MX2008007336A - Methods for preparing crystalline rapamycin and for measuring crystallinity of rapamycin compounds using differential scanning calorimetry - Google Patents

Abstract

Methods for purifying rapamycin are described. Methods for measuring particle quality, median particle size, and crystallinity of samples containing rapamycin or a derivative thereof are also provided.

Description

METHODS TO PREPARE CRYSTALLINE RAPAMICINE AND TO MEASURE THE CRYSTALLINATION OF RAPAMYCIN COMPOUNDS USING DIFFERENTIAL SCANNING CALORIMETRY FIELD OF THE INVENTION Methods for preparing crystalline rapamycin compounds and measuring the stability and particle quality of the samples containing a rapamycin compound are described. Rapamycin (the drug Rapamune®) is an immunosuppressant derived from nature, having a novel mechanism of action. CCI-779 (42-ester of rapamycin with 3-hydroxy acid -2- (hydroxylmethyl) -2-methylpropionic acid) is an ester of rapamicma, which has shown important inhibitory effects on the growth of tumors in in vitro and in vivo models Numerous routes for rapamycin compounds and rapamycin purification have been described in the literature, some resulting in rapamycin compounds that have specifications required by regulatory agencies such as the United States Food and Drug Administration. (FDA) However, samples of rapamycin compounds prepared using these routes may contain crystals of varying quality What is needed in the art are methods for preparing crystalline rapamycin and for measuring the particle and particle quality of the samples of the rapamycin compounds BRIEF DESCRIPTION OF THE INVENTION In one aspect, methods are provided for measuring the quality of rapamycin compound particles. In another aspect, methods for measuring the substance of a rapamycin compound are provided. In even a further aspect, methods for preparing crystalline rapamycin are provided. Other aspects and advantages of the present invention are further described to the following detailed description of the preferred embodiments thereof BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides peak temperatures obtained from DSC charts for twenty-five (25) samples of CCI-779 as a function of the particle category. A category of particle 1 refers to crystalline samples of CCI-779 that have large particles, a category of particle 2 refers to crystalline samples of CCI-779 that have small particles, a particle category 3 refers to semicpstalin aggregates of CCI-779; and the category of particle 4 refers to non-crystalline CCI-779 Figures 2A and 2B provide DSC graphs that highlight peak temperatures for five (5) rapamycin samples of varying variability In Figure 2A, the upper graph corresponds to a sample containing crystalline rapamycin, the average graph corresponds to a sample containing rapamycin semicpstahna, and the lower graph corresponds to a sample containing amorphous rapamicma. In Figure 2B, the upper graph corresponds to a sample containing crystalline rapamycin maintained at 25 to 60 ° C for 2 months, the mean graph corresponds to a sample containing crystalline rapamycin maintained at 25 to 60 ° C for 4 months, and the lower graph corresponds to the sample containing crystalline rapamycin identified in figure 2A Figure 3 provides a graph illustrating the relationship between the degree of cstastaity and thermal parameters including heat of fusion (J / g), temperature of initiation of casting (° C) and peak temperature (° C) for six (6) samples of CCI-779 Shaded triangles (A) illustrate the ratio of peak temperature to crystallinity, shaded diamonds (+) illustrate the relationship of heat of fusion and the crystallinity, and the shaded (") shaded squares illustrate the relationship of the start temperature on the stability.

DETAILED DESCRIPTION OF THE INVENTION Methods for preparing crystalline rapamycin and measuring samples of rapamycin compounds in terms of particle quality, particle size and crystallinity are described. The term "particle quality" as used herein refers to the quality of the crystals of a rapamycin compound. Typically, the particle quality refers to most of the crystals in a sample that contain a rapamycin compound. The quality of the particle can indicate a variety of factors including the crystal size, the distribution and size of the crystals, the homogeneity / chemical purity of the crystals and the morphology of most crystals. In one example, a high particle quality can refer to crystals in which the majority of the crystals in a sample are large. In another example a high particle quality can refer to a sample in which most of the crystals have the same morphology. In a further example, a high particle quality can refer to a sample in which the majority of the crystals are not contaminated by impurities. In yet another example, a high particle quality can refer to a sample in which most of the crystals are large, have the same morphology and are not contaminated by impurities. The term "crystallinity" as used herein refers to the degree of structural order in a sample containing a compound of Rapamycin Typically, the crystallinity is represented by a fraction or percentage as a measure of how likely the atoms or molecules will be arranged in a regular pattern as in a crystal. The crystallinity of a rapamycin compound contributes to the overall quality of the particle and is affected by impurities, such as atoms and molecules or by crystallization conditions or the presence of defects. In one example, a sample having a higher crystallinity will have a powder diffraction pattern with X-rays having well-defined peaks. In one example, a sample having a crystallinity of about 0% contains a solid that is substantially amorphous. In another example, a sample having a crystallinity of about 100% contains a solid that is highly crystalline. In a further example, a sample having a crystallinity of about 50% contains a solid that is semi-crystalline. The term "particle size" as used herein refers to the size of most crystals in a sample. Typically, "particle size" refers to the average size of the crystals in a sample as determined by the measurement of the longest linear dimension. The particle size of a rapamycin compound contributes to the overall quality of the particle.

A. Methods for preparing crystalline rapamycin. In one embodiment, the methods for preparing crystalline rapamycin are described. These methods are especially useful for preparations on a large scale and offer highly crystalline rapamycin. These methods are also convenient since the crystalline rapamycin thus prepared is more stable, thus resulting in the presence of less impurities of oxidative degradation and / or hydrolysis. The term "oxidant" or "hydrolysis" degradation impurities refers to impurities formed by the oxidation and / or hydrolysis of the triene region of the rapamycin molecule. The term "rapamycin" is a term used in the art and in the present to describe the following compound.

RAPAMICINE The term "raw rapamycin" as used herein refers to a sample of rapamycin that is substantially crystalline but contains less than about 20% impurities. In one example, crude rapamycin contains less than about 15% impurities. In another example, raw rapamycin contains less than about 10% impurities. In a further example, raw rapamycin contains less than about 5% impurities. There are a variety of methods for preparing crude rapamycin and includes the US patent. No. 3,993,749, which is incorporated herein by reference. Alternatively, the rapamicma can be purchased commercially (for example with Wyeth) The raw rapamicma can be micronized or non-micronized as described in US Patent No. 5,985,325, which is incorporated herein by reference. The first step of this method includes heating raw rapamycin. in ethyl acetate at an elevated temperature. In one embodiment, the rapamycin / ethyl acetate solution is heated from about 52 to about 58 ° C. In another embodiment, the rapamycin / ethyl acetate solution is heated to about 55. ° C After this, the heated solution of ethyl acetate is filtered A variety of filtration instruments can be used as easily understood by one skilled in the art The filtered solution is then maintained at an elevated temperature In one embodiment, the solution of rapamycin / ethyl acetate is maintained at a temperature of about 50 to about 60 ° C. In another embodiment, the rapamycin / acetase solution The ethyl ester is maintained at a temperature of about 54 ° C to about 57 ° C. A solvent containing a hydrocarbon solvent is then added to the heated solution. In one embodiment, the hydrocarbon solvent is heptane. In another embodiment, the solvent of hydrocarbon is hexane. In a further embodiment, the hydrocarbon solvent is pentane. The hydrocarbon solvent is preferably added at a rate that results in the formation of crystalline rapamycin, preferably by gradual crystallization. The hydrocarbon solvent may then be added in a constant speed or non-linear speed. Preferably, the speed of the addition of the hydrocarbon solvent maintains the temperature of the heated solution. More preferably, the rate of addition of the hydrocarbon solvent maintains the temperature of about 54 to 57 ° C. A person skilled in the art will readily be able to adjust the rate of addition of the hydrocarbon solvent to avoid premature precipitation of rapamycin. The hydrocarbon solvent is typically added over a period of at least about 20 minutes. In one example, the hydrocarbon solvent is added in a period of at least about 30 minutes. In another example the hydrocarbon solvent is added over a period of about 60 minutes. In a further example, the hydrocarbon solvent is added over a period of about 60 minutes at constant speed. A person skilled in the art will readily be able to adjust the period required to add the hydrocarbon solvent to avoid the premature precipitation of rapamycin. The temperature of the ethyl acetate / hydrocarbon solvent solution is then maintained at an elevated temperature. In one example, the ethyl acetate / hydrocarbon solvent solution is maintained for about 30 minutes at a temperature of about 55 to about 57 ° C. The agitation speed is then reduced to the minimum speed that is required to achieve a solid suspension. A person skilled in the art will be easily able to adjust the agitation speed based on the teachings that are provided here, the specific reactor used and especially the power per reactor volume. In one example, the stirring speed is reduced to equal to or less than about 100 revolutions per minute (RPM). In another example the stirring speed is about 45 to about 100 RPM. After reducing the stirring speed, the solution is cooled non-linearly at a decreasing cooling rate. In one example, the solution is cooled to around a first reduced temperature using a first cooling rate; it is cooled to a second reduced temperature using a second cooling rate; and is further cooled using a third reduced temperature at a third cooling rate. Generally, the third reduced temperature is less than the second reduced temperature, which is less than the first reduced temperature. In one example, the first reduced temperature is from about 38 to about 42 ° C; the second reduced temperature is around 23 to about 27 ° C; and the third reduced temperature is from about 5 to about 10 ° C. In another example, the first reduced temperature is around 40 ° C; the second reduced temperature is around 25 ° C; and the third reduced temperature is around 9 ° C. Typically, the third rate of cooling is faster than the second rate of cooling, which is faster than the first rate of cooling. In one example, the first cooling speed is around 4 to about 7 ° C / hour; the second cooling rate is around 5 to about 9 ° C / hour and the third cooling speed is around 7 to 10 ° C. In a further example, the first cooling rate is around 5 ° C / hour; the second cooling speed is around 7.5 ° C / hour; and the third cooling speed is around 9 ° C. In one embodiment, the solution is cooled to about 40 ° C at a rate of about 5 ° C / hour; it is further cooled to a temperature of about 25 ° C at a rate of about 7.5 ° C / hour; and even further cooled to a temperature of about 7 to 8 ° C at a rate of at least about 9 ° C / hour. This solution is then maintained at that temperature for about 2 to about 6 hours. In an example, the solution is maintained at this temperature of about 2 hours. The inventors also found that the rate of addition of the hydrocarbon solvent influenced the crystallinity of rapamycin. For example, when heptane is added at a rate of 60 minutes or less, the morphology of the resulting crystals is orthorhombic. However, when heptane is added over a period of at least about 60 minutes, the morphology of the resulting crystals is acicular. However, the slower cooling rate, preferably non-linearly, after the heptane addition resulted in crystals with a more uniform size distribution. By controlling these parameters, the precipitation of fine particles of rapamycin is controlled and / or prevented more easily, resulting in crystalline rapamycin with a uniform size distribution The resulting crystalline rapamycin is collected by filtration. An additional wash of rapamycin is then performed with a solution containing ethyl acetate and the hydrocarbon solvent, preferably heptanes, and the crystalline rapamycin is dried. Preferably, a surplus of the hydrocarbon solvent is used on the ethyl acetate. In one example, a 2 1 ratio of hydrocarbon / ethyl acetate solvent is used. uses a 2 1 ratio of heptane / ethyl acetate. Rapamycin is washed using a solution of hydrocarbon / ethyl acetate solvent at reduced temperatures. In one example, the rapamicma is washed at a temperature of about 6 to about 10 ° C. In another example, rapamycin is washed at a temperature of about 8 ° C. Typically, rapamycin dries on a low shear dryer, but other drying techniques may be used as determined by one skilled in the art. In pring the crystalline rapamycin in accordance with the method described herein, rapamycin and crystallized are obtained in which the cpsta nity is substantially maintained. for a period of up to 4 months with up to 60% relative humidity In one example, the stability is maintained for a period of about 2 months. In another example, the stability is maintained for a period of about 4 months.

Further example, the crystallinity is maintained up to about 60% relative humidity. Specifically, the DSC profiles for the crystalline rapamycin pred as described herein stored for up to 4 months with up to about 60% relative humidity showed a minimal change in the cast endotherm. In one example, the DSC profile for the crystalline rapamycin showed a change in the casting endotherm of less than about 1%. In one example, the DSC foundry endotherm showed a change of less than about 0.5%. In another example, the DSC casting endotherm showed a change of less than about 0.3%. In a further example, the DSC casting endotherm showed a change of less than about 0.1%. In one embodiment, a method for purifying rapamycin is provided and includes (i) heating raw rapamycin in ethyl acetate at about 55 ° C; (ii) filtering the product of step (i); (iii) maintaining the temperature of step (i) from about 54 ° C to about 57 ° C; (iv) adding heptanes to the product of step (ii) for a period of about 60 minutes at constant speed; (v) maintaining the product of step (iv) at the same temperature for about 30 minutes; (vi) reducing the stirring speed of step (v); (vii) cooling the product from step (vi) to about 40 ° C at a rate of about 5 ° C / hour; (viii) cooling the product of step (vii) to a temperature of about 25 ° C at a rate of about 7.5 ° C / hour; (X) cooling the product of step (viii) to a temperature of about 7 to 8 ° C at a rate of at least about of 9 ° C / hour; (x) maintaining the product of step (ix) at the same temperature for about 2 hours; and (xi) filtering the product of step (x) to obtain crystalline rapamycin. In a further embodiment, a method for purifying rapamycin is provided and includes (i) heating crude rapamycin in ethyl acetate at about 55 ° C; (I) filtering the product of (i) (iii) maintaining the temperature of step (ii) from about 54 ° C to about 57 ° C; (iv) adding heptanes to the product of step (iii) for a period of about 60 minutes at constant speed; (v) maintaining the product of step (iv) at this temperature for about 30 minutes; (vi) reducing the stirring speed of step (v); (vii) cooling the product from step (vi) to around 40 ° C at a rate of about 5 ° C / hour; (viii) cooling the product of step (vii) to a temperature of about 25 ° C at a rate of about 7.5 ° C / hour; (ix) cooling the product of step (viii) to a temperature of about 7 to 8 ° C at a rate of at least about 9 ° C / hour; (x) maintaining the product of step (ix) at this temperature for about 2 hours; (xi) filtering the product of step (x) to obtain crystalline rapamycin; (xii) wash the crystalline rapamycin with ethyl acetate and heptane at about 8 ° C; and (xii) drying the product of step (xii).

B. Methods for Analyzing Rapamycin Compounds Methods for analyzing rapamycin compounds are also described and are typically performed using differential calorimetry.

Sweep (DSC). Other techniques can be used in conjunction with DSC and include X-ray diffraction (XRD) and Raman spectroscopy, without restriction. A variety of DSC instruments are known in the art and can be used. In one embodiment, the DSC instrument is the Q1000 ™ instrument (TA Instruments), among others. The term "rapamycin compound" defines a class of immunosuppressive compounds that contain the basic rapamycin nucleus shown above. The rapamine compounds of this invention include compounds that can be chemically or biologically modified as derivatives of the rapamycin nucleus, while maintaining immunosuppressive properties. Accordingly, the term "rapamycin compound" includes esters, ethers, oximes, hydrazones and hydroxylamines of rapamycin, as well as rapamycin in which the functional groups in the rapamycin nucleus have been modified, for example by reduction or oxidation. The term "rapamycin compound" also includes pharmaceutically acceptable salts of rapamycin, and are capable of forming such salts, either by virtue of containing an acidic or basic portion. Examples of rapamycin compounds that can be analyzed as described herein include, without restriction, rapamycin, 42-esters of rapamycin including CCI-779 (temsirolimus), norrapamycin, deoxorapamycin, desmethylpamycin, or demetoxirapamycin or pharmaceutically acceptable salts, prodrugs or metabolites of the same and those described in the United States Patent Application Publication Nos. US-2005- 0272702, US-2006-013550, US-2006-0040971, US-2006-0036091, US-2005-0014777, US-2006-0199834, US-2005-0234086, and US-2003-0114477 and US patent. Nos. 5,358,908; 5,358,909; 5,362,718; 5,302,584, which is incorporated herein by reference. In one example, a rapamycin compound includes rapamycin that can be purchased commercially or can be prepared using a variety of methods available in the art. In another example, a rapamycin compound includes CCI-779. The term "CCI-779" as used herein refers to the 42-ester of rapamycin with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid. A variety of methods for preparing CCI-770 are known in the art and include those described in U.S. Patent Nos. 5,362,718 and 6,277,983, which are incorporated herein by reference. Alternatively, CCI-779 can be purchased commercially (for example with Wyeth). CCI-779 may be micronized or non-micronized, as described in U.S. Patent Application Publication No. US-2005-0152983-A1, which is incorporated herein by reference.

CCI-779 The term 'desmethylrapamycin' refers to the class of rapamicma compounds that do not have one or more methyl groups. Examples of desmethyl rapamycins that may be used in accordance with the present invention include 3-demet? Rapam? Cna (US Patent No. 5,358,969), 7-O-desmet? L-rapam? C? Na (U.S. Patent No. 6,399,626), 17-demethylrapamycin (U.S. Patent No. 6,670,168), and 32-0-demethylrapamycin, among others. The term "demethoxyrapamycin" "refers to the class of rapamycin compounds that do not have one or more methoxy groups and includes, without restriction, 32-desmetox? rapam? c? na The rapamicma compounds measured in the methods described herein include samples in solid state and can they are crystalline, semicpstalline, non-crystalline and aggregates. Crystalline rapamycin is preferably prepared according to the procedures described in Sehgal et al, J Antibiotics, 28 (10) 727-732 (1975), Swindells et al, Canadian J Chem, 56 ( 18) 2491-2492 (1978), and the pub US Patent Application No. US-2006-040971 CCI-779 crystalline is preferably prepared by recrystallization from diethylether and heptane as described in U.S. Provisional Patent Application No. 60 / 748,006, which are incorporated by reference into US Pat. present Samples containing rapamycin compounds may contain low levels of impurities, including oxidant and / or hydrolysis impurities, solvents or the like In one example, the samples of CCI-779 they contain only trace amounts of acetone, preferably less than about 0.3% w / w acetone. Similarly, samples of CCI-779 contain less than about 0.3% w / w phenylboronic acid and less than 1.5% p oxidant / hydrolysis decomposition products from CCI-779. The term "crystalline" as used herein refers to solid samples of rapamycin compounds that have a definite crystalline structure. The term "semi-crystalline" as used herein refers to solid samples of rapamycin compounds having crystalline regions dispersed within amorphous regions. The term "non-crystalline" and "amorphous" are used interchangeably and refer to solid samples of rapamycin compounds that do not have crystalline regions dispersed therethrough and therefore have no crystalline form. The term "aggregate" as used herein refers to the grouping of crystals that grow internally or fuse into a particle of a rapamycin compound. It is known that crystal quality influences the stability of the sample containing a rapamycin compound. For example, the amorphous or semicrystalline compounds of rapamycin undergo rapid oxidative degradation. Additionally, the average particle size of the rapamycin compounds determines the flow property, with a larger particle size desired. The method thus includes determining / calculating the quality of the particle, its crystallinity, particle size or a combination of these from a sample containing a rapamycin compound, ie the test sample. The method is thus performed by analyzing the DSC heat flux signal of the rapamycin compound. The heat flux signal of the rapamycin compound is then compared to the heat flux signal of a predetermined standard. A series of useful parameters can be obtained from the heat flow signal and include melting temperature and including the start and peak temperature melting temperature, as well as heat of melting. These parameters can also be used in the determination of particle quality, crystallinity or particle size. The term "melting temperature" as used herein includes the temperature at which a solid, i.e. a rapamycin compound, melts. The melting temperature may include the initial melting temperature or the peak melting temperature. Typically, the melting temperature is the peak melting temperature. The term "heat of fusion" as used herein describes the total heat released by a rapamycin compound during melting or melting. The heat of fusion is obtained by integrating the area under the heat flow signal graph and is typically expressed in calories / gram or Joules / gram. However, other conventions may be used to express the fusion heat units by those skilled in the art. Desirably, the DSC peak temperature, i.e. the melting temperatures, of the heat flux signal of the compound of Rapamycin is measured and compared to the heat flow signal of the predetermined standard. As used herein, the term "predetermined standard" refers to one more solid samples of a highly crystalline rapamycin compound in which the average particle size and crystallinity is known and related to a peak DSC temperature. More desirably, the predetermined standard contains a crystalline rapamycin compound. More desirably, the predetermined standard contains a 100% crystalline rapamycin compound. The heat flux signal of the rapamycin compound can be compared with the heat flux signal of the predetermined standard by an individual point ratio or by using a calibration curve. By doing this, the crystallinity, particle size or particle quality of the rapamycin compound being analyzed can be determined. In a modality, the heat flux signal of the test sample containing the rapamycin compound is compared to the heat flux signal of the predetermined standard containing the crystalline rapamycin compound using a one point ratio. Typically, the heat of fusion obtained from the heat flow signal is used for comparison. In one example, the heat of fusion is used in an individual point ratio to determine the crystallinity of a compound of Rapamycin In another example, the crystallinity of a rapamycin compound was calculated using an individual point ratio. In an individual example, the crystallinity of the rapamycin compound can be calculated using the following equation: crystallinity 1 00 x heat of fusion of the test sample demonstrates test = - - - - - heat of fusion of the predetermined standard In another embodiment, the heat flux signal from the test sample containing the rapamycin compound is compared to the heat flux signal and the predetermined standard containing the crystalline rapamycin compound used in the calibration curve. One skilled in the art will readily be able to prepare a calibration curve using the teachings of the description and knowledge in the art. Typically, the calibration curve is prepared for the predetermined standard using multiple samples containing crystalline rapamycin compounds. Desirably, at least 3 samples are necessary to generate the calibration curve. However, more samples can be used as determined by one skilled in the art to prepare the calibration curve. In one example, the heat of fusion of a test sample containing a rapamycin compound is used in combination with a calibration curve to determine the crystallinity of the rapamycin compound. The calibration curve is prepared by plotting the heat of fusion, peak temperature or start temperature for each of the multiple samples against the crystallinity of each of the multiple samples for obtain the calibration curve. A curve or line with better fit is then drawn and the formula of the best fit line is calculated. In another example, the calibration curve is prepared by plotting the heat of fusion against the crystallinity. In a further example, the calibration curve is prepared by plotting the peak temperature against the crystallinity. In still a further example, the calibration curve is prepared by plotting the start temperature against the crystallinity. In yet another example, the calibration curve is calculated by plotting the heat of fusion for each of multiple samples containing a crystalline rapamycin compound of a known crystallinity against the crystallinity for each of multiple samples containing the rapamycin compound. Typically, the calibration curve is specific to the type of the DSC instrument and to the experimental conditions and the procedure used to obtain the values of heat of fusion. However, one skilled in the art will be able to determine whether a calibration curve obtained from a DSC method and instrument can be used when using data obtained from another DSC instrument using the same procedure. Once the calibration curve is prepared, it can then be used to determine the crystallinity of test samples containing a rapamycin compound. Specifically, test samples containing a rapamycin compound are analyzed to determine one or more of the heat of fusion, peak temperature or start temperature of the rapamycin compound in the test sample. These values, is say the heat of fusion, peak temperature or start temperature can then be used using the formula of the line of best fit of the predetermined standard to determine the likelihood, among other factors, and the rapamycin compound in the test sample. When doing this, an accurate determination of the density of samples containing rapamycin compounds can be obtained. The inventors found the tendency in the DSC heat flux signal and thus the melting temperature, for the samples of the rapamycin compound. In particular, the signal The heat flux of the samples containing a rapamycin compound appears to vary depending on the amount of the rapamycin compound. In one example, the sample crystalness of the rapamycin compound is proportional to the melting temperature of the heat flow signal. In one embodiment, the samples containing higher crystalline rapamycin had higher particles and higher melting temperatures of at least about 188 °. C, preferably about 188 ° C to about 190 ° C Samples containing less crystalline rapamycin had smaller particles and lower melting temperatures of less than about 183 ° C, preferably less than about 180 less than about 183 ° C See figure 2 In another embodiment, the samples containing CCI-779 higher crystalline had higher particles and higher melting temperatures of at least about 168 ° C, preferably around 168 ° C to around 170 ° C. Samples containing less crystalline CCI-779 had smaller particles, lower melting temperatures of at least about 166 to less than about 168 ° C. Samples containing semicrystalline aggregate of CCI-779 had lower melting temperatures than crystalline samples, ie casting temperatures of at least about 164 ° C to less than about 166 ° C. Additionally, samples containing non-crystalline CCI-779 had glass transition temperatures but had no melting temperatures. See figure 3. As noted above, the melting temperature of DSC is to provide the size and crystallinity of the particles of the rapamycin compound. For samples containing CCI-779, a large particle size includes particles that have an average particle size greater than about 30 μm in length for the longest axis of the particle and more preferably around 30 μm to about 250 μm in order to the longest axis of the CCI-779 particle. Alternatively, a small particle size includes particles having an average particle size of less than about 30 μm for the longest axis of the CCI-779 particle. The inventors also found that the X-ray diffraction pattern of a less crystalline rapamycin compound contained broad peaks. Furthermore, when the samples containing amorphous and crystalline rapamycin compounds are analyzed by XRD, the XRD pattern showed sharp peaks of the crystalline rapamycin compound and a displacement of the baseline or "amorphous halo" for the amorphous compound of rapamycin. In one embodiment, a method for measuring the particle quality of a rapamycin compound using differential scanning calorimetry is described, including analyzing the heat flux signal of a sample containing a rapamycin compound; and comparing the heat flow signal with a predetermined standard, wherein the particle quality is to provide the melting temperature of a sample. In another embodiment, a method for determining the particle size of a rapamycin compound using differential scanning calorimetry is described, including analyzing the heat flux signal of a sample containing a rapamycin compound and comparing the heat flux signal. with a predetermined standard, where the particle size is proportional to the melting temperature of the sample. In a further embodiment, a method for determining the particle quality of a rapamycin compound using differential scanning calorimetry is provided, including analyzing the heat flux signal of a sample containing a rapamycin compound and comparing the flow signal of heat with a predetermined standard, wherein a large particle size of a rapamycin compound is characterized by a high melting temperature and a small particle size is characterized by a low melting temperature.

The following examples are provided to illustrate the invention and do not limit the scope thereof. One skilled in the art will observe that although specific reagents and conditions are delineated in the following examples, modifications can be made that are intended to be encompassed by the essence and scope of the invention.

EXAMPLES EXAMPLE 1 General procedure for analyzing particles from samples of CCI-779 In this example, DSC peak temperatures were measured and used to evaluate the particle categories for test samples containing CCI-779. Samples containing CCI-779, which are obtained by crystallizing CCI-779 from ether / heptane using the procedure set forth in U.S. Provisional Patent Application No. 60 / 748,006 were analyzed using the DSC Q Series ™ Q1000- 0450 (TA Instruments) using the parameters in Table 1. Once the DSC peak temperatures were obtained, they were compared to predetermined standards containing crystalline CCI-779 and placed in particle categories. See Figure 1 in which the peak temperatures for the 25 samples were grouped according to a particle category. As there was an overlap in peak temperatures for certain samples, 25 different samples are not visible. A category of particle 1 refers to crystalline samples of CCI-779 having large particles; a category of particle 2 refers to the crystalline samples of CCI-779 that have small particles; a category of particle 3 refers to semicrystalline aggregates of CCI-779; and the category of particle 4 refers to CCI-779 non-crystalline. TABLE 1 From these data it is determined that higher peak temperatures of DSC indicate samples of CCI-779 that are more crystalline.

EXAMPLE 2 Analysis of sample particles CCI-779 In this example, the particle quality, crystallinity and melting temperature of twenty-five (25) samples of CCI-779 obtained by crystallizing CCI-779 from ether / heptane using the procedure established in the provisional patent application of the Member States were measured. United No. 60 / 748,006. The solid samples were analyzed for the DSC peak temperatures using the Q Series ™ Q1000-0450 DSC instrument (TA Instruments) using the parameters in Table 1 as stated above. The degree and size of crystallinity of the sample is then analyzed by optical microscopy. In summary, optical microscopy was performed using a Nikon ™ Eclipse E600 microscope capable of 5x to 100x magnification, equipped with a Nikon ™ DXM 1200 digital camera and a Nikon ™ calibrated image acquisition system ACT-1 v 2.12. The measurements were obtained by dispersing about 0.05 mg of the sample in a glass holder. The sample was then covered with a drop of Resolve® microscope immersion oil (Richard-Alian Scientific) and a coverslip was added. Care was taken that the particles did not undergo friction during image acquisition. The images of the samples were acquired about one to two minutes after sample preparation. Fresh samples were prepared if re-capture of the images was required. The "class" of the sample was determined by relating the DSC temperature to the "degree" and "crystallinity size" of the sample. Specifically, if it was determined that a sample containing CCI-779 is crystalline by optical microscopy with large crystals, a class 1 sample was assigned; if it was determined by optical microscopy that a sample containing CCI-779 was crystalline with small crystals, it was assigned a class 2 sample; and if it was determined by optical microscopy that a sample containing CCI-779 was semicpstalin, regardless of crystal size, was assigned a class 3 sample. Then, the sample class was related to the DSC peak temperature obtained for the same sample TABLE 2 'No peak DSC temperature was obtained for these samples. These results illustrate that, in general, the CCI-779 crystalline samples had higher peak DSC temperatures than the semicrystalline samples of CCI-779. In addition, the CCI-779 samples containing larger crystals generated peak temperatures of DSC than the CCI-779 samples containing smaller crystals.

EXAMPLE 3 Rapamycin crystallization A crude rapamycin is suspended in ethyl acetate and heated to 55 ° C. The heated solution is then filtered using a lightening filter in a crystallization vessel and the solution is maintained at 54 to 57 ° C. Heptanes are then added to the vessel for a period of 60 minutes at constant speed. After the addition of heptanes, the solution is maintained at 55 to 57 ° C for 30 minutes. The agitation speed is then reduced in an effort to achieve a solid suspension. The solution is then cooled to 40 ° C for a period of 3 hours at a rate of 5 ° C / hour; then it is cooled to 25 ° C for a period of 2 hours at a speed of 7.5 ° C / hour; and then further cooled to 7 to 8 ° C for a period of at least 60 minutes, preferably over a period of 2 hours at a speed of 9 ° C. The solution is then kept at 7 to 8 ° C for 2 hours and then filtered. The solid obtained from the filtration is then washed using a solution containing ethyl acetate / heptanes maintained at 8 ° C. The washed solid is then dried using a low shear dryer to obtain crystalline rapamycin.

EXAMPLE 4 Analysis of the crystallinity of samples containing different content of crystalline rapamycin In this example, samples containing rapamycin were analyzed (Table 3) by DSC and optical microscopy. The morphology and average particle size were determined by optical microscopy. A DSC standard sample rate increase of 10 ° C / min and a tray was used. of hermetically sealed aluminum. The DSC graphs were obtained and are given in Figures 2A and 2B. Sample 1 contained crystalline rapamycin and was prepared by suspending crude crystalline rapamycin (1 g) in 10 mL of methox? -2-propanol and heating the suspension to 40 ° C. to obtain a transparent solution. The solution was cooled from 40 ° C to 1 5 ° C over a period of 2 hours and resulted in the gradual cpstalinization of rapamycin. The crystallized solid was collected by filtration at room temperature and air dried at room temperature Sample 2 contained crystalline rapamycin and about 2 to about 3% oxidant degradation / hydrolysis impurities and was prepared using the procedure of Example 3 A portion of the batch was maintained at 25 ° C and at 60% relative humidity for 2 months. Sample 3 contains crystalline rapamycin and around 2 to about 3% oxidant degradation / hydrolysis impurities and was prepared using the procedure of Example 3 It was subjected to a batch portion at 25 ° C and 60% relative humidity for 4 months TABLE 3 These results illustrate that low levels of impurity and samples containing crystalline rapamycin do not affect the DSC melting temperature of a crystalline rapamycin sample over time EXAMPLE 5 Calculation of a calibration curve to determine the relationship between the heat of fusion and the crystallinity This example was made to prepare a calibration curve to establish the relationship of the heat of fusion with the stability. Samples containing CCI-779 known crystalline were analyzed, that is, a predetermined standard, using DSC and the parameters observed in table 1. Each sample contained a known percentage of CCI-7709 crystalline and amorphous The results were given in Table 4 TABLE 4 The crystallinity is then plotted against each of the heat of fusion, start and peak temperature. See figure 3. The graph illustrates that all three parameters were linearly related to the amount of crystalline CCI-779 in the samples. The graph also illustrates that the best linear relationship is achieved using the heat of fusion. Not only was the ratio error of the melting heat measurement lower than in the two parameters, it also has a higher sensitivity. The highest sensitivity was determined by monitoring the slope of the line, whose slope is around twice (006049) the slope of the start temperature (0.0875). Specifically, by using the heat of fusion obtained from each sample and the degree of crystallinity, the relationship between crystallinity and heat of fusion for this particular instrument was determined as illustrated by the following equation. Degree of crystallinity = 1.6465 x heat fusion + 3.5988 EXAMPLE 6 Determination of the crystallinity of samples containing various amounts of crystalline CCI-779 This example was performed to determine the precision of the equation set forth in Example 4 Specifically, the heats of fusion were determined for four (4) samples containing known amounts of crystalline CCI-779. Once determined, the coefficients were calculated using the equation of example 4 The results are shown in table 5 TABLE 5 These data illustrate that by using the heat of fusion, the station can be calculated accurately with an error of less than 3% EXAMPLE 7 Example of sample weight in the heat of fusion In this example, the effect of the weight of the sample was measured when determining the crystallinity of 3 samples of CCI-779. Samples 1 and 2 were obtained by crystallizing CCI-779 from ether / heptane using the procedure set forth in U.S. Provisional Patent Application No. 60/748, 006. Sample 3 was obtained using the procedure set forth in United States Provisional Patent Application No. 60 / 7478,143. Samples were analyzed for the DSC start temperature, peak temperatures and heat of fusion values using the instrument DSC Q Senes ™ Q1000-0450 (TA Instruments) using the parameters set forth in Table 2. The results were given in Tables 6-8.

TABLE 6 TABLE 7 TABLE 8 Using the average heat of fusion established in Tables 6-8 above, the degree of cpstance of each batch was calculated using the equation set forth in Example 3 The results were given in Table 9 TABLE 9 These data illustrate that the weight of the sample does not substantially affect the crystallinity of a sample or the use of heat of fusion to predict the crystallinity of a sample containing CCI-770.

EXAMPLE 8 Determination of the crystallinity of samples containing CCI-779 Nineteen (19) samples containing crystalline CCI-779 were prepared and analyzed using the DSC parameters set forth in Table 2. The crystallinity of each sample was calculated using the average heat of fusion obtained for each sample using DSC and the equation of the example 6 and which is reproduced below. The results are given in table 10.

TABLE 10 The stability of the samples was analyzed separately after a period of 6 months at (i) 5 ° C or (n) 25 ° C at a relative humidity of 60%. The results indicate that the batches that have a higher CCI-779 crystalline content were more stable than the samples containing a lower crystalline CCI-779 content.

EXAMPLE 9 Variation of heating speed over fusion heat and crystallinity Six samples containing crystalline CCI-779 were analyzed by DSC using the parameters set forth in Table 2. Samples 1, 4, and 7 contained 7 mg crystalline CCI-779 and were heated in DSC at a temperature of 7 ° C / min. Samples 2, 5, and 8 contained 10 mg of crystalline CCI-779 and were heated in DSC at a rate of 10 ° C / min. Samples 3, 6, and 9 contained 20 mg crystalline CCI-779 and were heated in DSC at a rate of 20 ° C / min. The peak temperature, the start temperature and the heat of fusion of DSC were obtained and are given in table 11.

TABLE 11 The data illustrated that increasing the heating rate during DSC analysis did not significantly alter the heat of fusion. All publications cited in this description are incorporated herein by reference herein. Although the invention has been described with reference to a particularly preferred embodiment, it will be noted that the modifications can be made without departing from the essence of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims (3)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for measuring the particle quality of a rapamycin compound using differential scanning calorimetry comprising: analyzing the heat flux signal of a sample comprising a rapamycin compound; and comparing the heat flow signal and said sample with the heat flow signal of a predetermined standard; wherein said particle quality is proportional to the melting temperature of said heat flow signal of said sample. 2. The method according to claim 1, further characterized in that the higher melting temperature corresponds to a particle of higher quality. 3. The method according to claim 1 or 2 further characterized in that a higher particle quality corresponds to a higher crystallinity of said rapamycin compound. 4. The method according to claim 1, further characterized in that said melting temperature is proportional to the average particle size of crystals of said rapamycin compound. 5. The method according to claim 4, further characterized in that a large average particle size is characterized by a high melting temperature. 6. - The method according to claim 5, further characterized in that said sample comprises CCI-779 and said large particle mean size is less than 30 μm. 7 - The method according to claim 6, further characterized in that said large average particle size is from about 30 μm to about 250 μm. 8. The method according to claim 5, further characterized in that said sample comprises CCI-779 and said high melting temperature is at least about 168 ° C. 9. The method according to claim 8, further characterized in that said high melting temperature is from about 168 to about 170 ° C. 10. The method according to claim 5, further characterized in that said sample comprises rapamycin and said high melting temperature is at least about 188 ° C. 11. The method according to claim 10, further characterized in that said high melting temperature is from about 188 ° C to about 190 ° C. 12. The method according to claim 1, further characterized in that a lower melting temperature corresponds to a particle of lower quality. 13 -. 13 - The method according to claim 12, further characterized in that a lower particle quality corresponds to a lower capacity 14 - The method according to claim 1, further characterized in that a small average particle size of said rapamycin compound has a low melt temperature 15 - The method according to claim 12 or 14, further characterized in that said sample comprises CCI-779 and said low melting temperature is less than about 166 ° C. according to claim 15, further characterized in that said low melting temperature is from about 164 to about 166 ° C. The method according to claim 12 or 14, further characterized in that said sample comprises rapamicma and said low melting temperature is less than about 183 ° C 18 - The method according to claim 12 or 14, further characterized in that said sample comprises rapamycin and said low melting temperature is less than about 180 to about 183 ° C. The method according to claim 14, further characterized in that said sample comprises CCI-779 and said Small average particle size is less than about 30 μm 20. - The method according to claim 1, further characterized in that said sample comprises semicrystalline aggregates and has a melting temperature lower than a crystalline sample. 21. The method according to claim 1, further characterized in that said sample comprises a non-crystalline rapamycin compound and has a melting temperature lower than a sample comprising a semicrystalline rapamycin compound. 22. The method according to claim 1, further characterized in that said sample comprises a non-crystalline rapamycin compound and has a melting temperature lower than a sample comprising a crystalline rapamycin compound. 23. The method according to claim 1, further characterized in that said rapamycin compound is purified from the same solvent as the predetermined standard. 24. A method for determining the average particle size of a sample containing crystals of a rapamycin compound using differential scanning calorimetry comprising: analyzing the melting temperature of a sample comprising a rapamycin compound; and comparing the melting temperature with a predetermined standard; wherein said average particle size is proportional to the melting temperature of said sample. 25 -. 25 - The method according to claim 24, further characterized in that a large average particle size has a high melting temperature and a small average particle size, a low melting temperature 26 - A method for determining the cnstalinity of a compound Rapamycin comprises analyzing the heat flux signal of a test sample comprising a rapamycin compound, and calculating the crystallinity of said test sample by comparing said heat flux signal with the heat flux signal of a predetermined standard. comprising a crystalline rapamycin compound 27 - The method according to claim 26, further characterized in that said calculation is performed by using a single calculation point 28 - The method according to claim 27, further characterized in that said predetermined standard comprises a 100% crystalline rapamycin 29 - The method according to claim 28, further characterized in that said crystallinity of said test sample is calculated according to the following

Test sample stability = 100 x heat of fusion of said test sample Heat of fusion of said predetermined standard 30. - The method according to claim 26, further characterized in that said calculation is made by using a calibration curve. 31. The method according to claim 30, further characterized in that said predetermined standard comprises multiple samples comprising crystalline rapamycin compound. 32. The method according to claim 31, further characterized by additionally comprising plotting the heat of fusion, the peak temperature or the initial temperature for each of said multiple samples against the crystallinity of each of said multiple samples to obtain a calibration curve that has a better adjustment line; calculate a formula of said best adjustment line; analyzing the heat of fusion, the peak temperature or the initial temperature of said rapamycin compound in said test sample; and calculating the crystallinity of said rapamycin compound in said test sample using said melting heat, peak temperature or initial temperature of said test sample and said formula. 33. The method according to claim 31, further characterized in that said calibration curve is prepared by plotting said heat of fusion for each of the multiple samples comprising a crystalline rapamycin compound of a known crystallinity against the crystallinity for each one of the multiple samples comprising said rapamycin compound.

3. 4 - . 34 - The method according to any of claims 1 or 24 to 33, further characterized in that said sample comprises rapamycin 35 - The method according to any of claims 1 or 24 to 33, further characterized in that said sample comprises CCI-779 36 - A method for purifying rapamycin comprises, (i) heating the crude rapamycin in ethyl acetate at about 55 ° C, (n) filtering the product from step (i), (ni) maintaining the temperature of the step (n) ) at about 54 ° C to about 57 ° C, (iv) adding heptanes to the product of step (ni) over a period of about 60 minutes at a constant rate, (v) keeping the product from step (iv) to said temperature for about 30 minutes, (vi) reducing the stirring speed of step (v), (vn) cooling the product of step (vi) to about 40 ° C at a rate of about 5 ° C / hour, (vin) cooling the product of stage (vn) to a at a temperature of about 25 ° C at a rate of about 7 5 ° C / hour, (ix) cooling the product of step (vm) to a temperature of about 7 to 8 ° C at a rate of at least about 9 ° C / hour, (x) maintaining the product of step (ix) at said temperature for about 2 hours, and (xi) filtering the product of step (x) to obtain said crystalline rapamycin 37 - The method according to claim 36, further characterized by additionally comprising (XII) washing said crystalline rapamycin with ethyl acetate and heptane at about 8 ° C; Y (xiii) drying the product of step (xii).