CA2188529C - Crystal of pyrrolidylthiocarbapenem derivative, lyophilized preparation containing said crystal, and process for producing the same - Google Patents

Abstract

The present invention provides a crystal of (1R,5S,6S)-6-[(1R)-1-hydroxyethyl]-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid represented by the following formula, a lyophilized preparation comprising the crystal, and a process for producing the same. The production process comprises the steps of freezing an aqueous solution containing the above-mentioned compound by cooling to -20°C or lower; warming the frozen solution to 0 to -10°C; and cooling and warming the frozen solu-tion at least twice in a temperature range of 0 to -10°C.

Description

DESCRIPTION

CRYSTAL OF PYRROLIDYLTHIOCARBAPENEM DERIVATIVE, LYOPHILIZED PREPARATION CONTAINING SAID CRYSTAL, AND PROCESS FOR PRODUCING THE; SAME
TECHNICAL FIELD
The present invention relates to a crystal of pyrrolidylthiocarbapenem derivative excellent in storage stability and solubility, a lyophilized preparation containing the crystal and a process for producing the same.
BACKGROUND ART
(1R,5S,6S)-6-[(1R)-1-hydroxyethyl]-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid represented by the following formula (hereinafter referred to as S-4661 in the specification) is a pyrrolidylthiocarbapenem deriva-tive. S-4661 is a useful compound as an antimicrobial drug, and orally or parenterally administered.
COzH H
O
\ S ~NHSOZNHz . HOC H ~NH
H ~H H CHa S-4661 itself is described in Japanese Laid-Open Patent Publication (Kokai) No. 5-294970. However, no crystal of S-4661 is described in the Publication. When z ~ 8~~z9 S-4661 is produced, conventionally, a process for produc-ing it in an amorphous form only is known. However, an amorphous solid of S-4661 has insufficient stability during storage. Therefore, when S-4661 is stored under ordinary storage conditions for a long period, the color thereof is changed, and the purity disadvantageously deteriorates. Thus, it is desired to prepare a crystal-line preparation of S-4661 having a higher storage stability than an amorphous preparation. In particular, among the crystalline preparations, a preparation con-taining an inner salt is more preferable in that it is unnecessary to use an additive in a large amount in order to form a salt.
Furthermore, among the crystalline preparations of S-4661, a crystalline lyophilized preparation is preferable, because it is generally easy to ensure sterility of a lyophilized preparation and to remove particulate matters from the lyophilized preparation.
As a general process for producing a lyophilized preparation, a variety of processes are conventionally known. For example, Japanese Laid-Open Patent Publica-tion (Kokai) Nos. 61-172878, 63-174927 and 4-59730 disclose that NaCl is added to a solution before lyophi-lization in order to improve solubility and stability of an amorphous lyophilized preparation. Japanese Laid-Open Patent Publication (Kokai) No. 4-338332. discloses that Na2C03 or NaHC03 is added to a drug in a form of a hydro-chloride salt so that an insoluble free base is not deposited.
The following processes are known as a process 2 ~ b~~~9 for obtaining a crystalline lyophilized preparation: for example, Japanese Patent Publication (Kokoku) No. 4-66202 discloses that an aqueous solution of a drug is allowed to be in a supercooled state without freezing, and crystal nuclei are generated in the supercooled state;
Japanese Patent Publication (Kokoku) No. 60-19758 dis-closes that an aqueous solution of a drug containing 2 to 25 v/v o of alcohol is gradually cooled to freeze water and crystal nuclei are generated in a state of a concen-trated alcohol solution; and Japanese Patent Publication (Kokoku) Nos. 60-19759, 03-74643 and Japanese Laid-Open Patent Publication (Kokai) No. 5-271241 disclose that an aqueous solution of a drug is frozen at a predetermined temperature, then warmed to a predetermined temperature, and retained at a constant temperature.
Conditions suitable for lyophilization are varied depending on the drugs to be lyophilized. Therefore, the aforementioned known processes are not necessarily suit-able for a process for producing a lyophilized prepara-tion containing the crystal of S-4661.. For example, since the process for lyophilization by freezing an aqueous solution containing S-4661 at a predetermined temperature and retaining it at a constant temperature takes a very long time, it is not suitable as an indu-strial process.
As described above, no process suitable to obtain a crystal of S-4661, in particular, an :inner salt crys-tal, and no lyophilization process suitable to obtain a lyophilized preparation containing the crystal have been found yet.

21 ~3~~9 DISCLOSURE OF THE INVENTION

A crystal of S-4661 of the present invention is a crystal of pyrrolidylthiocarbapenem derivative S-4661 represented by the following formula.
o co2H H
~NHSOzNHz to Hs~ = ~ NH
H H
OH
A lyophilized preparation containing the above mentioned crystal is also in the scope of the present invention.
The process for producing a lyophilized prepara-tion according to the present invention comprises the steps of freezing an aqueous solution containing S-4661 by cooling to -20°C or lower; warming the frozen solution to 0 to -10°C; and cooling and warming the frozen solu-tion at least twice in the temperature range of 0 to -10°C.
The present invention provides a crystal of S-4661 excellent in storage stability and solubility, and having a high industrial applicability. Furthermore, the present invention provides a lyophilized preparation containing the crystal of S-4661, excellent in storage stability and reconstitution property.
BRIEF DESCRTPT'T(1N (1F mHF nunrartrr_c Figure 1 is a powder X-ray diffraction chart of a lyophilized product of S-4661 obtained in Example 3.
Figure 2 is a powder X-ray diffraction chart of a lyophilized product of S-4661 obtained in Example 8.
Figure 3 is a powder X-ray diffraction chart of a lyophilized product of S-4661 obtained in Example 13.
Figure 4 is a powder X-ray diffraction chart of a lyophilized product of S-4661 obtained in Example 14.
BEST MODE FOR CARRYING OUT THE INVENTION
A crystal of S-4661 of the present invention is a crystal of pyrrolidylthiocarbapenem derivative S-4661 represented by the following formula.
cot H H

~ NHSOzNHz S t HOC H' NH
H ~H H CHI
The crystal of S-4661 is preferably an inner salt crystal. The inner salt crystal of S-4661 is believed to be a betaine structure represented by the following formula.
3o O G02 H
~NHS02NHz - ~- NH2 H H ' H. O H

Such an inner salt crystal is mare preferable in that it has a pure form containing no counter ion other than a desired component, unlike the case of an Na salt and the like.
The results of measurements by a powder X-ray diffraction method reveal that two different crystal forms exist for a crystal of S-4661. Hereinafter, the two crystal forms are referred to as Type I and Type II.
Type I crystal and Type II crystal are identified by characteristic peaks obtained by the powder X-ray dif-fraction method. Diffraction angles (28) of the charac-teristic main peaks of the respective crystal forms are shown below.
1. 5 Type I: 7.32, 14.72, 16.62, 20.42, 21.1, 22.18, 23.88 and 29.76 (degree) Type II: 6.06, 12.2, 14.56, 17.0, 18.38, 20.68, 24.38, 24.60, 25.88 and 30.12 (degree) (X ray-diffraction measurement conditions: CuKa line, 1.54 ~1 (monochromator), a tube voltage 40 kV, a tube current 40 mA) The crystal structure of S-4661 described above is novel knowledge.
In the case where the crystal of the present invention is used as an aqueous solution, for example, used for an injection, Type II crystal is more preferable to Type I crystal in that the dissolution rate of Type II
crystal is higher.
The crystal of S-4661 of the present invention ~, ,,:_ can be obtained by a method such as recrystallization, but can also be obtained in a form contained in a lyophi-lized preparation and the like.
In order to obtain the crystal of S-4661 of the present invention by recrystallization, S-4661 is crys-tallized from an organic solvent such as alcohol and acetone, water, or a mixed solution thereof. Examples of alcohol used herein include methanol, ethanol, isopropa-:10 nol and the like. In the case where a mixed solvent of an organic solvent and water is used, a. mixing ratio of water to the organic solvent is preferably 1:1 to 1:5 (v/v). In order to obtain the crystal of the present invention, S-4661 is dissolved in the organic solvent, water, or the mixed solvent to prepare an S-4661 solu-tion. A concentration of the S-4661 solution is prefera-bly about 5 to 40 percent by weight. In order to deposit the crystal of S-4661 from the solution, any crystalliza-tion operation such as cooling and/or stirring can be performed. Crystals of S-4661 can be preferably obtained by stirring the solution while cooling it to about 0 to 10°C.
The crystals of the present invention may be polymorphism, but can be obtained in the form of a single crystal by controlling crystallization conditions. In order to obtain Type I crystal, for example, S-4661 is crystallized from water or a water/ethanol system. On the other hand, Type II crystal can be obtained by crystallization from water, but preferably crystallized from a water/isopropanol system, and more preferably, the water/isopropanol system is a mixed solution of water and isopropanol in proportion of 1 . 3 (v / v).

21$529 _ g _ Although a water content of the crystals of the present invention is not necessarily constant but varied depending on drying conditions and storage conditions, crystals of Type I generally tend to be stabilized with water content in the range of about 0 to 5 . 0 o at room temperature. Type II crystal may be stabilized with water content in a ratio of up to about loo at room temperature. In either case of any water content, however, the characteristic diffraction angles ( 28 ) in X-ray diffraction patterns are not changed, but the charac teristic peaks described above are present. An amount of an organic solvent which remains in the crystals is not constant in either type of the crystal, but varied depending on the crystallization method and drying conditions.
The crystal of S-4661 of the present invention can be obtained as a lyophilized preparation. Hereinaf-ter, the lyophilized preparation containing the crystal of S-4661 may be simply referred to a.s a crystalline lyophilized preparation. The crystalline lyophilized preparation of the present invention can contain either one of the two types of crystal, i.e., Type I crystal and Type II crystal, or a mixture thereof. In the case where the crystalline lyophilized preparation of the present invention contains the two types of crystals as a mix-ture, each crystal can be contained :in an arbitrary proportion.
A lyophilized preparation containing the crystal of S-4661 can be preferably obtained by the following process. The process comprises the steps of freezing an aqueous solution containing S-4661 by cooling to -20 ° C or lower; warming the frozen solution to 0 to -10°C; and cooling and warming the frozen solution at least twice in the temperature range of 0 to -10°C. This process is one aspect of the present invention.
In order to obtain a crystalline lyophilized preparation of S-4661 by the process of the present invention, first, an aqueous solution of S-4661 is prepared. S-4661 can be synthesized by a known process and be in any state such as a crystal, amorphous, a hydrate, a solvate, and a mixture thereof. The aqueous solution of S-4661 can contain S-4661 at: a concentration of about 8 to 17 percent by weight. The pH of the aqueous solution is about 5.8 to 6.2, and preferably, 5.8 to 6.0 in terms of stability of S-4661. In order to adjust the pH to be in the aforementioned pH range, any base and/or basic salt can be added to the aqueous solution as a pH adjusting agent. The base and/or basic salt is preferably at least one compound selected from 2.0 the group consisting of NaOH, NazC03 and NaHC03. Although an addition amount thereof is varied depending on the compound to be added and the concentration of S-4661 and the like, for example, in the case of NazC03, an appropri-ate amount is about 0 .1 percent by weight: ( about 0. 005 in molar ratio) to S-4661, for example, about 0.67 mg for 500 mg of S-4661.
The aqueous solution can further contain an inorganic salt. Examples of the inorganic salt include alkali metal halide salts such as NaCl, NaBr, KC1, and alkali metal sulfate salts such as NaZS04. NaCl and NaZS04 are preferable, and NaCl is more preferable. Preferably, the inorganic salt can be added to the aqueous solution 21 ~$52~ 50029 in an amount of about 0.01 to 0.22 moles, and more preferably about 0.02 to 0.13 moles, per mole of S-4661.
In the case where the inorganic salt is NaCl, the addi-tion amount is preferably about 0.15 to 5 parts by weight, and more preferably about 0.3 to about 3 parts by weight to 100 parts by weight of S-4661. For example, preferably about 0.75 to 25 mg, and more preferably about 1.5 to about 15 mg of NaCl is added to ~i00 mg of S-4661.
The thus prepared aqueous solution containing 5-4661 is frozen by cooling to -20°C or lower, and prefera-bly -30 to -40°C. Such cooling step is performed, preferably over 1 to 2 hours. This process is herein referred to as first freezing or a first freezing step.
Prior to the first freezing step, the prepared aqueous solution of S-4661 may be allowed to stand at room temperature for several hours, preferably 1 to 2 hours.
A frozen solution obtained in the first freezing step is then warmed to 0 to -10°C, preferably -2 to -10°C, and more preferably -3 to -5°C. Such warming step is performed preferably over 0.5 to 1.5 hours. Then the warmed frozen solution is further cooled and warmed repeatedly in the range of 0 and -10°C. The operation of warming and cooling is performed at least: twice, prefera-bly 2 to 10 times. The expression "warming and cooling"
refers to not only the case where the solution is first warmed and then cooled, but includes thE~ case where the solution is first cooled and then warmed. Preferably, the solution is warmed and cooled in the temperature range of 1 to 5 K, more preferably 1 to 3 K. The warming rate and cooling rate is preferably 8 to 12 K/hour. The repetition of warming and cooling is preferably performed in a continuous manner, and preferably over 5 hours to 10 hours. Preferably, after the step of warming and cool-ing, the frozen solution is further allowed to stand at about -3 to -10 ° C, preferably -5 to -7.0 ° C for 1 to 10 hours, preferably 3 to 8 hours. Crystals of S-4661 are deposited by the steps of warming the frozen solution, repeating the operation of warming and cooling, and optionally allowing the frozen solution to stand. The :LO process including these steps all together after the first freezing is herein referred to as crystallization or a crystallization step.
The thus obtained crystalline frozen product is subjected to vacuum drying according to an conventional method, so as to obtain a crystalline lyophilized prepa-ration of S-4661.
In the case where S-4661 is lyophilized without being crystallized, in general, a collapse temperature of a frozen phase is about -15°C. On the other hand, in the case where S-4661 is lyophilized after crystals of S-4661 are deposited, an eutectic point of the frozen phase is about -0.5°C. In other words, by crystallizing S
4661, lyophilization can be caused at a higher tempera ture. When stability of the obtained lyophilized prepa ration was tested, it was confirmed that the crystalline lyophilized preparation has a higher stability than an amorphous lyophilized preparation.
As described above, in the proce::,s of the present invention, the pH of an aqueous solution containing 5-4661 is preferably adjusted to about 5.8 to 6.2. By '~~::.~:

doing this, the stability of S-4661 in the aqueous solution can be improved. An addition amount of a pH
adjusting agent is as small as about 0.005 to 0.01 moles per mole of S-4661. Thus, even if a base is added as the pH adjusting agent, S-4661 is not converted to a salt corresponding to the base (e. g., an Na salt in the case where NaOH is added). Accordingly, the pH adjusting agent can be added even when an inner :salt of S-4661 is desired. The aforementioned Japanese Laid-Open Patent Publication (Kokai) No. 4-338332 discloses the addition of Na2C03 or NaHC03. However, these bases are added to a drug which is a hydrochloride salt for t:he purpose of not depositing the drug in the form of a free base. For this reason, the addition amount is as relatively large as 0.5 :15 to 5 equivalents to the drug. Therefore, in this case, the drug is believed to be in the form of an Na salt.
Thus, the base is used for a different purpose than in the process of the present invention where the base is used as a pH adjusting agent.
Furthermore, according to the present invention, an inorganic salt :is preferably added to an aqueous solution containing S-4661 in order to facilitate crys-tallization. zn the conventional lyophilization process where the condition of warming and cooling is not de-fined, when an inorganic salt is added, an adverse effect is caused in crystallization in that an amorphous portion in a resulting lyophilized preparation i.s increased. In the process of the present invention, however, the addi-tion of the inorganic salt does not cause such an adverse effect in crystallization. Since the inorganic salt is added at a significantly low concentration, the conver-sion of S-4661 to the inorganic salt does not occur. On the other hand, in Japanese Laid-Open Patent Publication (Kokai) Nos. 61-172878, 63-174927 and 4-59730 which disclose the addition of the inorganic salt, the inorgan-ic salt is added in order to improve solubility and stability of an amorphous lyophilized preparation. Thus, the inorganic salt is added at a high concentration, i.e, a molar ratio of 0.2 or more in all the Publications.
The pH adjusting agent described above is believed to have the same function as the inorganic salt as well as the function of adjusting pH.
Furthermore, according to the present invention, a lyophilized preparation can be obtained without using an organic solvent, so that no problems such as a residu-1.5 al solvent arise, thus leading to an advantage of safety.
The present invention provides a crystalline lyophiled preparation containing either one of the two types of crystals of S-4661, i.e., Type I crystal and Type II crystal, or a mixture thereof.. Which of the crystal the lyophilized preparation contains, or in what ratio the respective crystal forms are present in the crystalline lyophilized preparation containing the mixture of Type I and Type II crystals .is determined by various conditions such as the pH adjusting agent, the types of added inorganic salt, the concentration and temperature conditions in each step. I:n order to more selectively obtain Type II crystal, for example, NaCl is added, preferably in an amount of 0.6 percent by weight or more, more preferably 2 percent by weight or more, and still more preferably 3 percent by weight: or more. Then, crystallization is performed by raising and lowering the temperature in a temperature range of -2°C to -10°C, preferably -3°C or lower. Alternatively, in the case where crystallization is performed after an aqueous solution of S-4661 is prepared and allowed to stand at room temperature for several hours, it a.s preferred that the solution with NaCl added in an amount of 2 percent by weight or more is used and the crystallization is per-formed at -2°C or lower.
In the case where Type I crystal is desired, for example, conditions can be as follows: mannitol is added in place of the inorganic salt; or the pH is adjusted to be in the vicinity of 6 by using a phosphate buffer without adding the inorganic salt.
The lyophilized preparation of the present invention can be used as an aqueous solution for use as an injection or the like. In this case, as described above, Type II crystal is preferable to Type I crystal in that Type II crystal has a higher dissolution rate in water. The dissolution rate can be further improved by adding mannitol to the preparation. The mannitol is added to an aqueous solution for preparing a lyophilized preparation, in an amount of 5 parts by weight or more, preferably 10 parts by weight or more, more preferably 15 parts by weight or more, and most preferably 15 to 50 parts by weight on the basis of 100 parts by weight of 5-4661.
Examples Hereinafter, the present invention will be described by way of examples.
Example 1 Preparation of Type I crystal A 25 wt% aqueous solution of S-4661 was prepared.
The aqueous solution was stirred in an ice bath to obtain a targeted product by recrystallization. The product was dried under reduced pressure at room temperature for 20 hours in a desiccator. As a result of powder X-ray diffraction measurement, it was confirmed that the obtained crystal is Type I crystal of an inner salt. For the obtained crystal, element analysis,, water quantity J.0 measurement (Karl Fischer's method), and IR measurement were performed. The results are shown below.
Element analysis ( calculated as C15H24N4OE,SZ ~ 1/2Hz0 ) Calculated values: C,41.950; H,5.870; N,13.040;
5,14.93%
Analytical values: C,41.94$; H,5.950; N,13.14$;
5,14.75$
HZO(KF method): 2.140 (calculated value 2.10$) IR(cm-1)(Nujol): 3460,3255,3120,1763,1620,1555,1378,1325 Example 2 Preparation of Type II crystal Isopropanol was added in an amount about three times as much on the basis of volume to a 10 wt% aqueous solution of S-4661. The solution was stirred in an ice bath to obtain a targeted product by recrystallization.
The product was dried under reduced pressure at room temperature for 40 hours in a desiccator. As a result of powder X-ray diffraction measurement, it was confirmed that the obtained crystal is Type II crystal of an inner salt. For the obtained crystal, element analysis, water quantity measurement (Karl Fischer's method), and IR

2 ~ gg5~9 So029 measurement are performed. The results are shown below.
Element analysis ( calculated as C15Hz4N406Sz ~ 1. 65H20 ) Calculated values: C,40,00%; H,6.ll0; N,12.440;
5,14.23%
Analytical values: C,39.76%; H,6.15%; N,12.56%;
S,14.16%
Hz0(KF method): 6.650 (calculated value 6.600) IR(cm-1)(Nujol): 3540,3465,3180,1748,1625,1560,1457,1150 Hereinafter, preparation examples of the crystal line lyophilized preparation of the present invention are shown in Examples 3 to 14.
Example 3 NaOH and NaCl were added to a 10 wt% aqueous solution of S-4661 so as to be contained in an amount of 0.07 percent by weight and 0.6 percent by weight on the basis of S-4661, respectively. The resulting aqueous solution was cooled to -30°C over 1 hour to be frozen (the first freezing step). The frozen solution was warmed, and the operation of cooling and warming was performed 4 times in the temperature range of -3.5 to -6°C over 4 hours. The operation of cooling and warming was performed at a rate of about 10 K/hour. After the repeated operation of cooling and warming, the frozen solution was allowed to stand at -5.5°C for 5 hours (crystallization step). After crystallization, vacuum drying was performed according to a conventional method to obtain a crystalline lyophilized product of S-4661.
Herein, vacuum drying conditions are as follows:

First drying conditions: shelf temperature +20°C, 19 hours, degree of vacuum 0.1 hPa.
Second drying conditions: shelf temperature +40°C, 5 hours, degree of vacuum 0.02 hPa.
Rxamr~l c d NaOH and NaCl were added to an 8 wto aqueous solution of S-4661 so as to be contained in an amount of 0.07 percent by weight and 0.6 percent by weight on the basis of S-4661, respectively. The resulting aqueous solution was cooled to -30°C over 1 hour to be frozen.
The frozen solution was warmed, and the operation of cooling and warming was performed 5 times in the tempera-ture range of -3.5 to -6°C over 5 hours. The repeated operation of cooling and warming was performed at a rate of about 10 K/hour. After the repeated operation of cooling and warming, the frozen solution was allowed to stand at -7°C over 5 hours. After the crystallization, vacuum drying was performed in the same manner as in Example 3 to obtain a crystalline lyophilized product of S-4661.
Example 5 NaOH and NaCl were added to a 10 o aqueous solution of S-4661 so as to be contained in an amount of 0.07 percent by weight and 0.6 percent by weight on the basis of S-4661, respectively. The resulting aqueous solution was cooled to -30°C over 1 hour to be frozen.
The frozen solution was warred, and the operation of cooling and warming was performed 5 times in the tempera-ture range of -4.0 to -8°C over 5 hours. The repeated operation was performed at a rate of about 10 K/hour.
M

After the repeated operation of cooling and warming, the frozen solution was allowed to stand at --5 ° C for 5 hours .
After crystallization, vacuum drying was performed in the same manner as in Example 3 to obtain a lyophilized product of S-4661.
Examples 6 to 14 A crystalline lyophilized product of S-4661 was obtained in the same manner as in Example 3 except that the concentration of S-4661, the types .and the addition amount of a pH adjusting agent, the addition amount of an inorganic salt, and crystallization conditions shown in Table 1 were adopted.

Table 1 ExampleS-4661 pH controlling Salt CrystallizationCrystal agent Concentration(Addition amount)(Addition conditions form amount) 3 10 % NaOH NaCI -3.5~'-6C 4hoursII

(0.07 %) (0.6 %) -5.5C 5hours 4 8 % NaOH NaCI -3.5~--6C 5hoursII

(0.07 %) (0.6 %) -7C 5hours 10 % NaOH NaCI -~4.0~--8C II
5hours (0.07 %) (0.6 %) -5C 5hours 6 8 % Na2C03 NaCI -1.5~--6C 5hoursI

(0.13 %) (0.3 %) ~-5C 5hours 7 10 % Na2C03 NaCI -1.5~--6C 5hoursI

(0.13 %) (0.6 %) -5C 5hours 8 10 % NaOH not added -3.5~--6C 5hoursI

(0.07 %) -5C 5hours 9 10 % NaHC03 not added -5.5~~-7.5C I
5hours (0.3 %) -5C 5hours 10 % NaHC03 NaCI -3.5~--6C 5hoursI

(0.3 %) (0.6 %) -5C 5hours 11 8 % Na2C03 NaCI -3.5~--6C 5hoursI

(0.13 %) (0.3 %) -5C 5hours 12 10 % Na2C03 NaCI ;3.5~'-6C 5hoursI

(0.13 %) (0.6 %) -10C 5hours 13 10 % NaHC03 NaCI -2.5~--4.5C I
5hours (0.3 %) (1 %) -4C 5hours 14 10 % NaHC03 NaCI -2.5~~-4.5C II
5hours (0.3 %) (2 %) -4C 5hours 2'1$~52g 50029 Comparative Example 1 NaCl was added to an 8 wt% aqueous solution of 5-4661 so as to be contained in an amount: of 5 percent by weight on the basis of S-4661. The resulting aqueous solution was cooled to -30°C over 1 hour to be frozen.
After freezing, vacuum drying was performed according to a conventional method to obtain an amorphous lyophilized product of S-4661. Herein, vacuum drying conditions are as follows:
First drying conditions: shelf temperature -20°C, 83 hours, degree of vacuum 0.08 hPa.
Second drying conditions: shelf temperature 60°C, :L5 5 hours, degree of vacuum 0.02 hPa.
Comparative Example 2 An amorphous lyophilized product of S-4661 was obtained in the same manner as in Comparative Example 1 except that NaCl was added so as to be contained in an amount of 15 percent by weight on the basis of S-4661.
Comparative Example 3 An amorphous lyophilized products of S-4661 was obtained in the same manner as in Comparative Example 1 except that NaCl was not added.
Comparative Example 4 S-4661 was dissolved in a 3 wto aqueous solution of ethyl acetate so as to be contained in an amount of 20 percent by weight. The resulting solution was rapidly cooled by acetone dry ice to be frozen at -70°C. After freezing, crystallization was performed at -5°C for 10 hours, and after the crystallization, freezing was again performed at -70°C. After freezing, vacuum drying was performed according to a conventional method to obtain a lyophilized product of S-4661. Drying at this time was performed by spontaneously warming the shelf temperature to room temperature for 16 hours at a degree of vacuum of 0.02 hPa.
Comparative Example 5 A lyophilized product of S-4661 was obtained in the same manner as in Comparative Example 3 except that a 2 wto aqueous solution of ethanol was used.
Comparative Example 6 A lyophilized preparation of S-4661 was obtained in the same manner as in Comparative Example 3 except that a 2 wt% aqueous solution of isopropanol was used.
Example 15 X-ray diffraction measurement Powder X-ray diffraction measurement was per-formed for the lyophilized products obtained in Examples 3 to 14 and Comparative Examples 1 to 6. X-ray dif-fraction charts of the crystalline lyophilized products of Examples 3, 8, 13 and 14 are shown in Figures 1, 2, 3 and 4. X-ray diffraction angles of the lyophilized products obtained in Examples 3 to 5 and 8 are shown in Table 2. Type II crystal was obtained in Examples 3 to 5 and 14, whereas Type I crystal was obtained in Examples 6 to 13. The identified crystal form is shown in Table 1 above. Whether the contained crystal_ was Type I or Type II was identified by the characteristic peaks shown below.

Type I: 28 - 7.32, 14.72, 16.62, 20.42, 21.1, 22.18, 23.88 and 29.76 (degree) Type II: 2A - 6.06, 12.2, 14.56, 17.0, 18.38, 20.68, 24.38, 24.60, 25.88 and 30.12 (degree) These peaks were matched with the X-ray diffrac tion patterns of crystals of Type I and II obtained in Examples 1 and 2. The obtained lyophilized products were :LO all inner salt crystals.

x'.188529 Table 2 Exam le Diffraction intensiDiffraction angleC stal form strong 6.0 12.2 14.5 3 medium-weak 17.3 Type II

20.8 24.4 24.5 30.5 strong 6.1 12.2 14.4 4 medium-weak 17.3 Type II

18.4 20.7 24.4 24.6 30.4 strong 6.1 12.2 14.6 5 medium-weak 17.3 Type II

18.4 20.8 24.4 24.6 30.5 strong 7.3 14.7 16.6 8 medium-weak 19.9 Type I

20.4 21.0 22.2 29.6 Example 16 Relationship between a pH adjusting agent and the addition amount of salts, and the resulting crystal form As shown in Tables 3 to 5 below, crystalline lyophilized products of S-4661 were obtained by varying the types and the addition amounts of the pH adjusting agent and the added salts. In Tables 3 to 5, "addition concentration" refers to a molar concentration relative to a 10 wto aqueous solution of S-4661, and "addition amount" refers to wto to S-4661. Crystallization was performed by raising and lowering the temperature in the range of -3 to -4°C over 5 hours, and further allowing the solution to stand at -5°C for 5 hours. The crystal form thereof was confirmed by X-ray diffraction measure-ment of the obtained lyophilized products. In the Tables, II > I indicates a mixture containing Type II
crystal in a larger amount, II » I indicates a mixture containing crystals mainly consisting of Type II, and II
? I indicates a mixture containing Type II crystal in a slightly larger amount. The relationship between the amount of Type I crystal and that of Type II crystal was evaluated from a peak area ratio of the respective peaks at 28 = 7.3 and 6Ø

Table 3 Inorganic salt NaCI Na2S04 NaBr pH controlling agent Addition concentration10 mM 7.5 mM 10 mM

(pH 6) Addition amount0.6 % 1.1 % 1.0 Addition concentration4.0 mM 4.0 mM 4.0 mM

Sodium sulfiteAddition amount0.5 % 0.5 % 0.5 Crystal form II > I I > II I ~ II

Addition concentration2.5 mM 2.5 mM 2.5 mM

Disodium phosphateAddition amount0.4 % 0.4 % 0.4 Crystal form II > I = I I z II

Addition concentration45.7 mM ~ 45.'7 45.7 mM
mM

Sodium acetateAddition amount3.8 % 3.8 % 3.8 Crystal form II ( III II

Addition concentration12.3 mM 12.3 mM 12.3 mM

Disodium succinateAddition amount2.0 % ( 2.0 % 2.0 Crystal form II I > II I ~ II

Addition concentration4.8 mM 5.8 mM 4.8 mM

Trisodium Addition amount1.3 % 1.5 % 1.3 citrate Crystal form III I III

Addition concentration156.1 mM 156.1 mM 138.8 mM

Sodium benzoateAddition amount22.5 % 22.5 % 20.0 Crystal form II II > I III

Addition concentration2.1 mM 2.1 mM 2.1 mM

Lysine Addition amount0.3 % 0.3 % 0.3 Crystal form II II:I III

Addition concentration2.5 mM 2.5 mM 2.1 mM

Tris-aminomethaneAddition amount0.3 % 0.3 % 0.3 Crystal form II I~>II III

Table 4 Inorganic salt NaCI KCI

pH controlling agent Addition concentration10 mM 10 mM
(pH 6) Addition amount0.6 % 0.7 Addition concentration2.7 mM 5.2 mM

Potassium hydroxideAddition amount0.2 % 0.3 Crystal form II II

Addition concentration1.5 rnM 1.1 mM

Potassium carbonateAddition amount0.2 % 0.15 Crystal form II z I II

Addition concentration2.9 mM 2.3 mM

Dipotassium phosphateAddition amount0.5 % 0.4 Crystal form 11 II

Addition concentration61.1 mM 51.0 mM

Potassium acetateAddition amount6 % 5 Crystal form II II

Sodium bisulfiteAddition concentration4~8 mM

(pH 5.5) Addition amount0.5 % -Crystal form II

Sodium salicylateAddition concentration15.6 mM

(pH 5.6) Addition amount2.5 % -Crystal form I

Table 5 pH controlling agent Added salts NaCI
(pH 6) Addition concentratioOrnM 5mM lOmM l7mM 34mM 86mM

not added Addition amount0 0.3% 0.6% 1 % 2% 5%
%

Crystal form II

Addition concentratiol.BmMl.BmMl.BmM l.BmMl.BmMl.BmM

Sodium hydroxideAddition amount0.07%0.07%0.07% 0.07%0.07%0.07%

Crystal form I III II II II II

Addition concentratiol.3mMl.3mMl.3mM - - -Sodium carbonateAddition amount0.13%0.13%0.13% - - -Crystal form I I I > II - - ->
II

Addition concentratio45.7mM -45.7mM
45.7mM
45.7mM
45.7mM

Sodium acetateAddition amount3.75%3.75%3.75% 3.75%3.75%-Crystal form p Addition concentratio4.8mM4.8mM4.8mM 4.8mM4.8mM-Sodium citrateAddition amount1.25%1.25%1.25% 1.25%1.25%-Crystal form III

~' 188529 Example 17 Stability test of crystal An accelerated test was conducted at 50 ° C for the crystals obtained in Examples 1 and 2 and the amorphous lyophilized products obtained in Comparative Example 3.
Thereafter, a residual percentage of S-4661 was deter-mined by HPLC method using an absolute correlation curve method. The crystal structure of the samples, amounts of the samples and residual percentage of S-4661 after the accelerated test ( 1 to 6 months later ) are shown in Table 6. In the table, RH indicates a relative humidity, and lux indicates illuminance. It was confirmed that com-pared with the amorphous lyophilized products, the crys-tal of the present invention had a higher residual percentage after storage and, thus, more satisfactory storage stability. For example, under the condition of a high temperature, the maximum difference in the residu-al percentage between the crystal of the present inven-tion and the amorphous lyophilized products was about 30%. Furthermore, in the crystal of the present inven-tion, a change in appearance was not substantially observed even after the accelerated test.

Table 6 Inner salt Amorphous lyophilized crystal preparation Example 13 Example 14 Comparative example Storage conditionPeriodResidual percentageResidual percentageResidual percentage In hermetic 1 98.6 % 99.1 % 70.5 M

container 2 98.1 99.1 at 50C 3 97.2 99.5 In hermetic 1 98.6 g9.7 78.p M
~

container 2 97.8 99.5 76.4 at 40C 3 98.5 99.8 4 99.1 99.8 6 98.0 99.4 40C RH 0 1 99.5 99.4 88.1 % M

2 98.7 99.6 83.4 3 97.7 99.5 4 99.7 99.6 6 98.7 99.1 1800 lux 1 99.7 100 97.4 M

2 99.3 100 3 99.0 99.0 - 30 - ~ ~ ~ g 5 2 9 S0029 Example 18 Stability test An accelerated test was conducted at 50°C for the crystalline lyophilized products obtained in Examples 3 to 8 and 10 to 14 and the amorphous lyophilized products obtained in Comparative Examples 1 to 3. Thereafter, a residual percentage of S-4661 was determined by an HPLC
method using an absolute correlation curve method. The crystal structures of the samples, the amounts of the 7.0 samples and the residual percentages after the acceler ated test (after 0.5 month and after 1 month) are shown in Table 7. It was confirmed that compared with the amorphous lyophilized products, the lyophilized products of the present invention have more satisfactory storage 1.5 stability.

~188~29 Table 7 Sample Crystal structureSample amountResidual (Example No.) (mg/vial) percentage (stora a at 50C) 0.5 month 1 month Example 3 Type II 250 99.1 % 99.6 %

Example 4 Type II 250 99.9 % 98.0 Example 5 Type II 250 ~ 100 % 99.3 Example 6 Type I 250 98.0 % 99.3 Example 7 Type I 250 100 % 100 Example 8 Type I 250 98.6 % 98.7 Example 10 Type I 250 98.5 % 98.5 Example 11 Type I 250 97.9 % 97.2 Example 12 Type I 250 98.3 % 97.8 Example 13 Type I 250 99.4 % 99.9 %

Example 14 Type II 250 98.9 % 99.5 l Comparative exampleAmorphous 100 90.9 % 86.8 Comparative exampleAmorphous 250 96.7 % 93.7 Comparative exampleAmorphous 100 83.6 % 74.6 Examples 19 to 39 Effect of the addition of mannitol on reconstitution time and storage stability In Examples 19 to 39, the effect of the addition of mannitol on reconstitution time and storage stability of the lyophilized preparation of the present invention was evaluated. Crystalline lyophilized products of 5-4661 were obtained by using 500 mg of S-4661 (10 wto aqueous solution) and varying the addition amount of mannitol as shown in Table 8. In each Example, NaCl was added in an amount of 15 mg (3 wt% wit;h respect to S-4661). NaOH was added in an appropriate amount so that pH of the aqueous solution was 6. The first freezing step was performed by cooling the aqueous solution from 1.5 +5°C to -40°C over 1 hour. The crystallization step was performed by repeating twice to 10 times the operation of cooling and warming the frozen solution in the range of -3°C to -5° at a rate of about 10 K/hour over 5 to 10 hours, and then allowing the solution to stand at -5°C or -10°C for 5 to 10 hours. Vacuum drying was performed in the same manner as in Example 3.
The reconstitution time of the obtained crystal-line lyophilized products was measured. The measurement of the reconstitution time was performed by adding the samples to 10 ml of water for injection, shaking the resulting mixture at 200 times/min., and measuring the period of time until the samples were completely dis-solved. The results are shown in Table 8. Furthermore, each sample was tested with respect to storage stability at 50°C in the same manner as in Example 18. The results are shown in Table 8. In the table, a change in appear-ance of each sample compared with the samples stored at -2~ 88529 20°C is shown in a parenthesis. - indicates no change, +- indicates a slight change, + indicates some change, ++
indicates a considerable change, and +++ indicates a significantly large change. It was confirmed that when mannitol was added, solubility was improved, and storage stability was not deteriorated.

Table 8 Exam Mannitol 0.5 month1 month 2 months 3 months le Reconstitution residual residual residual residual No. addition percentage(96)percentage(9o)percentage(~'~)percentage(~'o)period amount 19 99.5 (-) 99.5 (-) 97.6 (-) 97.4 1 '02"

20 98.8(-) 98.8(-) 98.0(-) 97.3 50"

0 mg 21 98.7(-) 98.8(-) 99.7(-) 99.5 22 * 98.9(_~-+_)98.1 (--r+-;I97.6 1'02"

23 96.5(+) 95.6(+) 95.9(+) 94.5 44"

24 96.5 (+) 95.2 (++)94.5 (+++)93.8 36"

25 mg 25 96.9(+) 95.7(+) 94.8(+) 93.8 28"

26 96.5(+) 94.6(+) 93.9(+~-++)93.8 35"

27 99.1 (-) 98.2 (-) 98.3 47"
98.3 (-) 28 98.8(-) 97.4(-) 97.2 34"
97.6(-) 29 96.0 (-) 96.0 (+-) 93.1 30"
94.3 (+) 50 mg 30 g8.7(-) 99.2(-) 96.5 25"
96.8(+-) 31 97.1 (+) 96.3(++) 93.9 31"
94.9(++~-+++) 32 97.7(-) 96.2(+-) 93.3 24"
96.7(+-) 33 96.5(+) 94.7(+) 94.7 30"
94.9(+) 34 99.9 (-) 99.5 (-) 98.0 23"
G 98.7 (-) 35 75 mg 98.6(-) 97.8(-~~+-) 96.7 35"
96.6(+-~+)~

36 100.7(-) 100.2(-) 99.1 25"
99.0(-) 37 100 rng 99.2 (-) 98.0 (-) 97.7 26"
98.7 (-) 38 150 mg 99.5(-) 99.0(-) 99.0 26"
99.3(-) 39 200 mg 99.3(-) 98.9(-) 99.4 26"
98.8(-) * No data INDUSTRIAL APPLICABILITY

According to the present invention, crystal of S-4661 excellent in storage stability and solubility and having a high industrial applicability can be obtained.
Furthermore, the present invention also provides a lyophilized preparation containing the crystal. It is not only easy to ensure sterility and remove particulate matters in the lyophilized preparation, but also the lyophilized preparation is excellent in storage stability and solubility. S-4661 is useful as an antimicrobial drug, and orally or parenterally administered. The crystal of S-4661 of the present invention and the lyophilized preparation containing the crystal are particularly useful as an injection.

Claims (15)

The embodiments of the invention in which an exclusive property ar privilege is claimed are defined as follows:

1. A Type II crystal of (1R,5S,6S)-6-[(1R)-1-hydroxyethyl]-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid represented by the following formula:
wherein a powder X-ray diffraction pattern of the crystal has a primary peak at a diffraction angle (28) - 6.06, 12.2, 14.56, 17.0, 18.38, 20.68, 24.38, 24.60, 25.88 and 30.12 (degree).

2. A crystal according to claim 1, which forms an inner salt.

3. A lyophilized preparation comprising the crystal according to claim 1 or 2.

4. A process for producing the lyophilized preparation according to claim 3, the process comprising the steps of:
freezing an aqueous solution containing (1R,5S,6S)-6-[(1R)-1-hydroxyethyl]-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid represented by the following formula by cooling to -20°C or lower warming the frozen solution to 0 to -10°C; and cooling and warming the frozen. solution at least twice in a temperature range of 0 to -10°C.

5. A process according to claim 4, wherein pH of the aqueous solution is in a range of 5.8 to 6.2.

6. A process according to claim 4, wherein the aqueous solution comprises at least one compound selected from the group consisting of NaOH, Na2CO3 and NaHCO3.

7. A process according to claim 4, wherein the aqueous solution further comprises an inorganic salt.

8. A process according to claim 7, wherein the inorganic salt is present in an amount of 0.02 to 0.13 moles on the basis of 1. mole of (1R,5S,6S)-6-[(1R)-1-hydroxyethyl)-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid.

9. A process according to claim 7, wherein the inorganic salt is NaCl.

10. A process according to claim 4, wherein the aqueous solution further comprises mannitol.

11. A process according to claim 10, wherein the mannitol is present in an amount of at least 15 parts by weight on the basis of 100 parts by weight of (1R,5S,6S)-6-[(1R)-1-hydroxyethyl]-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid.

12. A process according to claim 11, wherein the mannitol is present in an amount of 15 to 50 parts by weight on the basis of 100 parts by weight of (1R,5S,6S)-6-[(1R)-1-hydroxyethyl]-2-[(3S,5S)-5-sulfamoylaminomethyl-1-pyrrolidin-3-yl]thio-1-methyl-1-carba-2-penem-3-carboxylic acid.

13. A process according to claim 4, wherein the frozen solution is cooled and warmed in a temperature range of 1 to 3 K.

14. A process according to claim 4, wherein the frozen solution is cooled and warmed twice to 10 times.

15. A process according to claim 4 further comprising the step of allowing the frozen solution to stand at -3 to -10°C for 1 to 10 hours.