JP6936747B2 - Injectable solution formulation containing palonosetron - Google Patents

Description

The present invention relates to an injectable solution preparation in which the stability of palonosetron is maintained.

Palonosetron has the chemical name (3aS) -2-[(3S) -Quinuclidine-3-yl] -2,3,3a, 4,5,6-hexahydro-1H-benzo [de] -isoquinolin-1-one. It is used as an antiemetic agent for gastrointestinal symptoms (nausea, vomiting) including the late onset associated with administration of antineoplastic agents.
Palonosetron hydrochloride is currently provided as a vial preparation and a bag preparation as a solution preparation for intravenous injection for a single dose under the product name of ALOXI (ALOXI registered trademark). As a vial preparation in Japan, there is a preparation containing 5 mL per vial at a concentration of 0.15 mg / mL as palonosetron. On the other hand, as a vial preparation sold overseas, there is a preparation containing 5 mL or 1.5 mL of palonosetron at a concentration of 0.05 mg / mL per vial. The vial preparation is a preparation that is slowly intravenously administered over 30 seconds before administration of the antineoplastic agent.

The stability of palonosetron in an aqueous solution is known to be concentration-dependent, and it is known that the lower the concentration of palonosetron, the higher the chemical stability. Patent Document 1 and Patent Document 2 report that a solution preparation having a concentration of palonosetron of 0.01 to 0.2 mg / mL is an aqueous solution preparation in which stability is guaranteed.

Special Table 2006-508977 Japanese Patent Application Laid-Open No. 2006-516583

The palonosetron preparation is used by intravenous bolus administration or by instillation after appropriately diluting it. Here, in the case of bolus administration, in order to reduce the burden on the patient, a preparation having a reduced amount of administered liquid is required.
An object to be solved by the present invention is to provide an injectable solution preparation containing palonosetron, which maintains storage stability while increasing the concentration of palonosetron to reduce the dose amount.

The present invention is a formulation containing palonosetron or a salt thereof at a concentration higher than 0.2 mg / mL in terms of palonosetron, which contains tartrate acid or a salt thereof at a concentration of 2.0 mg / mL or more in terms of tartrate. Based on the finding of an injectable solution formulation containing a high concentration of palonosetron, which suppresses the decomposition of the active ingredient palonosetron. The gist of this application is the invention described in the following [1] to [3].
[1] In an injectable solution preparation containing palonosetron as an active ingredient, palonosetron or a salt thereof is contained in a value higher than 0.2 mg / mL and less than 1.0 mg / mL in terms of palonosetron, and palonosetron or a salt thereof is 2.0 mg in terms of tartrate. An injectable solution formulation containing palonosetron, which is a solution containing more than / mL and less than 20 mg / mL.
[2] The injectable solution preparation according to the above [1], which contains mannitol.
[3] The injectable solution preparation according to the above [1] or [2], wherein the solution has a liquid volume of 1.5 mL or more and 4.0 mL or less per unit preparation.

The injectable solution preparation containing palonosetron of the present invention can suppress the production of related substances which are decomposition products of palonosetron even in a preparation having a high concentration of palonosetron and a low liquid volume by adding palonosetron, and the efficacy of palonosetron In addition, it is possible to provide a preparation which can maintain safety for a long period of time, has excellent safety, and reduces the burden at the time of administration.

In the present invention, in an injectable solution preparation containing palonosetron as an active ingredient, palonosetron or a salt thereof is contained at a concentration higher than 0.2 mg / mL in terms of palonosetron, and palonosetron or a salt thereof is 2.0 mg / mL or more in terms of tartrate. It is an injectable solution preparation containing a high concentration of palonosetron that suppresses the decomposition of palonosetron, which is an active ingredient, and enables long-term storage. The details will be described below.

Palonosetron in the present invention is used as an active ingredient of an antiemetic agent, and has a chemical name of (3aS) -2-[(3S) -Quinuclidine-3-yl] -2,3,3a, 4,5,6- Hexahydro-1H-benzo [de] -isoquinolin-1-one, the pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salts include hydrochlorides, sulfates, phosphates, mesylates, tosylates, sodium salts, potassium salts, lithium salts, calcium salts, magnesium salts, ammonium salts, trimethylammonium salts, etc. Pharmaceutically acceptable alkali metal salts such as triethylammonium salt, monoethanolammonium salt, triethanolammonium salt, pyridinium salt, substituted pyridinium salt, alkaline earth metal salt, ammonium salt and substituted ammonium salt and the like can be mentioned. It is preferable to use hydrochloride. That is, in the present invention, it is preferable to use palonosetron hydrochloride.
The concentration of palonosetron in the present invention is a solution having a concentration higher than 0.2 mg / mL in terms of palonosetron. That is, it is a solution having a concentration higher than 0.2 mg / mL in terms of palonosetron and less than 1.0 mg / mL. The concentration in terms of palonosetron is preferably 0.25 mg / mL or more and 0.5 mg / mL or less, and more preferably higher than 0.25 mg / mL and 0.4 mg / mL or less.

The present invention contains tartaric acid or tartrate salt. The tartrate can be used as a pharmaceutically acceptable salt such as a sodium salt, a potassium salt, a lithium salt, an ammonium salt, an amine salt and a pyridinium salt. When preparing the solution preparation of the present invention, it is preferable to use tartaric acid or sodium tartrate salt.
Tartaric acid should be used at 2.0 mg / mL or higher, with an upper limit of 20 mg / mL or lower. The concentration of tartaric acid indicates the concentration converted into tartaric acid from the amount of tartaric acid or a salt thereof added. If the tartaric acid concentration is less than 2.0 mg / mL, it may not be possible to obtain a sufficient inhibitory effect on palonosetron related substances during the storage period. Preferably, the tartaric acid equivalent concentration is 2.0 mg / mL or more and 15.0 mg / mL or less. More preferably, the concentration is 2.5 mg / mL or more and 12.0 mg / mL or less.

In the injectable solution preparation of the present invention, additives for preparation other than tartaric acid or a salt thereof may be used as long as the stability of palonosetron is maintained. As other pharmaceutical additives, pH adjusters, isotonic agents, chelating agents, antioxidants, stabilizers and the like used in ordinary pharmaceutical preparations may be added.
The pH adjuster is generally used in pharmaceutical preparations, and is preferably used within a range that does not adversely affect the stability of the injectable solution preparation of the present invention. Examples thereof include acidic agents such as inorganic acids such as hydrochloric acid, phosphoric acid, boric acid and carbonic acid, and organic acids such as ascorbic acid, lactic acid and acetic acid. In addition, alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide and lithium hydroxide, and inorganic acids such as sodium dihydrogen phosphate, disodium monohydrogen phosphate, sodium carbonate and sodium hydrogen carbonate. Alkaline agents such as alkaline earth metal salts of the above can be mentioned. Further, a buffer agent obtained by mixing the acidic agent and the alkaline agent and adjusting the pH may be used. Examples of the buffer include a phosphate buffer, an acetate buffer, a lactic acid buffer, a succinic acid buffer, a TRIS buffer, a glycine buffer, a histidine buffer and the like. These pH adjusters may be used alone or in combination of two or more.
In the present invention, the pH adjuster is preferably hydrochloric acid, phosphoric acid or a salt thereof (sodium dihydrogen phosphate, disodium monohydrogen phosphate, etc.) and sodium hydroxide.
For example, in the injectable solution preparation containing a known paronosetron as an active ingredient described in JP-A-2006-516583, hydrochloric acid, sodium hydroxide, citric acid and sodium citrate are used as pH adjusters. .. However, the present inventors have found that citric acid or a salt thereof adversely affects the stability of the palonosetron solution. Therefore, the present invention should be 10 mg / mL or less when citric acid or a salt thereof is added. Preferably, it is 5.0 mg / mL or less when citric acid or a salt thereof is added, and more preferably, it is an injectable solution preparation which does not substantially contain citric acid or a salt thereof.

The pH adjuster is used in an appropriate amount as long as the pH of the solution can be adjusted to the desired pH when preparing the solution preparation for injection. The blending amount of the pH adjuster used in the production of the pharmaceutical composition of the present invention is not particularly limited as long as the pH can be adjusted within an appropriate range.
The injectable solution preparation of the present invention can improve the stability of palonosetron by adjusting the pH. In the present invention, the decomposition of palonosetron can be further suppressed by setting the pH in the range of 4.0 to 7.0. More preferably, the pH is 4.5-6.0.

In the present invention, the stability of the palonosetron solution preparation can be improved by adding a chelating agent. Examples of the chelating agent include disodium edetate (EDTA). The concentration of EDTA in which the production of palonosetron-related substances is suppressed is 0.005 mg / mL or more and 2.0 mg / mL or less, more preferably 0.3 mg / mL or more and 1.5 mg / mL or less, and further preferably. Is 0.8 mg / mL or more and 1.2 mg / mL or less.

Since the solution preparation for injection of the present invention is directly administered to the vein of a peripheral blood vessel, the osmotic pressure of the solution preparation should be adjusted to the same level as the osmotic pressure of plasma from the viewpoint of suppressing vascular pain during administration. preferable. The osmotic pressure of the solution preparation is preferably 150 to 450 mosm / L, more preferably 200 to 400 mosm / L, and further preferably 250 mosm / L to 350 mosm / L.
The osmotic pressure can be adjusted by adding an isotonic agent. Examples of the tonicity agent include sodium chloride, calcium chloride, magnesium chloride, glycerin, fructose, xylitol, sorbitol, trehalose, nicotine amide, glucose, purified sucrose, mannitol and the like. In particular, sodium chloride and mannitol are preferable.

In the present invention, it is preferable to add mannitol because the osmotic pressure of the solution preparation can be adjusted and the stability of palonosetron can be improved. The prescription concentration of mannitol is preferably 10 mg / mL or more and 80 mg / mL or less, more preferably 20 mg / mL or more and 60 mg / mL or less, and further preferably 25 mg / mL or more and 45 mg / mL or less.

In the present invention, it is preferable to add sodium chloride because the osmotic pressure of the solution preparation can be adjusted and the stability of palonosetron can be improved. The prescription concentration of sodium chloride is 1.0 mg / mL or more and 20 mg / mL or less, more preferably 2.0 mg / mL or more and 15 mg / mL or less, and further preferably 5.0 mg / mL or more and 10 mg / mL or less. Is.

Examples of the antioxidant include ascorbic acid, sodium sulfite, sodium hydrogen sulfite, sodium pyrosulfite, sodium thioglycolate, α-thioglycerin, sodium edetate, methionine, acetylcysteine and the like.
Stabilizers include, for example, acetyltryptophan, alanine, albumin, benzoic acid and its salts, inositol, fructose, sodium cardiamide, sodium carbazochrome sulfonate, creatinine, sodium chondroitin sulfate, diethylenetriamine pentaacetic acid, cystine, calcium bromide, Gelatin, sorbitol, potassium thiocyanate, dextran, calcium saccharide, sodium formaldehyde sulfosylate, lactose, urea, propyl paraoxybenzoate, methyl paraoxybenzoate, benzalconium, benzethonium, maltose, sodium pyrophosphate, maleic acid, meglumine , Sodium metasulfobenzoate, magnesium sulfate and the like.
However, these additives are optional components that are not essential.

The injectable solution preparation of the present invention contains paronosetron or a salt thereof at a concentration higher than 0.2 mg / mL in terms of paronosetron, and contains tartrate acid or a salt thereof at a concentration of 2.0 mg / mL or more in terms of tartrate acid, and contains water as a main component. This is an embodiment of a pharmaceutical preparation in which a solution dissolved in a solvent is sterilized and filled in a container for a pharmaceutical preparation such as an ampoule, a vial or an injection. Since the solution preparation has a higher concentration of the active ingredient than the conventional palonosetron preparation, the solution filling amount per unit preparation is smaller than that of the conventional product. That is, as a preparation in which the burden on the patient at the time of administration is reduced, it is preferable that the solution of the solution preparation has a liquid volume of 1.5 mL or more and 4.0 mL or less per unit preparation. More preferably, it is a unit preparation filled with a liquid amount of 1.5 mL or more and 3.5 mL or less.
The injectable solution preparation is a solution using a solvent containing water as a main component. Examples of solvents other than water that can be used include ethanol, polysorbate 80, macrogol, glycerin, polyoxyethylene hydrogenated castor oil, and the like, and water is used as a single solvent for these or a mixed solvent of two or more kinds. It may be mixed with and used. The solvent is preferably of a quality that can be orally administered. As a particularly preferable solvent, an embodiment in which only water is used may be used.

The solution preparation for injection of the present invention is preferably a unit preparation prepared as a sealed filling preparation that is aseptically filled in a container for a unit preparation such as a vial and sealed with an airtight rubber stopper.
The container to be used is not particularly limited in shape such as a vial or a syringe as long as it can be sealed with a rubber stopper or the like, but a vial-shaped container is preferable.
Examples of the material of the container include soda lime glass, borosilicate glass, cyclic polyolefin polymer, and cyclic polyolefin copolymer. From the container, it is preferable to use a vial in which elution of container components such as glass and resin is suppressed. As the glass vial made of glass and in which the elution of the container component is suppressed, it is preferable to use a container whose surface is treated according to the purpose such as suppressing the elution of the glass component. In particular, it is preferable to use a borosilicate glass vial whose inner surface is treated with silica. An example of a vial internally treated with silica is a Siricoat vial (manufactured by Fuji Glass Co., Ltd.). Other vials in which elution of glass components are suppressed include VIST vials (manufactured by Daiwa Special Glass Co., Ltd.), VIALEX (manufactured by Nipro Co., Ltd.), type1 plus (manufactured by SCHOTT Co., Ltd.), and the like.
Further, the solution preparation for injection can be prepared by sealing with an airtight rubber stopper. As the material of the rubber stopper, it is preferable to use a rubber stopper made of butyl rubber or a rubber stopper made of butyl rubber laminated with a fluororesin.
These sealable containers are preferably packaging materials that have been sterilized by an appropriate method for both the container and the rubber stopper.

The injectable solution preparation of the present invention is stored at room temperature. Moreover, it is preferable to store it under shading.

The injectable solution preparation containing palonosetron of the present invention can be produced by a process generally used in the production of the injectable solution preparation, as shown in the following steps. However, the present invention is not limited to the following manufacturing methods.
Step (a) Dissolve palonosetron or a pharmaceutically acceptable salt thereof, tartrate acid or a salt thereof, and optionally a pharmaceutically acceptable additive, in a solvent containing pharmaceutically acceptable water as the main component.
Step (b) Add a pharmaceutically acceptable pH regulator to the mixture as needed.
Step (c) The mixture of step (b) is optionally supplemented with a pharmaceutically acceptable solvent to fill the final volume.
Step (d) The mixture of step (c) is filtered.
Step (e) The mixture obtained in step (d) is filled in a pharmaceutically acceptable container.
Step (f) If necessary, the inside of the container is purged with a pharmaceutically acceptable inert gas.
Step (g) Plug the container with a rubber stopper and seal it with an aluminum cap if necessary.
Step (h) Perform pharmaceutically acceptable final sterilization as needed.
Through the above steps, the injectable solution preparation according to the present invention can be produced.

Hereinafter, the present invention will be further described with reference to Examples. However, the present invention is not limited to these examples.

[Example 1]
Dissolve 2076 mg of D-mannitol in an appropriate amount of water for injection, and add 200.0 mg of sodium tartrate dihydrate (130.5 mg in terms of tartrate) and 16.8 mg of palonosetron hydrochloride (15.0 mg in terms of palonosetron). The pH was adjusted to 5.0 with an appropriate amount of hydrochloric acid solution and an appropriate amount of sodium hydroxide solution, and water for injection was added to make the total volume 50 mL.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Example 1.

[Example 2]
Dissolve 2076 mg of D-mannitol in an appropriate amount of water for injection, and add 400.0 mg of sodium tartrate dihydrate (260.9 mg in terms of tartrate) and 16.8 mg of palonosetron hydrochloride (15.0 mg in terms of palonosetron). The pH was adjusted to 5.0 with an appropriate amount of hydrochloric acid solution and an appropriate amount of sodium hydroxide solution, and water for injection was added to make the total volume 50 mL.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Example 2.

[Example 3]
Dissolve 2076 mg of D-mannitol in an appropriate amount of water for injection, and add 800.0 mg of sodium tartrate dihydrate (521.9 mg in terms of tartrate) and 16.8 mg of palonosetron hydrochloride (15.0 mg in terms of palonosetron). The pH was adjusted to 5.0 with an appropriate amount of hydrochloric acid solution and an appropriate amount of sodium hydroxide solution, and water for injection was added to make the total volume 50 mL.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Example 3.

[Example 4]
Dissolve 9000 mg of D-mannitol in an appropriate amount of water for injection, 2700.0 mg of sodium tartrate dihydrate (1761.3 mg in terms of tartaric acid), 120 mg of tartaric acid, 300 mg of EDTA and 100.8 mg of paronosetron hydrochloride (90 in terms of paronosetron). 0.0 mg) was added, the pH was adjusted to 5.0 with an appropriate amount of hydrochloric acid solution and an appropriate amount of sodium hydroxide solution, and water for injection was added to make the total volume 300 mL.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Example 4.

[Comparative Example 1]
Dissolve 2076 mg of D-mannitol in an appropriate amount of water for injection, and add 186.0 mg of sodium citrate dihydrate, 78.0 mg of citric acid monohydrate and 16.8 mg of paronosetron hydrochloride (15.0 mg in terms of paronosetron). In addition, the pH was adjusted to 5.0 with an appropriate amount of a citric acid solution and an appropriate amount of sodium hydroxide solution, and water for injection was added to make the total volume 50 mL.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Comparative Example 1.

[Comparative Example 2]
Dissolve 2076 mg of D-mannitol in an appropriate amount of water for injection, and dissolve 186.0 mg of sodium citrate dihydrate, 78.0 mg of citric acid monohydrate, and 20.0 mg of sodium tartrate dihydrate (13. Add 0 mg) and 16.8 mg of paronosetron hydrochloride (15.0 mg in terms of paronosetron), adjust the pH to 5.0 with an appropriate amount of citric acid solution and an appropriate amount of sodium hydroxide solution, and add water for injection to make the total volume 50 mL. bottom.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Comparative Example 2.

[Comparative Example 3]
2076 mg of D-mannitol was dissolved in an appropriate amount of water for injection, and 186.0 mg of sodium citrate dihydrate, 78.0 mg of citric acid monohydrate, and 100.0 mg of sodium tartrate dihydrate (65. in terms of tartrate). 2 mg) and 16.8 mg of paronosetron hydrochloride (15.0 mg in terms of paronosetron) were added, the pH was adjusted to 5.0 with an appropriate amount of citric acid solution and an appropriate amount of sodium hydroxide solution, and water for injection was added to make the total volume 50 mL. bottom.
This solution was sterile-filtered using a membrane filter with a pore size of 0.22 μm, and 3 mL of the filtered solution was filled into a fluoropolymer glass vial (manufactured by Fuji Glass Co., Ltd.) with a capacity of 10 mL and an inner diameter of 12.5 mm. Then, it was stoppered with a D21-7 chlorinated butyl rubber stopper laminated with a fluororesin manufactured by Daikyo Seiko, Ltd. Then, it was wrapped with an aluminum cap to prepare an injectable solution preparation of Comparative Example 3.

Here, the concentrations and pH of palonosetron and additives in Examples 1 to 4 and Comparative Examples 1 to 3 are shown in Table 1.

[Table 1]

[Analytical conditions for palonosetron-related substances]
Related substances derived from the decomposition of palonosetron in Examples and Comparative Examples were analyzed under the following liquid chromatography (HPLC) conditions.
Column: Cosmosil πNap, 4.6 mm x 250 mm, 5 μm (manufactured by Nacalai Tesque)
Column temperature: 35 ° C
Mobile phase A: 15 mM triethylamine / phosphate buffer (pH 2.5)
Mobile phase B: Acetonitrile liquid feed rate: 1.0 mL / min.
Wavelength: 210 nm
Mobile phase liquid feed: Liquid was fed under the conditions shown in Table 2.

[Table 2]

[Test Example 1] Storage stability test at 60 ° C. The injectable solution preparations of Examples 1 to 4 and Comparative Examples 1 to 3 were stored under the condition of 60 ° C. for up to 8 weeks. For each preparation, related substances derived from palonosetron were evaluated. The evaluation results are shown in Table 2.

[Table 3]

[Test Example 2] 40 ° C. Storage Stability Test The injectable solution preparations of Examples 1 to 4 and Comparative Examples 1 to 3 were stored under the condition of 40 ° C. for up to 3 months. For each preparation, related substances derived from palonosetron were evaluated. The evaluation results are shown in Table 3.

[Table 4]

In Examples 1 to 4 in which the tartaric acid concentration is 2 mg / mL or more in terms of tartaric acid, 40 ° C. and 60 ° C. are compared with Comparative Examples 1 to 3 in which the tartaric acid concentration is lower or does not contain tartaric acid. The increase of related substances was greatly suppressed under both storage conditions. From this, by converting the tartaric acid concentration to 2 mg / mL or more in terms of tartaric acid, it is possible to suppress the production of related substances that are decomposition products of palonosetron even if the concentration of palonosetron is high, and the efficacy and safety of palonosetron can be improved. It became clear that it can be retained for a long period of time.

Claims (3)

In an injectable solution preparation containing palonosetron as an active ingredient, palonosetron or a salt thereof is contained in a amount higher than 0.2 mg / mL in terms of palonosetron and less than 1.0 mg / mL, and palonosetron or a salt thereof is contained in a amount of 2.0 mg / mL or more in terms of tartrate. An injectable solution formulation containing palonosetron, which is a solution containing 20 mg / mL or less. The injectable solution preparation according to claim 1, which comprises mannitol. The injectable solution preparation according to claim 1 or 2, wherein the solution has a liquid volume of 1.5 mL or more and 4.0 mL or less per unit preparation.