Powdery pharmaceutical composition of myeloperoxidase.

The present invention relates to a powdery pharmaceutical composition of myeloperoxidase comprising myeloperoxidase and an amount, enough to stabilize the myeloperoxidase, of citric acid or a salt thereof, as well as to process for producing said composition.

Myeloperoxidase (hereinafter, simply referred to as "MPO") is an enzyme which was first isolated in 1941 from human pus by Agner [Acta Physiol. Scand. 2, Suppl., 8 (1941)]. It is contained in a large quantity in myelogenous cells, particularly in polymorphonuclear leukocytes and monocytes, together with lysozyme, and its content amounts to 5% based on the weight of neutrophils. This enzyme is a basic hemo-protein having two iron atoms per one protein molecule and having a molecular weight of about 120,000 - 150,000 daltons. It belongs to the group of oxidoreductases. The physiological function of MPO is considered consisting in killing or inactivating the pathogenic micro-organisms harmful to animals, such as bacteria, fungi, vira and so on, in the presence of hydrogen peroxide and halide. Usefulness of MPO as a pharmaceutical is well known in the point that pharmaceutical compositions comprising MPO as a main ingredient exhibit a dramatic therapeutic effect when used for the treatment of infectious diseases with, for example, isonicotinic acid hydrazide (INH)-resistant mycobacterium tuberculosis.

Pharmaceutical compositions of MPO as usually offered as an injection or a topical composi÷ tion having a form of liquid or more preferably a freeze-dried powder, by sealing unit dosage into an ampoule or a dividual container. However, pharmaceutical compositions comprising MPO as main ingredient are often instable to freeze-drying, and they tend to decrease MPO activity either at the time of freeze-drying or in the lapse of time after freeze-drying.

The present inventors conducted many studies with the aim of retaining the stability of pharmaceutical compositions comprising MPO as main ingredient during a long-term storage. As the result, it was found that the drop in MPO activity at the time of freeze-drying and the time-dependent decrease of MPO activity in the course of storage can be prevented and, at the same time, solubility of MPO compositions can be enhanced by adding citric acid or a salt thereof to MPO compositions.

It is the object of the present invention to provide a powdery MPO composition to which an amount, enough to stabilize the MPO, of citric acid or a salt thereof is added, as well as a process for producing said composition.

The citric acid or salt thereof used in the present invention is not critical, so far as it is physiologically acceptable. As the salt of citric acid, alkali metal salts such as sodium salt, potassium salt and the like and alkaline earth metal salts such as calcium salt, magnesium salt and the like can be referred to, for example. Of these salt, sodium salt is particularly preferable.

The powdery MPO composition of the invention is preferably a freeze-dried product. The citric acid or salt thereof for stabilizing MPO is added before freeze-drying the aqueous solution of MPO, and it is not removed but is allowed to remain in the powder. Alternatively, it may also be added and mixed just after the freeze-drying. The amount of citric acid or salt thereof may vary depending on the form and concentration of MPO at the time of adding the citric acid or salt thereof. However, if it is added before the aqueous solution of MPO having a concentration of 20 to 10,000 units/ml is freeze-dried, it should be added so that its concentration reaches 0.1 through 4.0 W/V %, preferably 0.3 through 2.0 W/V %. If it is added to a powdery material comprising 200,000 to 500,000 units of MPO and a buffering salt, it should be added so that its concentration reaches 1.5 through 3,000 _W/W %, preferably 4.5 through 1,500 _W/W %. Proportion of citric acid or salt thereof in the powdery _MPO composition thus obtained is 0.1 through 500 µg per one unit of MPO.

Next, the stabilizing effect of MPO will be explained with reference to Experimental Examples 1 - 4. Activity of MPO was determined by an improvement of the method of B. Chance et al. [Method in Enzymology, II, 764 (1955)] using guaiacol.

Experimental Example 1

Trisodium citrate, as a stabilizer, was added to 5 ml of an aqueous solution of MPO having a concentration of 100 units/ml before freeze-drying the aqueous solution, so that concentration of trisodium citrate reached 0.1, 0.5, 2.0 or 4.0 W/V %. After the MPO solution was freeze-dried, residual titer of the resulting mixture was measured (A) just after freeze-dried and (B) after stored at room temperature for 6 months. The results are summarized in Table 1. <tables id="tabl0001" num="0001"><img file="EP0098073A2_D0001.tif" /></tables>

Experimental Example 2

Trisodium citrate, as a stabilizer, was added to 10 ml of an aqueous solution of MPO having a concentration of 10,000 units/ml before freeze-drying the solution, so that concentration of the citrate reached 0.1, 0.5, 2.0 or 4.0 W/V %. After the MPO solution was freeze-dried, residual titer of the mixture was determined (A) just after freeze-dried and (B) after stored at room temperature for 6 months. The results are summarized in Table 2. <tables id="tabl0002" num="0002"><img file="EP0098073A2_D0002.tif" /></tables>

Experimental Example 3

Five milliliters of an aqueous solution of M_PO having a concentration of 100 units/ml was freeze-dried, just after which trisodium citrate was added as a stabilizer in the amount shown in Table 3. The mixture was stored at room temperature for 6 months, and then its residual titer (%) was determined. The results are shown in Table 3. <tables id="tabl0003" num="0003"><img file="EP0098073A2_D0003.tif" /></tables>

Experimental Example 4

One milliliter of an aqueous solution of MPO having a concentration of 10,000 units/ml was freeze-dried, just after which trisodium citrate was added and mixed as a stabilizer in the amount shown in Table 4. The mixture was stored at room temperature for 6 months, and then the residual titer (%) was measured. The results are shown in Table 4. <tables id="tabl0004" num="0004"><img file="EP0098073A2_D0004.tif" /></tables>

It is apparent from the results of these Experimental Examples that addition of citrate exercises a marked effect on the stability of MPO at the time of freeze-drying and on the stability of freeze-dried MPO composition in the lapse of time.

Next, the invention will be explained with reference to the following examples in no limitative way.

Example 1

A dry powdery MPO composition was prepared by adding 0.5 W/V % of trisodium citrate to 5 ml of an aqueous solution of MPO having a concentration of 100 units/ml and then freeze-drying the mixture.

Example 2

A dry powdery MPO composition was prepared by adding 0.5 W/V % of trisodium citrate to 5 ml of an aqueous solution of MPO having a concentration of 10,000 units/ml and freeze-drying the mixture.

Example 3

A dry powdery MPO composition was prepared by freeze-drying an aqueous solution of MPO having a concentration of 100 units/ml and, just after it, adding 154 W/W % of trisodium citrate.

Example 4

A dry powdery MPO composition was prepared by freeze-drying an aqueous solution of MPO having a concentration of 10,000 units/ml and, just after it, adding 4.5 W/W % of trisodium citrate.