JP2006321728A - New anti-leishmania pharmaceutical composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel anti-leishmania pharmaceutical composition. <P>SOLUTION: The anti-leishmania pharmaceutical composition comprises komaroviquinone represented by the formula and a pharmaceutically acceptable carrier. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel anti-Leishmania pharmaceutical composition.

  Leishmaniasis is one of the WHO-designated tropical diseases that is prevalent in 88 countries mainly in the tropics. Currently, about 12 million people are infected worldwide, and 1.5 million people are newly infected every year, and the number has increased rapidly over the past decade. Leishmania protozoa are biphasic and proliferate in the reticuloendothelial cells of human rodents, particularly macrophages, as amastigotes (non-flagellate), and enter the midgut of the salamander, a blood-sucking insect that mediates infection. Then it grows in a promastigote (pre-flagellar stage) type. The location of the lesion differs depending on the species or subspecies of the protozoa, and it can be divided into three types, skin type, mucosal type, and visceral type, depending on the pathological condition. There are many cases of lymphadenopathy and death. Recently, this visceral type is frequently recognized in the route of HIV (human immunodeficiency virus) infection, which is a new problem. For this reason, the development of a new chemotherapeutic agent for Leishmania is considered to be an important issue that urgently requires humankind.

Drugs conventionally used for the treatment of leishmaniasis are (1) pentavalent antimony agents typified by pentostam and glucan time as first-line drugs. However, there are many problems in terms of stability due to differences in susceptibility, side effects such as liver dysfunction and kidney damage, high price, and stability. (2) A diamidine derivative typified by pentamidine is used as a second-line drug when treatment with the antimony agent is not sufficient. (3) In addition, although antibacterial agents such as imidazole, triazole compounds and amphotericin B are also used, they are usually not as effective as antimony agents in terms of efficacy.
Thus, there is an urgent need to discover a novel anti-Leishmania active ingredient having a structure different from that of conventional anti-Leishmania drugs and to develop a novel anti-Leishmania drug.

  For these purposes, attempts have been widely made to search for anti-Leishmania active ingredients from among many medicinal plants. For example, Non-Patent Document 1 has excellent anti-Leishmania activity in an extract containing a quinone compound obtained from Millettia pendula, a leguminous plant in Myanmar, and Non-Patent Document 2 has an anti-Leishmania activity. Pterocin and athycene compounds, and diallyl heptane compounds obtained from birch plants are effective as an anti-Leishmania active ingredient. Non-patent document 3 describes that natural or synthetically obtained lycochalcone A is an effective anti-Leishmania drug. Non-patent document 4 shows that the canthinone-based indole alkaloids contained in Zanthoxylum chiloperone var. Angustifolium, an anti-Leishmania activity, have anti-leishmania activity. Of sesquiterpenes in Elephantopus mollis, a medicinal plant in Brazil It is described that the compound has anti-Leishmania activity.

In addition, komaroviquinone, a quinone-type diterpene used in the present invention, is effective as an anti-trypanosoma active ingredient against trypanosoma cruzi, a pathogenic parasite of Chagas disease, a tropical disease that is prevalent in Central and South America. This is described in Non-Patent Document 6.
M. Takahashi, H. Fuchino et al., “In Vitro Screening of Leishmanicidal Activity in Myanmar Timber Extracts”, Biol. Pharm. Bull. 27 (6), 921-925 (2004). M. Takahashi, H. Fuchino et al., “In Vitro Leishmanicidal Activity of Some Scarce Natural Products”, Phytother. Res. 18, 573-578 (2004). M. Chen, SB Christensen et al., “Antileishmanial Activity of Licochalcone A in Mice Infected with Leishmania major and in Hamsters Infected with Leishmania donovani”, Antimicrob. Agents Chemother., 38, 1339-1344 (1994). ME Ferreiraa, A. Rojas de Ariasa et al., “Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone var. Angustifolium“, J. Ethnopharmacol., 80, 199-202 (2002). H. Fuchino, T. Koide et al., “New Sesquiterpene Lactones from Elephantopus mollis and Their Leishmania Activities”, 67, 647-653 (2001). N. Uchiyama, F. Kiuchi et al., “New Icetexane and 20-Norabietane Diterpenes with Trypanocidal Activity from Dracocephalum komarovi” J. Nat. Prod. 66, 128-131 (2003).

  An object of the present invention is to provide a novel anti-Leishmania agent.

で表されるコマロビキノンおよび医薬的に許容される担体を含む抗リーシュマニア医薬組成物に関する。 The present inventors have intensively studied to achieve the above object, and found that the quinone-type diterpene, komaroviquinone, has anti-leishmania activity, and based on this finding, completed the present invention. It has been made.
That is, the present invention provides compounds of formula I:

The anti-leishmania pharmaceutical composition containing the komabiquinone represented by these, and a pharmaceutically acceptable carrier.

  By using the anti-Leishmania pharmaceutical composition of the present invention, leishmaniasis in mammals including humans and dogs can be effectively treated.

  Hereinafter, in order to explain the present invention specifically, an example of producing komabiquinone, which is an active ingredient of the anti-Leishmania pharmaceutical composition of the present invention, is carried out to clarify the effect of the anti-Leishmania pharmaceutical composition of the present invention. Examples and comparative examples, and formulation examples of the pharmaceutical composition of the present invention will be described. However, the present invention is not limited to these examples.

Production Example 1
Production Method of Comalobiquinone The comalobiquinone can be prepared by extracting and isolating a plant, specifically, Drakocephalum komarovi, which is a medicinal plant of Uzbekistan, as a raw material. D. komarovi is called “buzbosh” in the field, and it is decocted and used as a blood lowering and anti-inflammatory action (see Non-Patent Document 6).
When isolating and preparing komabiquinone from D. komarovi, D. komarovi used as a raw material may be whole plant or a part thereof. The preparation of the comalobiquinone is not limited. For example, the whole plant of D. komarovi or a part thereof can be extracted and fractionated with a solvent containing an organic solvent (see Non-Patent Document 6).

1.6 kg of dried D. komarovi whole plant was finely divided and extracted three times for each solvent in the order of hexane and ethyl acetate overnight at room temperature. Thereafter, these were filtered and the filtrate was concentrated to obtain 22.2 g of a solid (hereinafter referred to as “hexane extract”) and 67.3 g of a solid (hereinafter referred to as “ethyl acetate extract”). Further, each of the hexane extract and the ethyl acetate extract was subjected to silica gel column chromatography, and eluted in the order of hexane-acetone (10: 1, 8: 1, 6: 1, 4: 1, 0: 1) and methanol. Six fractions were obtained from each extract. Among these, hexane-acetone (8: 1 and 6: 1) elution fraction (5.6 g) of hexane extract and hexane-acetone (6: 1) elution fraction (4.6 g) of ethyl acetate extract were used. These were combined and subjected to silica gel column chromatography again to obtain a fraction eluted with hexane-ethyl acetate (6: 1). Further, fractionation was repeated (chloroform-acetone (100: 1), benzene-ethyl acetate (30: 1), and HPLC (YMC Pack SIL-06)) and eluted with hexane-ethyl acetate (5: 1). ), An orange oily comalobiquinone (124 mg) was obtained.
The data results obtained for the obtained comalobiquinone are as follows.
[α] ²⁵ _D + 34.2 ° (c 1.86, CHCl ₃ ); UV (MeOH) λ _max (log ε) 366 (2.89), 272 (3.84) nm; IR (KBr) v _max 3410, 2947, 1651, 1600 , 1458 cm ^-1 .
・ EIMS : m / z 360 [M ⁺ ] (23), 342 (8), 316 (100), 301 (30), 273 (11), 247 (20), 221 (19).
・ HREIMS : m / z 360.1934 (calcd for C ₂₁ H ₂₈ O ₅ , 360.1929).
・ ¹ H NMR (CDCl ₃ , 500 MHz) δ: 5.99 (1H, s, O H ), 3.98 (1H, s, O Me ), 3.23 (1H, sep, J = 7.0 Hz, 15), 2.55 (1H , d, J = 19.6 Hz, 20b), 2.30 (1H, dd, J = 12.8, 7.76 Hz, 6a), 2.26 (1H, d, J = 19.6 Hz, 20a), 2.04 (1H, dd, J = 12.8 , 7.8 Hz, 6b), 2.03 (1H, overlap, 2b), 1.73 (1H, overlap, 2a), 1.71 (1H, t, J = 8.2 Hz, 5), 1.60 (1H, m, 1), 1.59 ( 1H, overlap, 3b), 1.18 (1H, d, J = 7.3 Hz, 16), 1.18 (1H, d, J = 7.0 Hz, 17), 1.15 (1H, dd, J = 11.3, 5.8 Hz, 3a) , 0.95 (1H, s, 18), 0.86 (1H, s, 19).
・ ¹³ C NMR (CDCl ₃ , 125 MHz) δc: 189.1 (14), 183.6 (11), 156.1 (12), 142.1 (8), 138.9 (9), 137.0 (13), 100.9 (7), 79.3 ( 10), 61.1 (OMe), 51.4 (5), 45.7 (6), 39.0 (20), 32.0 (4), 31.2 (3), 30.3 (18), 29.8 (2), 27.0 (19), 24.3 ( 15), 20.4 (16), 20.4 (17), 15.6 (1).

Example 1
Measurement of anti-Leishmania activity in vitro In order to evaluate the anti-Leishmania activity of a specimen, the promastigote (preflagellar stage) and amastigote (non-flagellar stage) of Leishmania major ATCC 50122 strain were used in vitro. The growth inhibition rate of Leishmania parasites was measured.
For the Promastigote (preflagellate stage) type of Leishmania parasite, the medium was prepared by adding 20% fetal bovine serum to Schneiders medium. The protozoa were cultured in an incubator at 26 ° C. For the amastigote (flagellar stage) type, mouse intraperitoneal macrophages were first added to a 24-well plate and cultured in an incubator at 37 ° C. for 4 days in an atmosphere of 5% carbon dioxide. Thereafter, macrophages were infected with promastigotes (pre-flagellar phase), and further cultured under the same conditions to change the morphology to amastigote (non-flagellar phase), and then washed three times. Furthermore, after adding a culture medium and recultivating, it used for the measurement.

Specifically, the growth inhibition rate of Leishmania parasites was measured as follows.
The specimen was dissolved in MeOH and diluted to various concentrations to prepare test samples. This test sample was added to a 24-well plate to which the culture medium of Leishmania parasite had been added, and the final concentration was 2.0, 1.0, 0.5, 0.25, 0.1, 0.05 μM. Promastigotes (pre-flagellar stage) were cultured at 26 ° C., and the number of viable protozoa was determined under a microscope every 2 hours and compared with a control. The amastigote (non-flagellar stage) was cultured in an incubator at 37 ° C. for 24 hours in an atmosphere of 5% carbon dioxide, then observed under a microscope and compared with a control.
As a result, comarobiquinone began to inhibit the growth of promastigote (preflagellate stage) after 4 hours at a concentration of 2.0 μM, and inhibited growth of protozoa by 50% after 8 hours and 95% after 24 hours.
In the case of amastigote (flagellar stage), the protozoa completely disappeared at a concentration of 1.0 to 2.0 μM, but at the same time, it also affected macrophages. At concentrations of 0.5 μM and 0.25 μM, the protozoa completely disappeared and had no effect on the cytoplasm of macrophages. On the other hand, at a concentration of 0.1 μM, the number of protozoa in macrophages was only reduced. At a concentration of 0.05 μM, there was no effect on the protozoa (FIG. 1).

Example 2
Measurement of in vivo anti-leishmania activity of specimens In order to evaluate the in vivo anti-leishmania activity of specimens, the therapeutic effect was examined using a mouse model of cutaneous leishmaniasis.
As a comparison with the sample, komabiquinone, a positive control, pentostam, was used.
Comalobiquinone and pentostam were dissolved in DMSO and sterilized purified water, respectively.
Infected the right footpad of the 8th week mouse (Balb / C, female, 20 mice) with the promastigote (preflagellar stage) of Leishmani major ATCC 50122 strain. The size of the left footpad was used as a control. Three weeks after the infection, it was confirmed that the site of infection of the right footpad of the mouse was enlarged, and 200 μl of each specimen was administered (comarobiquinone: 4.0 mg / kg, pentostam: 20 mg / kg, the recommended concentration for WHO for humans). . A 26 gauge hypodermic needle and a 1 ml syringe were used for intraperitoneal administration, and a round injection needle was used for oral administration.

In the administration schedule, after 4 consecutive days of administration, administration was stopped for 3 days, and thereafter, administration was continued for 3 days. The size of the non-administration group was subtracted from the size of the right footpad 14 days after the start of administration, the difference was compared, the growth inhibition rate% in the presence of the specimen was determined according to the following formula, and the anti-Leishmania activity of each specimen was evaluated.

Growth inhibition rate (%) = {1− (difference in footpad size after drug administration / difference in footpad size before drug administration)} × 100

The results are shown in Table 1 and FIG.

Comalobiquinone showed growth inhibitory action of about 60% by oral administration and 30-60% by intraperitoneal administration. Pentostam, which was used as a positive control test, was administered 4 times the amount of comarobiquinone. In both oral and intraperitoneal administrations, 4 out of 6 animals showed ulcers, and the remaining 2 animals compared to comarobiquinone. The growth inhibition rate was low.
From this result, anti-leishmania activity was observed for comalobiquinone in both in vitro and in vivo tests. That is, it can be seen that comalobiquinone can be an effective component of a new anti-Leishmania drug.

  Comarobiquinone represented by Formula I can be used in various dosage forms for administration purposes in view of its pharmacological action. The pharmaceutical composition of the present invention can be produced by uniformly mixing an effective amount of a specific compound in the form of a free or acid addition salt as an active ingredient with a pharmacologically acceptable carrier. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are preferably in unit dosage forms suitable for oral or injection administration. Any useful pharmaceutically acceptable carrier can be used in preparing the compositions in oral dosage form. For example, oral liquid preparations such as suspensions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, and alkyl paraffin. It can be produced using a preservative such as hydroxybenzoate, and flavors such as strawberry flavor and peppermint.

  Powders, pills, capsules and tablets are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxypropylcellulose, gelatin Etc., a surface active agent such as a fatty acid ester, and a plasticizer such as glycerin. Tablets and capsules are the most useful unit oral dosages because they are easy to administer. When manufacturing tablets and capsules, solid pharmaceutical carriers are used.

Moreover, the solution for injection can be prepared using a carrier comprising a salt solution, a glucose solution or a mixture of salt water and a glucose solution.
The medicament of the present invention is administered orally or by injection, and its effective dose is 4-8 mg / kg / day, and the frequency of administration is preferably about 3 times a day.

Formulation Example 1: Tablets Tablets are prepared according to the following composition by a conventional method.
Comalobiquinone 80 mg
Lactose 60mg
Potato starch 30mg
Polyvinyl alcohol 2mg
Magnesium stearate 1mg
Tar pigment Trace amount

Formulation example 2: Powder Powder comarobiquinone which is prepared by the usual method and having the following composition 80 mg
Lactose 275mg

Formulation example 3: syrup formulation 300 mg of active ingredient for preparing a syrup formulation with the following composition by a conventional method
40g refined white sugar
Methyl paraoxybenzoate 40mg
Propyl paraoxybenzoate 10mg
Strawberry flavor 0.1ml
Water is added to make a total volume of 1000 ml.

  The anti-Leishmania pharmaceutical composition of the present invention can be used as a pharmaceutical product.

The effect of comarobiquinone on Leishmania amastigote type is shown. The in vivo effect of comalobiquinone is shown.

Claims (1)

Formula I:

An anti-Leishmania pharmaceutical composition comprising komabiquinone represented by the formula: and a pharmaceutically acceptable carrier.

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