CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a U.S. National Phase Patent Application and claims priority to International Application Number PCT/CN2017/092199, filed on Jul. 7, 2017, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the field of pharmaceutical preparation, particularly relates to a preparation for injection withPulsatillasaponin B4 as the active ingredient.

BACKGROUND ART

Chinese herb medicine RadixPulsatiliae Chinese is a dry root ofPulsatilla chinensis(Bge.) Regel of family Ranunculaceae, it was first recorded inShennong's Classic of Materia Medica, and is a commonly used Traditional Chinese medicine. It owns the property of bitter and cold, and has the functions of clearing heat and detoxifying, cooling blood and checking dysentery, and drying dampness and killing insects, and is used to treat dysentery with blood stool due to heat-toxicity, warm malaria with chill and fever, epistaxis and hemorrhoids with blood stool. Modern pharmacological studies have demonstrated that Radix Pulsatiliae Chinese has a wide range of antibacterial activities, and has inhibitory effects onStaphylococcus aureus, Bacillus dysteriae, dermatophytes, yeast,Candida albicansand so on. The antitumor effect of Radix Pulsatiliae Chinese is also a research hotspot. In addition, Radix Pulsatiliae Chinese has the functions of anti-inflammatory and immune enhancement.

Radix Pulsatiliae Chinese is rich in triterpenoid saponins.Pulsatillasaponin B4 belongs to a lupine-type pentacyclic triterpenoid saponin having the structure of Formula 1.



Pulsatillasaponin B4 has strong activity. For example, Chinese patent publication No. CN105213410A (publication date: Jan. 6, 2016) discloses a use ofPulsatillasaponin B4 as an immunomodulator in the treatment of acute inflammation which includes acute renal injury, acute liver injury and acute lung injury caused by overexpression of inflammatory factors. For another example, Chinese patent publication No. CN105535004A (publication date: May 4, 2016) discloses a use of the compound as an EV71 virus inhibitor in the preparation of anti hand-foot-and-mouth disease drugs.

Pulsatillasaponin B4 has five glycosyl groups and has good water solubility. Research has demonstrated that if the compound is administered orally (such as by gavage in animal experiments), the effective dose is large, resulting in a narrow safety window. Therefore, it is necessary to develop a non-oral preparation ofPulsatillasaponin B4 in order to facilitate the active role in a safe dose range to meet the clinical medical needs. However, till now, there has been no report on thePulsatillasaponin B4 injection.

SUMMARY OF THE INVENTION

In order to overcome the disadvantages of the prior art, the present invention provides aPulsatillasaponin B4 preparation for injection. The preparation is stable and easy to prepare. Compared with oral administration, the effective dose of the preparation ofPulsatillasaponin B4 for injection to reverse cisplatin-induced renal injury is reduced by 20 times, thereby improving drug safety, therefore, this preparation is promising to provide a new choice for clinical treatment of renal injury and renal failure.

In order to achieve the aforesaid aim, the present invention provides the following technical solution:

APulsatillasaponin B4 preparation for injection, comprisingPulsatillasaponin B4 and a pharmaceutically acceptable excipient, wherein the preparation is an aqueous injection or a lyophilized powder for injection.

Preferably, thePulsatillasaponin B4 preparation for injection consists ofPulsatillasaponin B4, a pharmaceutically acceptable excipient and unavoidable impurities.

Preferably, thePulsatillasaponin B4 preparation for injection is an intramuscular injection and/or an intravenous injection; more preferably an intravenous injection.

Preferably, thePulsatillasaponin B4 preparation for injection is an aqueous injection, wherein the pharmaceutically acceptable excipient is water for injection.

Preferably, the mass percentage of thePulsatillasaponin B4 in the aqueous injection is 0.5%˜5%, more preferably 2˜3%.

The present invention also provides a method for preparing the aqueous injection, comprising dissolving, decolorizing by active carbon, filtrating, making up to the final volume, sub-packing and sterilizing.

Preferably, the specific method for preparing the aqueous injection is as follows:

Taking a prescribed amount ofPulsatillasaponin B4, weighing precisely, adding part of water for injection, stirring to dissolvePulsatillasaponin B4 completely, then adding 0.1%˜0.5% of active carbon based on the total mass of the aqueous injection to the solution, heating to boil, stirring for 15 minutes, making up to the final volume with water for injection, mixing well, filtering while hot with a 0.22 μm microporous filter membrane to remove the active carbon, thereby obtaining an intermediate solution; sub-packing and sterilizing to obtain the aqueous injection.

In the method for preparing the aqueous injection, the sub-packing and sterilizing operation are carried out according to the conventional method in the art. For example, the sterilizing operation may be sterilization by hot pressing, sterilization at 115° C. for 30 min or sterilization at 121° C. for 15 min.

More preferably, thePulsatillasaponin B4 preparation for injection according to the present invention is a lyophilized powder for injection, the pharmaceutically acceptable excipient is a lyophilized excipient, which is one or more (may be in any ratio) selected from the group consisting of lactose, sucrose and mannitol.

Preferably, the mass ratio of thePulsatillaSaponin B4 to the lyophilized excipient in the lyophilized powder for injection is 3:10˜3:16.

More preferably, the mass ratio ofPulsatillasaponin B4 to the lyophilized excipient in the lyophilized powder for injection is 3:14.

Further preferably, the lyophilized excipient is mannitol.

The present invention also provides a method for preparing the lyophilized powder for injection comprising the steps of the preparation and freeze-drying of the intermediate solution.

Preferably, the specific preparation of the intermediate solution is as follows:

Dissolving thePulsatillasaponin B4 into water for injection, stirring to dissolve, adding the lyophilized excipient according to the mass ratio, stirring, adjusting the pH to 6.0˜8.5, adding 0.05%˜0.1% of the active carbon based on the total mass of the intermediate solution, heating at 100° C. and stirring for 10˜20 min, cooling to room temperature, adding water for injection till the concentration of thePulsatillasaponin B4 is 0.5%˜5%, filtering with a 0.22 μm microporous filter membrane to obtain the intermediate solution;

More preferably, the specific preparation of the intermediate solution is as follows:

Dissolving thePulsatillasaponin B4 into water for injection, stirring to dissolve, adding the lyophilized excipient according to the mass ratio, stirring, adjusting the pH to 6.5˜8.0, adding 0.05% of active carbon based on the total mass of the intermediate solution, heating at 100° C. and stirring for 10˜20 min, cooling to room temperature, adding water for injection till the concentration of thePulsatillasaponin B4 is 2%˜3%, filtering with a 0.22 m microporous filter membrane to obtain the intermediate solution.

Preferably, the freeze-drying of the intermediate solution comprises pre-freezing, sublimating and desorption drying; the temperature for pre-freezing is −30° C.˜-15° C.; the vacuum pressure during the sublimating and desorption drying stage is 0.5˜10 Pa; the temperature for sublimating is −10° C.˜0° C.; and the temperature for desorption drying is 25° C.˜30° C.;

More preferably, the vacuum pressure during the sublimating and desorption drying stage is 5˜10 Pa.

More preferably, the temperature for sublimating is −10° C.

As a preferred embodiment, the specific freeze-drying operation is as follows:

Sub-packing the intermediate solution, transferring into a freezing equipment, reducing the temperature to −30° C.˜−15° C. at a rate of 3.0° C.˜5.0° C./min or 1.0° C.˜1.5° C./min, and freezing at this temperature for 8˜10 hours; then placing in a closed equipment, raising evenly the temperature to −10° C.˜0° C. at a rate of 25° C.˜30° C./3 h under a vacuum pressure of 0.5˜10 Pa, keeping at this temperature for 8˜10 h, raising the temperature to 25° C.˜30° C. at a rate of 1.0° C.˜1.5° C./min under the same vacuum pressure, and keeping at this temperature for 4˜6 hours;

As a more preferred embodiment, the specific freeze-drying operation is as follows:

Sub-packing the intermediate solution, transferring into a freezing equipment, reducing the temperature to −30° C.˜-20° C. at a rate of 3.0° C.˜5.0° C./min or 1.0° C.˜1.5° C./min, and freezing at this temperature for 8 hours; then placing in a closed container, raising evenly the temperature to −10° C. at a rate of 25° C.˜30° C./3 h under a vacuum pressure of 5˜10 Pa, keeping at this temperature for 8 h, raising the temperature to 25° C. at a rate of 1.0° C.˜1.5° C./min under the same vacuum pressure, and keeping at this temperature for 4 h.

The present invention also provides a lyophilized powder ofPulsatillasaponin B4 for injection obtained by the preparation method.

In addition, the present invention also provides a use of thePulsatillasaponin B4 preparation for injection, the aqueous injection ofPulsatillasaponin B4 or the lyophilized powder ofPulsatillasaponin B4 for injection obtained by the preparation method in the preparation of drugs for acute renal injury, chronic renal failure and/or renal insufficiency.

The “unavoidable impurities” in the description of the present invention include substances produced by the degradation of a small amount ofPulsatillasaponin B4, and a small amount of water in the lyophilized powder ofPulsatillasaponin B4 for injection. Although the unavoidable impurities exist, the content should meet the limit provision in the pharmacopoeia.

ThePulsatillasaponin B4 preparation for injection according to the present invention is administered to mammals, in particular to human beings. When the preparation is administered to a human being, the daily dose is 2-120 mg for an adult with a body weight of 60 kg.

The aqueous injection ofPulsatillasaponin B4 or the lyophilized powder ofPulsatillasaponin B4 for injection achieves the intramuscular injection or intravenous injection ofPulsatillasaponin B4 and ensures that the active ingredient enters the circulatory system in its original form and distributes to various organs in the body, especially kidney and lung etc, which enablesPulsatillasaponin B4 to work effectively at a much lower dose, thereby expanding the safety window for treatment and finally ensuring the safety of medication.

Pulsatillasaponin B4 has good water solubility and is easy to be prepared into an aqueous injection; but its stability changes with different solvents, therefore, the solvent of the aqueous injection needs to be carefully studied and selected.

Pulsatillasaponin B4 itself and its aqueous solution are sensitive to temperature and light; for example, after the aqueous solution ofPulsatillasaponin B4 was placed for 14 days at 60° C., the purity ofPulsatillasaponin B4 decreased from 98.90% at day 0 to 93%, the total impurities increased from 0.64% at day 0 to 0.94%. The lyophilized powder ofPulsatillasaponin B4 for injection is preferred because the low water content is more beneficial to the stability of the preparation.

However, in view of the complexity of lyophilized dispersion system, it is not easy to obtain the powder for injection efficiently and quickly that meets the injection requirements. Up to now, a mature theory for the selection of lyophilized formulation has not been formed yet, and individual or joint investigations of various factors that may affect the lyophilized behavior and lyophilized effect are required. Excipients and other additives in the lyophilized formulation will interact with active ingredients, and the composition and concentration of the formulation will significantly affect the freeze-solidification and sublimation dehydration behavior of the system, such as glass transition temperature at the maximum freezing concentration (an important indicator for measuring whether the collapse occurs during drying), the minimum freezing temperature, the cooling rate and the freezing annealing time etc. In addition, the type and concentration of additives will significantly affect the time consumed during drying stage and the properties of the final product. In the present invention, the parameters such as the type and amount of lyophilized excipients, the pH of the intermediate solution, the pre-freezing temperature, the pre-freezing method, the sublimation temperature, the vacuum pressure, and the desorption drying temperature are investigated separately or in combination to obtain the most preferred parameters, thereby aPulsatillasaponin B4 lyophilized powder for injection with desirable appearance, good re-solubility and stable quality is obtained.

