures 13a and

13b give an expanded and labelled reflectance spectra of those regions for a control and test sample. The most dramatic variations are a shift in the carboxylate asymmetric stretching band from 1593 to 1611 cm" ¹ ; overall band shifts from 1205 to 1135 cm" ¹ ; overall band shifts from 1205 to 1135 cm ^-1 ; and peak shifts and band intensity changes in the aromatic C-H vibration region from 970 to 830 cm" ¹ . These data show a different crystalline form for the material stored at 84% relative humidity for four weeks.