PREPARATION AND PREPARATION PROCESS THEREOF

[i]

TECHNICAL FIELD

[2] The present invention relates to an orally administrable antimalarial combined preparation containing artemisinine or its derivatives, and pyronaridine or its salts, as active ingredients, with pharmaceutically acceptable carriers, and to a preparation process thereof.

[3]

BACKGROUND ART

[4] According to the World Health Organization (WHO), approximately 3 to 5 hundred millions of malarial patients occur every year all over the world, and 90% of them are concentrated in Africa . Also, 1.5 to 2.7 millions of people die of malaria every year, and particularly at least one million among them are African children under 5. Specifically, the southern region of the Sahara shows the highest infection rate, morbidity rate, and death rate. Today, malaria has become more serious due to resistance to antimalarial agent by wars, environmental disruption, re-colonization, climatic change, and so on, and particularly, to increase of resistance to chloroquine and pyrimethamine/sulfadoxine.

[5]

[6] The treatment and prophylactic of malaria have depended on the chloroquine drug which was provided by WHO. However, as resistance to chloroquine increases, there has been urgent need for development of new drugs effective against P. falciparum and P.vivax which have resistance to chloroquine. After developing a new drug, it must be considered in which form the drug will be prepared. The first line of substitute for chloroquine is an orally administrable form because it is easily accessible by the public medical service organizations like WHO or inhabitants of outlying areas, easily ad¬ ministrable, effective, and safer than other forms.

[7]

[8] However, unfortunately, not many developing drugs are available for human experiment. One of them is pyronaridine, which was synthesized as blood sch- izonticide in China in 1970. It has a chemical property common with the existing drugs, but Mannich bases including quinoline type such as amodiaquine and a mopyroquine; and acridine type such as pyronaridine and pyraquine have significant advantages in terms of activity, resistance rate, and stability to resistance (Basco and

Le Bras, 1992; Peters and Robinson, 1992). Today, pyronaridine (orally administrable form and injectable form) is listed in the Chinese pharmacopoeia, and is in the market as the name of Malaridine. However, it is commercially vailable only in China , does not exhibit its effect quickly in the beginning of administration, and is very expensive. Thus, it is not appropriate for the public in the developing countries.

[9]

[10] Among artemisinine derivatives, artesunate is most widely used in China and

Vietnam . It is effective against P. falciparum and P.vivax which have resistances to other antimalarial agents. Now, 50mg tablet of Artesunate is in the markets of Vietnam (Mediplantex and Khanh Hoa) and China , and is also restrictively sold under the name of Arsumaχ ^R in some areas of Africa . However, this has short in vivo half-life, and so has a limit in completely curing the malaria infection.

[H]

[12] Synergy effect was confirmed when administering pyronaridine in combination with artemisinine to mouse infected with Pasmodium yoelii which has resistance to some chloroquine. Also, for the treatment of P. falciparum which has resistance to many antimalarial agents, a clinical pilot study was performed on the combined admin¬ istration of pyronaridine and artemisinine derivatives. As a result, when pyronaridine was administrated in combination with dihydroartemisinin or artemether, no recurrence was occurred in 7 days and 28 days of follow-up studies, even though the patient number of each group was limited (40 persons including adults and children). Also, in a study to compare effects between administration of each of dihydroartemisinin and pyronaridine and combined administration of these two, it is reported that the admin¬ istration of 32mg/kg of pyronaridine alone or the combined administration of 16mg/kg of pyronaridine with 6mg/kg of dihydroartemisinin are very effective and have superior tolerability (Lit et al., 2002). However, this combined administration was merely to confirm the treatment effect in administering each individual drug together.

[13]

[14] Therefore, a brand-new antimalarial drug that is effective for resistant strains, but has little economic burden has been seriously needed because resistance to the existing antimalarial agents is increasing, and the outbreak areas are mainly the underdeveloped countries such as Africa .

[15]

DISCLOSURE OF THE INVENTION

[16] The present inventors have searched safe, effective, quality, and inexpensive an¬ timalarial agents to treat uncomplicated P. falciparum and P.vivax malaria by oral ad¬ ministration in Africa and Asia. The inventors have tried various combinations of py¬ ronaridine or salts thereof and artemisinine or derivatives thereof which have the most

effect on the resistant strains, and finally, confirmed that the combination in the weight ratio of 6:1 ~ 1: 1, especially 3:1, is the most effective in the efficacy and toxicity. Further, the inventors discovered that when the two drugs are simply mixed together, artemisinine or derivatives thereof are resolved by direct contact because pyronaridine salts have physicochemical properties of acidic salts. Thus, the present inventors solved the problems of preparation stability, solubility, and dissolution rate through primarily formulating artemisinine or derivatives thereof by using pharmaceutically acceptable carrier such as preparing them into microcapsule or eutectic mixture, or coating them with coating agents, and then mixing them with pyronaridine acidic salts to prepare a combined preparation. The present invention was completed on the basis of the above discovery.

[17]

[18] Therefore, the purpose of the invention is to provide an orally administrable an¬ timalarial combined preparation containing artemisinine or its derivatives and py¬ ronaridine or its salts, as active ingredients, with the pharmaceutically acceptable carriers, and a preparation process thereof.

[19]

BRIEF DESCRIPTION OF THE DRAWINGS

[20] Fig. 1 is a graph showing the dissolution test result of artesunate of the combined preparation of Example 1 in simulated intestinal fluid, water, and pH 4.0 of phosphate buffer; and

[21] Fig. 2 is a graph showing the elution test result of pyronaridine tetraphosphate of the combined preparation of Example 1 in simulated gastric fluid, simulated intestinal fluid, water, and pH 4.0 of phosphate buffer.

[22]

BEST MODE FOR CARRYING OUT THE INVENTION

[23] The present invention relates to an orally administrable antimalarial combined preparation containing artemisinine or its derivatives, and pyronaridine or its salts, as active ingredients, with the pharmaceutically acceptable carriers, and to preparation process thereof.

[24]

[25] In the present invention, artemisinine derivatives include dihydroartemisinine, artesunate,, artemether, and arteether, but not limited thereto. Preferably, artemisinine derivative is artesunate.

[26]

[27] Also, pyronaridine salts include acidic addition salts with phosphoric acid, sulfuric aicd, hydrochloric acid, acetic acid, methansulfonic acid, toluenesulfonic acid, maleic

acid, or fumaric acid, but not limited thereto. Preferably, pyronaridine salts are py- ronaridine phosphate.

[28]

[29] The composition of the present invention contains artemisinine or its derivatives, and pyronaridine or its salts, preferably in the weight ratio of 1:1 ~ 1:6, more preferably 1:3.

[30]

[31] Further, in the present invention, it is preferable to formulate artemisinine or its derivatives not to contact with pyronaridine or its salts. To do so, artemisinine or its derivatives can be formulated by primarily formulating them into microcapsule, particulate form of eutectic mixture, or coated preparation with pharmaceutically acceptable carrier, and then mixing them with pyronaridine or its salts added thereto in a certain ratio to artemisinine or its derivatives. Doing so can solve those problems resulted from the direct contact of the two drugs during the combination with py¬ ronaridine salts in acidic form, including preparation unstability, low solubility, and low dissolution rate, thereby providing antimalarial agent showing remarkable treatment effect on the resistant strains, with securing the drug stability and having synergic effect of the two drugs.

[32]

[33] Any conventional carriers used in the pharmaceutical field can be used as the phar¬ maceutically acceptable carriers for the present invention. Some representative examples thereof are lactose, dextrine, sugar, microcrystalline cellulose, hydroxy propylmethyl cellulose, hydroxy propyl cellulose, hydroxy ethyl cellulose, ethyl cellulose, methyl cellulose, polyethylene glycol, silicon dioxide, hydrotalcite, aluminium magnesium silicate, aluminium hydroxide, aluminium silicate, magnesium aluminium metha silicate, bentonite, and mixture thereof. For the present invention, the preferable melting dispersing carrier is polyethylene glycol, and the preferable weight ratio of artemisinine or its derivatives to polyethylene glycol is 1:0.1 ~ 1:2. Par¬ ticularly, the weight ratio of artesunate to polyethylene glycol is 1:1.

[34]

[35] Also, the composition of the present invention can further comprise surfactants so that the components can quickly disintegrate and dissolve at the time of contacting with aqueous medium during in vivo administration, in addition to carriers. Repre¬ sentative examples of surfactants include sodium lauryl sulfate and its derivatives; poloxamer and its derivatives; saturated polyglycorized glyceride (gelucire); labrasol; all sort of polysorbate such as polyoxyethylene sorbitan mono laurate (hereinafter, Tween 20), polyoxyethylene sorbitan monopalmitate (hereinafter, Tween 40), poly¬ oxyethylene sorbitan monostearate (hereinafter, Tween 60), and polyoxyethylene

sorbitan monooleate (hereinafter, Tween 80); sorbitan esters such as sorbitan mono laurate (hereinafter, Span 20), sorbitan monopalmitate (hereinafter, Span 40), sorbitan monostearate (hereinafter, Span 60), sorbitan monooleate (hereinafter, Span 80), sorbitan trilaurate (hereinafter, Span 25), sorbitan trioleate (hereinafter, Span 85), and sorbitan tristearate (hereinafter, Span 65); cremophor; PET-60 hydrogenated castor oil; PET-40 hydrogenated castor oil; disodium cocoamphodiacetate; and so on, but not limited thereto. The present composition can be formulated in the forms of powder, granule, tablet, capsule, dry syrup, coating agent, and so on.

[36]

[37] The following examples illustrate the present invention; but are not intended to limit the scope of the present invention in any way.

[38]

[39] Preparative Example 1 : Preparation of Artesunate Microcapsule (1)

[40]

Ingredient Content

Artesunate 3g

Polyethylene glycol 10000 3g

Liquid paraffin 396g

Tween 80 4g

[41] 3g of artesunate and 3g of polyethylene glycol were dissolved in 9g of acetonitrile.

This organic solution was dispersed in 396g of liquid paraffin (containing 1% Tween 80), and was stirred with a digital stirrer (IKA, EUROSTAR). After preparing an ap¬ propriate emulsion therefrom, the solvent was evaporated until 40 to 60% of ace¬ tonitrile was completely removed therefrom. Thus resulted microcapsule suspension was centrifuged at 41 x g for 10 minutes. The supernatant was decanted, and then the microcapsule was resuspended with 50 to 100ml of washing water, and was slowly filtered off. During the filtration, the microcapsule was maintained in the suspension, with stirring slowly and continuously adding a washing water thereto to remove ace¬ tonitrile which remains within the microcapsule, and to harden microcapsule.

[42]

[43] Preparative Example 2 : Preparation of Artesunate Microcapsule (2)

[44]

Ingredient Content

Artesunate 3g

Polyethylene glycol 10000 1.5g

Liquid paraffin 396g

Tween 80 4g

[45] Using the above ingredients, the microcapsule was prepared according to the same procedure as the Preparative Example 1.

[46]

[47] Preparative Example 3 : Preparation of Ar

[48]

Ingredient Content

Artesunate 3g

Polyethylene glycol 10000 6g

Liquid paraffin 396g

Tween 80 4g

[49] Using the above ingredients, the microcapsule was prepared according to the same procedure as the Preparative Example 1.

[50]

[51] Preparative Example 4 : Preparation of artesunate eutectic i

[52]

Ingredient Content (mg/preparation)

Artesunate 40

Polyethylene glycol 10000 40

Microcrystalline Cellulose 140

[53] Polyethylene glycol as melting dispersing carrier, and Artesunate as active ingredient were mixed, to which about 20mg of ethanol was added, and the mixture was suspended at room temperature. After heating the mixture to about 80 ⁰ C for melting, it was quickly frozen and finely ground. Microcrystalline cellulose was added thereto and mixed. The mixture was aggregated in the method of dry granulation to prepare the desired eutectic mixture granule containing artesunate.

[54]

[55] Preparative Example 5 : Preparation of artesunate eutectic r

[56]

Ingredient Content (mg/preparation)

Artesunate 40

Polyethylene glycol 10000 40

Poloxamer F127 15

Microcrystalline Cellulose 125

[57] Polyethylene glycol as melting dispersing carrier and Artesunate as active ingredient are mixed, and then about 20mg of ethanol is added thereto, and the mixture is suspended at room temperature. After heating the mixture to about 80 ⁰ C for melting, Poloxamer was added thereto, which was quickly frozen and solidified. After finely grinding the mixture, microcrystalline cellulose was added thereto, and the mixture was aggregated in the method of dry granulation to prepare the desired eutectic mixture granule containing artesunate.

[58]

[59] Preparative Example 6 : Preparation of artesunate eutectic mixture granule (3)

[60]

Ingredient Content (mg/preparation)

Artesunate 40

Polyethylene glycol 10000 40

Poloxamer F 127 15 hydroxypropylmethyl cellulose 20

Microcrystalline Cellulose 105

[61] Polyethylene glycol as melting dispersing carrier and artesunate as active ingredient are mixed, and then about 20mg of ethanol is added thereto, and the mixture is suspended at room temperature. After heating the mixture to about 80 ⁰ C for melting, Poloxamer and hydroxy propylmethyl cellulose were added thereto, which was quickly frozen and solidified. After finely grinding the mixture, microcrystalline cellulose was added thereto, and the mixture was aggregated in the method of dry granulation to prepare the desired eutectic mixture granule containing artesunate.

[62]

[63] Preparative Example 7 : Preparation of artesunate eutectic mixture granule (4)

[64]

Ingredient Content (mg/preparation)

Artesunate 40

Polyethylene glycol 10000 40

Poloxamer F 127 15 ethyl cellulose 20

Microcrystalline Cellulose 105

[65] Polyethylene glycol as melting dispersing carrier and artesunate as active ingredient are mixed, and then about 20mg of ethanol is added thereto, and the mixture is suspended at room temperature. After heating the mixture to about 80 ⁰ C for melting, Poloxamer and ethyl cellulose were added thereto, and the mixture was quickly frozen and solidified. After finely grinding the mixture, microcrystalline cellulose was added thereto, and the mixture was aggregated in the method of dry granulation to prepare the desired eutectic mixture granule containing artesunate.

[66]

[67] Example 1 : Preparation of artesunate/pyronaridine tetraphosphate (1:3) combination tablet (1)

[68]

Ingredient Content (mg/preparation) eutectic mixture granule of Preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 120 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[69] Hydroxypropyl cellulose in ethanol was mixed with pyronaridine tetraphosphate, aggregated in the method of wet granulation, dried, and sieved, and then eutectic mixture granule of Preparation Example 4, low-substituted hydroxypropyl cellulose , crospovidone, sodium lauryl sulfate, and silicone dioxide were added thereto and mixed uniformly to have the above composition. Magnesium stearate was added thereto to prepare a tablet.

[70]

[71] Example 2 : Preparation of artesunate/pyronaridine tetraphosphate (1:3) combination tablet (2) [72]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 5 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 120

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[73] Using the above ingredients, the tablet was prepared according to the same procedure as the Example 1. [74] [75] Example 3 : Preparation of artesunate/pyronaridine tetraphosphate (1:3) combination tablet (3) [76]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 6 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 120

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[77] Using the above ingredients, the tablet was prepared according to the same procedure as Example 1.

[78]

[79] Example 4 : Preparation of artesunate/pyronaridine tetraphosphate (1:3) combination tablet (4) [80]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 6 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 120

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[81] Using the above ingredients, the tablet was prepared according to the same procedure as Example 1. [82] [83] Example 5 : Preparation of artesunate/pyronaridine tetraphosphate (2:3) combination tablet [84]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 60

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[85] Using the above ingredients, the tablet was prepared according to the s procedure as Example 1.

[86]

[87] Example 6 : Preparation of artesunate/pyronaridine tetraphosphate (1:4) combination tablet [88]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 160

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[89] Using the above ingredients, the tablet was prepared according to the same procedure as Example 1. [90] [91] Example 7 : Preparation of artesunate/pyronaridine tetraphosphate (1:7) combination tablet [92]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 240

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[93] Using the above ingredients, the tablet was prepared according to the same procedure as Example 1. [94] [95] Example 8 : Preparation of artesunate/pyronaridine tetraphosphate (1:3)

combination hard capsule [96]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 120

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[97] Hydroxypropyl cellulose dissolved in ethanol and pyronaridine tetraphosphate were mixed, aggregated in the method of wet granulation , dried, and sieved. Then, to have the eutectic mixture granule of Preparation 4, low-substituted hydroxypropyl cellulose , crospovidone, sodium lauryl sulfate, and silicone dioxide were added thereto and mixed uniformly to have the above composition. Magnesium stearate was added thereto, mixed, and filled in hard capsule to prepare the capsule.

[98]

[99] Example 9 : Preparation of artesunate/pyronaridine tetraphosphate (2:3) combination hard capsule

[100]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 60

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[101] Using the above ingredients, the capsule was prepared according to the same procedure as Example 8. [102] [103] Example 10 : Preparation of artesunate/pyronaridine tetraphosphate (1:4) combination hard capsule [104]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 160

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[105] Using the above ingredients, the capsule was prepared according to the same procedure as Example 8. [106] [107] Example 11: Preparation of artesunate/pyronaridine tetraphosphate (1:6) mixture hard capsule [108] Using the above ingredients, the capsule was prepared according to the same procedure as Example 8. [109]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 240

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[HO]

[111] Example 12: Preparation of artesunate/pyronaridine tetraphosphate (1:1) combination tablet [112]

Ingredient Content (mg/preparation) eutectic mixture granule of preparation 4 220

Low-substituted hydroxypropyl cellulose 25 pyronaridine tetraphosphate 40

Hydroxypropyl cellulose 4 crospovidone 30 sodium lauryl sulfate 15 silicone dioxide 3 magnesium stearate 3

[113] Using the above ingredients, the tablet was prepared according to the same procedure as Example 1. [114]

[115] Experimental Example [116]

[117] Experimental Example 1: Dissolution test [118]

[119] ( 1 ) Dissolution Test of Artesunate in a combination tablet

[120] The tablet prepared according to the method of Example 1 was tested according to

the paddle method (apparatus 2) described in the United State Pharmacopeia (USP).

After 30minutes, the amount of Artesunate using filtered portions of the solution under the test was determined according to the following analysis method. [121]

[122] Dissolution instrument: SR8PLUS (HANSON)

[123] Medium temperature: 37 ⁰ C +0.5 ⁰ C

[124] Medium: simulated intestinal fluid, water, pH 4.0 phosphate buffer

[125] Rotation speed: IOORPM

[126] Analysis Method: Lipid Chromatography

[127] Column: 4.6mm x 25cm, ODS, 5-10 μm in diameter

[128] Column temperature: 40 ⁰ C

[129] Mobile phase - pH 3.0 buffer: acetonitrile (50:50)

[130] Flow rate: 0.2ml/min.

[131] pH 3.0 buffer: 1.36g of Potassium dihydrogenphosphate was dissolved in IL of water and adjusted to a pH 3.0+0.05 with phosphoric acid. [132]

[133] Fig. 1 shows the result of the above-Dissolution test.

[134]

[135] (2) Dissolution Test of pyronaridine tetraphosphate in a combination tablet

[136] [137] The tablet prepared according to the method of Example 1 was tested according to the paddle method (apparatus 2) described in the United State Pharmacopeia (USP).

After 30minutes, the amount of Artesunate using filtered portions of the solution under the test was determined according to the following analysis method. [138]

[139] Dissolution instrument: SR8PLUS (HANSON)

[140] Medium temperature: 37 ⁰ C +0.5 ⁰ C

[141] Medium: simulated gastric fluid, simulated intestinal fluid,

[142] water, pH 4.0 phosphate buffer

[143] Rotation speed: IOORPM

[144] Detector: Ultraviolet absorption spectrometer(wavelength:278nm)

[145] Column: 4.6mm x 25cm, ODS, 5-10 μm in diameter

[146] Column temperature: 40 ⁰ C

[147] Mobile phase : 10% acetonitrile solution adjusted with phosphoric acid to a pH

2.15

[148] Flow rate: l.Oml/min.

[149] [150] Fig. 2 shows the result of the above-dissolution test.

[151]

[152] Experimental Example 2: Storage Stability test

[153]

[154] Using the tablet prepared according to the method of Example 1, storage stability test was conducted under the conditions of temperature of 40 ⁰ C +2 ⁰ C and relative humidity of 75%+5%RH. That is, samples were taken for 48 weeks at a regular interval from the beginning, and the tablets' properties were observed. The assays of artesunate and pyronaridine in a combined preparation were conducted according to the following method, and the dissolution test was conducted according to Ex¬ perimental Example 1.

[155]

[156] (1) Assay of Artesunate in a combined preparation

[157] About 20 tablets of the tablets prepared according to the method of Example 1 were taken, weighed accurately, and powdered. A portion of the powder, equivalent to about 4.0mg of artesunate, was exactly measured, and mixed in 2ml of acetone, and the mixture was dissolved well, filtered, evaporated and dried. ImL of acetonitrile was added to this residue and dissolved. The resulting product was used as test solution. On the other hand, ImL of acetonitrile was added to 4.0mg of artesunate RS, and dissolved to obtain a standard solution.

[158]

[159] The chromatograph was conducted to each of 20μL of the test solution and the standard solution. The procedure was conducted according to general guidelines on the chromatography described in USP to calculate the peak areas of artesunate, A and A :

[160]

[161] Amount of Artesunate (C H O )(mg) = Amount of Artesunate standard

19 29 8 preparation converted into anhydride(mg) x A /A

T s

[162]

[163] Operation condition

[164]

[165] Detector: Ultraviolet absorption spectrometer (wavelength : 216nm)

[166] Column: 4.6mm x 25cm, ODS, 5-10 μm in diameter

[167] Column temperature: 40 ⁰ C

[168] Mobile phase - pH 3.0 buffer : acetonitrile(50:50)

[169] Flow rate: l.Oml/min.

[170] pH 3.0 buffer: 1.36g of Potassium dihydrogenphosphate was dissolved in lLof water, and phosphoric acid was added thereto to adjust to pH 3.0+0.05. [171]

[172] (2) Assay of pyronaridine tetraphosphate in a combined preparation

[173]

[174] About 20 tablets of the tablets prepared according to the method of Example 1 were taken, weighed accurately, and powdered. A portion of the powder, equivalent to about lOmg of pyronaridine tetraphosphate, was exactly measured, and was put into a yellowish brown flask. Phosphate buffer (pH 7.0) was added thereto to make the mixture to 100ml and mixed well. The mixture was promptly filtered off, and the first filtrate was discarded, and 5ml of the next filtrate was taken, and phosphate buffer (pH 7.0) was added thereto to make it to 50ml of test solution. On the other hand, about lOmg of pyronaridine tetraphosphate RS, which was dried at 105 ⁰ C to obtain a constant amount, was exactly measured, and was put into a yellowish brown flask. Phosphate buffer (pH 7.0) was added thereto to make the mixture to 100ml and dissolved. 5ml of the mixture was taken therefrom and phosphate buffer (pH 7.0) was added thereto to make 50ml of standard solution. According to the ultraviolet absorption spectrometry among the general methods described in USP, absorbance A and A test was conducted to the test solution and the standard solution at 260nm to measure absorbance A and A [175] [176] Amount of pyronaridine tetraphosphate (C H ClN O :4H PO ) (mg) = Amount of pyronaridine tetraphosphate standard preparation converted into dried product (mg) x

A T /A s

[177]

[178] Operation condition

[179] Detector : Ultraviolet absorption spectrometer (wavelength : 278nm)

[180] Column: 4.6mm x 25cm, ODS, 5-10 μm in diameter

[181] Column temperature: 40 ⁰ C

[182] Mobile phase : 10% acetonitrile solution adjusted with phosphoric acid to a pH

2.15

[183] Flow rate: l.Oml/min. [184]

[185] The result thereof is shown in Table 1. [186]

[187] Table 1

[188] Storage stability test result of artesunate and pyronaridine tetraphosphate in a combined preparation (Example 1)

[189] [190] The results shown in Table 1 show 1.3% of decrease in the content of Artesunate in a combined preparation and 2.2% of decrease in that of pyronaridine tetraphosphate after 48 weeks of storage. Therefore, they confirm that the storage stability of the combined preparation is very good since both pyronaridine and artesunate showed change of less than 5% in the 48-week content and dissolution tests.

[191] [192] Experimental Example 3: In vitro antimalarial activity test [193] [194] P. falciparum used in this Experiment was obtained from MR4 (Malaria Research and Reference Reagent Resource Center, Virginia, USA) [3D7 (Netherlands); drug- sensitive clon, Kl (Thailand); chloroquine/pyrimethamine/cycloguanyl-resistance subspecies, VSl (Vietnam); chloroquine/pyrimethamine-resistance subspecies, FCR3(Gambia); chloroquine/pyrimethamine-resistance subspecies, FCB (Colombia); chloroquine-resistance subspecies, Tm90 C2A (Thailand); chloroquine/ pyrimethamine/mefloquine-resistance]. The protozoa was put into in a tissue culture flask containing the culture media (RPMI- 1640:Rh+ red blood cell, 5% of Hematocrit, 25mM of HEPES, 24 mM of NaHCO , 0.2% of glucose, 0.03% of L-glutamine, 150μM of H ypoxantin, and 0.5% of albumax II), and cultured in a tissue culture incubator (at 37 ⁰ C with supplying 5% CO 2 /95% air).

[195] [196] Drug-sensitivity test was conducted according to a modified method from Deszardin et al. (Antimicorb. Agents Chemother. 1979, 16, 710). That is, the drug was dissolved in 100% of DMSO, and repeatedly diluted with the culture media (RPMI- 1640: 0.5% of albumax II, 0.2% of glucose, 0.03% of L-glutamine, and 150μM of H ypoxantin) in 96 well-plate. The ratio of the two drugs was fixed to 1 :0, 4: 1 , 3 :2,

2:3, 1:4, and 0:1 for the combination test. The same amount of P. falciparum parasite suspension (containing 0.5% of parasitemia and 1.5% of Hematocrit ) or non-infected erythrocytic solution was added thereto. The plate was cultured in incubator (37 ⁰ C and 5% of CO ) for 24 hours, 10 D of ^H] H ypoxantin (3.7 Bq/well) was added to each well, the plate was further cultured for 24 hours, and then put into a freezer of -80 ⁰ C to complete the reaction. The plate was de-frozen again and centrifuged to harvest the granulated product. The radioactivity was measured with scintillation spectrometer. The regression curve was obtained from the Concentration-Reaction Data, and 50% Inhibitory Concentration (IC ) to each product was calculated. FIC (FIC= Fractional

50 Inhibitory Concentration) value was obtained as follows:

[197] [198] Sum FIC = IC of Drug A in Combined Agent/ IC of Drug A + IC of Drug B

50 50 ^&& 50 ^& 50 ^& in Combined Agent/ IC of Drug B

[199] [200] The results are shown in Tables 2 and 3. [201] [202] Table 2 [203] Antimalarial activities of pyronaridine, artesunate, dihydroartemisinine and chloroquine

[204] [205] Each numerical value represents the average value from three tests, [206] nd: not determined. [207] [208] As shown in Table 2, IC of pyronaridine was similar to that of artesunate as 1.2-4. lmg/ml in six subspecies, and that of dihydroartemisinine was more efficient as 0.2~0.9mg/ml.

[209] [210] Table 3 [211] Drug sensitivity of the combination of pyronaridine and artesunate to various malarial protozoa

[212] [213] As shown in Table 3, when the mixture of pyronaridine and artesunate was ad¬ ministered, most FIC values were 1-2 to show some antagonistic activity. However, this is in vitro test results without considering pharmacodynamic parameters. Thus, further analysis is required in connection with in vivo test result.

[214] [215] Experimental Example 4: In vivo antimalarial activity test [216] [217] (1) Dose-response assay [218] To observe the dose response of the combined preparation of pyronaridine and artesunate obtained in Example 1, the suppressive/curative activity of the combined preparation at high dose ( pyronaridine + artesunate: 12mg/kg + 4 mg/kg) and low dose ( pyronaridine + artesunate: 0.9mg/kg + 0.3 mg/kg) were compared with that of each single drug by using P. chabaudi ASS protozoa. In short, Naϊve (meaning "no drug administered") TFW mice were intravenously inoculated with 2 x 10 P. chabaudi ASS parasitised erythrocytes, and then the curativeness test was conducted by a three-day course of treatment starting 2 days after the infection as follows: the mice were infected with P. chabaudi protozoa; pyronaridine/ artesunate (3:1) obtained in Example 1 was subcutaneously administrated; the infection process was observed for 28 days; every day, blood was taken from each animal, and thin blood film thereof was prepared; after Giemsa stains, it was examined whether the animal was infected or not with a microscope; the blood of mice which showed negative reaction after 28 days was pooled and sub-inoculated into naϊve mice ; pooled blood was diluted with 0.9% of saline solution to 2-fold volume; 0.2mL thereof was intravenously administrated to 5 naϊve mice to re-infect them; and observation was continued for 28 days more.

[219] As a result, when artesunate alone was administrated at the low dose of 0.3mg/kg

or the high dose of 4mg/kg, they did not affect the infection process. Also, the dose of 0.9mg/kg pyronaridine alone was administrated, it had no effect. However, the dose of 12 mg/kg completely suppressed parasitosis for 28 days. No infection was recurred when naϊve mice were observed for 28 more days after they were re-infected by the blood taken from mice which had not shown any infection till then. When a combination of pyronaridine/artesunate (3:1) was administrated at a low dose of 4 mg/ kg, at the medium dose of 8 mg/kg, or at a high dose of 16 mg/kg, all of them showed excellent and effective treatment effect. In particular, in a dose of more than 8mg/kg (pyronaridine + artesunate : 6 + 2), it completely suppressed parasitosis for 28 days, showing highly good treatment effect. No infection was recurred when naϊve mice were observed for 28 more days after they were re-infected by the blood taken from mice which had not shown any infection till then. These results suggest that the dose of artesunate showing the antimalarial activity could be reduced through the combined administration.

[220]

[221] (2) Full suppressive test (4-day test)

[222] On Day 0, mice were infected intravenously with 2 x 10 of infected erythrocytes.

Two hours post-infection treated groups received the first treatment at the doses specified. Mice were administered once a day on Days 1 to 3. At day 4, blood was taken from their tail vein, and thin blood film was prepared therefrom. After Giemsa stains, it was examined whether they were infected or not, with a microscope. The lines used therein were P. berghei NY- drug sensitiveness, P. berghei PNY- py¬ ronaridine resistance, and P. berghei SANA- Artesunate resistance. The next day after completing the administration, the bloods were taken, thin blood film was prepared therefrom, and the frequency of occurrence was measured by a microscope to observe bacteriostatic effect. The bacteriostatic activity was assessed by comparing the level of parasitemia between the drug-treated group and the control group. The 50% and 90% effective levels (ED , ED ) were calculated from the log-drug concentration and bac-

50 90 ^&& teriostatic activity graph, and the resistance factor at the level of ED was calculated as follows: [223] [224] Resistance factor, I = ED / ED

90 90 drug resistance 90dmg sensitivity

[225]

[226] The results are shown in the following Tables 4 - 6.

[227]

[228] Table 4

[229] Bacteriostatic activity of artesunate on malaria protozoa in blood

[230] [231] Table 5 [232] Bacteriostatic activity of pyronaridine on malaria protozoa in blood

[233] [234] Table 6 [235] Bacteriostatic activity of pyronaridine/artesunate on malaria protozoa in blood

[236] [237] As shown in Tables 4 to 6, the administration of pyronaridine, and the combined administration of pyronaridine/artesunate (3:1) were highly effective on P. berghei. W hen pyronaridine alone was administrated to pyronaridine resistant strains, ED was 18 mg/kg/day. However, in the combined administration, ED was reduced to 11 mg/ kg/day. Also, when artesunate alone was administrated to pyronaridine resistant strains, ED was 60 mg/kg/day. However, in the combined administration, ED was

90 90 reduced to 11 mg/kg/day. These results confirmed possibility that the combined admin¬ istration enables use of a smaller dose of pyronaridine and artesunate against py¬ ronaridine resistant strains.

[238] [239] Experimental Example 5: Repeated- Dose Toxicity Test [240] [241] A 4 weeks repeated-dose toxicity test in rats was performed based on the Toxicity Test Standard in the Korea Institute of Toxicology, which is the toxicity test GLP or¬ ganization authorized by the OECD. As a result, the NOAEL (No Observed Adverse Effect Level) of pyronaridine was 23mg/kg, and the clear effect level was 210m/kg, in both male and female . And, the NOAEL of artesunate was 3 0mg/kg in both male and

female. However, in the combined administration of Example 1, the NOAEL was 40mg/kg in both male and female, and the clear effect level was 360mg/kg. These results show that the repeated-dose toxicity for 4 weeks could be diluted by combination administration.

[242]

INDUSTRIAL APPLICABILITY

[243] The pharmaceutical composition of the present invention can provide antimalarial agent showing remarkable treatment effect on the resistant strains, with securing the drug stability and having synergic effect of the two drugs.