WO2011132943A9

WO2011132943A9 - Composition for treatment of acute pseudomembranous colitis

Info

Publication number: WO2011132943A9
Application number: PCT/KR2011/002845
Authority: WO
Inventors: Jae Sam Hwang; Eun Yeong Yun; Seong Ryeol Kim; Seong Hui Nam; Mi Yeong Ahn; Yeong Cheol Choi; Ho Kim; Hyo Jung Nam; Jin Ku Kang; In Woo Kim
Original assignee: Republic Of Korea(Management : Rural Development Administration); Daejin University Center For Educational Industrial Cooperation
Priority date: 2010-04-22
Filing date: 2011-04-20
Publication date: 2012-04-05
Also published as: KR101021226B1; WO2011132943A3; WO2011132943A2

Abstract

Disclosed is a composition for treatment of pseudomembranous colitis including a coprisin peptide derivative CopA3 (HL) having the following amino acid sequence, as an active ingredient: L-L-C-A-L-R-K-K. According to the present disclosure, the coprisin peptide derivative CopA3 (HL) does not show antibiotic effects upon effective colon microorganisms such as Bifidobacterium, Lactobacillus, or the like, while having antibiotic effects specific to Cd bacteria as a pathogenic microorganism. Effects of CopA3 (HL) for inhibiting growth of Cd bacteria have been verified through animal testing, and Cd bacteria-derived intestinal inflammation and villous necrosis processes are effectively relieved.

Description

COMPOSITION FOR TREATMENT OF ACUTE PSEUDOMEMBRANOUS COLITIS

Embodiments of the present invention relate to use of a coprisin peptide derivate CopA3 (HL) as a novel drug for treatment of Clostrdium difficile (Cd) bacteria-derived acute pseudomembranous colitis which is generally treated using limited number of conventional antibiotics such as metronidazole and vancomycin.

L-L-C-I-A-L-R-K-K

The present invention suggests that a coprisin peptide derivate CopA3 (HL) obtained from Copris tripartitus exhibits very strong antibiotic effects upon Cd bacteria inducing acute pseudomembraneous colitis. Cd bacteria have been discovered as pathogenic bacteria of acute pseudo-membraneous colitis since late 1970s (Kelly CP and LaMont JT. N Engl J Med, and LaMont JT. N Engl J Med 2008; 359: 1932). Among antibiotic related diarrhea, it was demonstrated that about 15 to 20% is caused by Cd bacteria as pathogenic bacteria and symptoms thereof vary from silent bacteria carrier conditions to serious pseudomembranous colitis (Pothoulakis C and LaMont JT. Gastroenterol Clin North Am 1993; 22:623). It was also reported that Cd bacteria are found in about 5% of healthy adults, about 30% of healthy children and about 30% of resident hospital patients, from stool culture. Until now, with regard to a pathogenic mechanism for diarrhea and pseudomembranous colitis caused by Cd bacteria, decrease in number of normal intestinal bacteria flora due to use of antibiotics has been considered a primary cause of such diseases. It has been known that decrease in number of effective intestinal microorganisms such as lactic-acid bacteria allows abnormal proliferation of Cd bacteria, enables colonized Cd bacteria to secrete toxic proteins (toxin A and toxin B), in turn causing cell death (apoptosis) of colon epithelial cells or increasing cell permeability, and ultimately causing continuous or chronic inflammation (Lamont JT. Trans Am Clin Climatol Assoc 2002; 113:167-80). Toxin A having a molecular weight of 300 kDa was verified to cause intestinal mucosa damages when injected into animal and/or human genografts, and was also demonstrated to cause abnormality in cytoskeletons including, for example, acute inflammatory response and actin. Although toxin B is known to be more than 100 times more cytotoxic than toxin A, it was reported that, when injected into the colon of a rat or rabbit, toxin B does not cause increase in cell permeability or apoptosis of intestinal epithelia, unlike toxin A (Castagliuolo I and Kelly CP. Am J Physiol 1997; 273: G333-41). However, recent documents have reported that toxin B injection into human colon tissues causes acute inflammation.

This means that both toxins A and B may act as a major regulator of pseudomembranous colitis within the human colon.

Clinical symptoms of pseudomembranous colitis may include high fever and mucinous or watery diarrhea. Serious abdominal pain and decreased bowel movement may also occur, and it is well known that leukocytosis of more than 50,000/m² and hypoalbuminemia caused by protein-losing enteropathy may be monitored during blood test. It was also reported that shock, pleural effusion, ascites, megacolon, colon perforation and peritonitis, secondary sepsis, or the like may be caused in serious cases and, occasionally, severe morbidity and death may result (Kelly CP and LaMon JT. N Engl J Med 2008; 359: 1932). A diagnostic method of pseudomembranous colitis may include detection of toxic protein in stool by cell culture. Since toxin A or B is detected in 95% of patients suffering from pseudomembranous colitis, Cd bacteria may be easily identified as pathogenic bacteria. In addition, enzyme immunoassay (EIA) having economic advantages and enabling rapid confirmation of results may also be applied.

Treatment of pseudomembranous colitis generally needs conservative treatment such as fluid therapy, correction of electrolyte imbalance, etc. combined with stopping administration of antibiotics causing this disease. In the case where the patient is in serious condition, vancomycin is often administered. In particular, it has been disclosed that, if the disease is due to toxins generated by intestinal bacteria rather than cell invasion by pathogenic bacteria, oral administration of metronidazole or vancomycin may provide beneficial results (Kelly CP and LaMont JT. N Engl J Med 2008; 359: 1932). Recent documents report that vancomycin exhibits excellent treatment efficiency, compared to metronidazole (Kelly CP and LaMont JT. N Eng. J Med 2008; 359: 1932). If oral administration is not possible, rectal injection may be recommended. Moreover, it was reported that yogurt or Lactobacilli administered through intestinal injection to normalize Escherichia coli flora may be an effective treatment.

However, about 30% of various bacteria have recently been reported to be tolerant to metronidazole as a clinically selective antibiotic, thus encountering problems. Although vancomycin may be used as an antibiotic for treatment, this drug is believed to be a final solution to treat all infectious diseases derived from gram-positive bacteria, thus being restrictedly used in clinical applications. In consideration of the foregoing problems, there is a strong need to develop novel antibiotics.

An aspect of the present invention is to provide a novel antibiotic for treatment of Cd bacteria-derived acute pseudomembranous colitis, by verifying whether an antibacterial peptide, that is, a coprisin peptide derivative CopA3 (HL) extracted from insects shows selective antibiotic effects upon Cd bacteria, and further confirming non-toxicity upon general cells of mammals including colon epithelial cells.

That is, an object of the present invention is to use a coprisin peptide derivative CopA3 (HL) for treatment of Cd bacteria-derived pseudomembranous colitis, which is now cured using limited number of antibiotics such as metronidazole and vancomycin.

In order to accomplish the foregoing objects, the present invention provides a composition for treatment of pseudomembranous colitis, including a coprisin peptide derivative CopA3 (HL) having the following amino acid sequence, as an active ingredient.

L-L-C-I-A-L-R-K-K

According to the present invention, Cd bacteria causing pseudomembranous colitis are cultured and treated using a coprisin peptide derivative CopA3 (HL), followed by confirmation of antibiotic effects thereof. For this purpose, Cd bacteria are inoculated and incubated (at 37℃ for 5 days) using a meat broth based culture solution, the incubated material is treated using an L-type coprisin peptide derivative CopA3 (HL) in a concentration of 1㎍/ml, followed by measuring variation in growth rates at an absorbance of 600 nm. Since orally administered peptide is easily degraded by digestive enzymes, D-type peptide of the coprisin peptide derivate CopA3 is additionally synthesized and examined. In order to determine a minimum concentration of the coprisin peptide derivative CopA3 expressing strong antibiotic effects upon Cd bacteria, the cultured material is treated using the coprisin peptide derivative CopA3 (HL) at concentrations of 1㎍ and 5㎍, respectively, while maintaining the foregoing conditions, followed by measuring decrease in growth rates. Thereafter, effects of the coprisin peptide derivative CopA3 (HL) are compared with those of existing antibiotics such as clindamycin, kanamycin, vancomycin, or the like.

General drugs having strong antibiotic effects upon normal intestinal bacteria flora (i.e., lactic-acid bacteria) are substantially less suitable as medical agents, although they exhibit excellent antibiotic efficiency to pathogenic microorganisms. Accordingly, attempts to identify that a coprisin peptide derivative CopA3 (HL) showing strong antibiotic effects upon Cd bacteria is non-toxic to lactic-acid bacteria have been conducted. For this purpose, colon microorganisms are isolated from a mouse and treated using 1㎍/ml of coprisin peptide derivative CopA3 (D-type) during growth thereof. From toxicity test, the treated microorganisms are verified to be non-toxic to normal intestinal bacteria flora. Specific lactic-acid bacteria such as Bifidobacterium, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus bulgaricus, etc. are respectively cultured and treated using the coprisin peptide derivative CopA3, followed by repeated toxicity testing. In this regard, vancomycin, clindamycin and kanamycin are also used to treat the microorganisms, parallel with treatment using the coprisin peptide derivative CopA3 (HL). Effects of these drugs are compared to the coprisin peptide derivative CopA3 (HL). Next, using a model in which pre-treatment using composite antibiotic and infection of Cd bacteria are executed to induce pseudomembranous colitis, animal testing is implemented. A composite antibiotic (including 0.4mg/ml of kanamycin, 0.035mg/ml of gentamicin, 850U/ml of colistin, 0.215mg/ml of metronidazole and 0.045mg/ml of vancomycin) is mixed with water and fed to Cd bacteria-treated mice (n=12) at 4 weeks of age for 3 days, followed by providing water alone for 1 day. Then, clindamycin is administered i.p. in an amount of 10mg/kg to the mice.

Following this, Cd bacteria (1×10⁵ cells) are directly fed into the stomach of each mouse using a sonde. The coprisin peptide derivative CopA3 (HL) (1㎍/ml) is added to drinking water and provided to the mice, followed by tracing weight loss of the mice and lethal dose.

From the dead mice, the intestines are extracted and compared in relation to damage of brush border membrane and variation in inflammatory cytokine secretion.

TABLE 1 illustrates a process of establishing a pseudomembranous colitis model by continuously exposing an antibiotic composite and Cd bacterial infection to mice.

Table 1

Table 2

Peptide	Amino acid sequence (9)
CopA3 (HL)	L-L-C-I-A-L-R-K-K

The coprisin peptide derivative CopA3 (HL) of the present invention may noticeably reduce acute enteritis in the colon of mice and exhibit selective antibacterial effects upon only pathogenic microorganisms, that is, Cd bacteria, other than normal intestinal bacteria flora such as Bifidobacterium, Lactobacillus, etc. Intestinal villous necrosis and intestinal bleeding caused by C. difficile may be inhibited by treatment using the coprisin peptide derivative CopA3 (HL).

These and/or other aspects of the invention will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 illustrates strong antibiotic effects of a coprisin peptide derivative CopA3 (HL) upon Cd bacteria;

FIG. 2 illustrates antibiotic effects depending upon concentrations of a coprisin peptide derivative CopA3 (HL) upon Cd bacteria;

FIG. 3 illustrates verified results of antibiotic effects of a coprisin peptide derivative CopA3 (HL) as well as existing antibiotics (vancomycin, clindamycin, kanamycin), respectively, upon Cd bacteria;

FIG. 4 demonstrates non-toxicity of a coprisin peptide derivative CopA3 (HL) upon intestinal microorganisms isolated from the colon of a mouse;

FIG. 5 demonstrates non-toxicity of a coprisin peptide derivative CopA3 (HL) upon effective microorganisms;

FIG. 6 demonstrates non-toxicity of a coprisin peptide derivative CopA3 (HL) upon a variety of intestinal lactic-acid bacteria (Bifidobacterium, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus bulgaricus, etc.);

FIG. 7 illustrates results of treatment using a coprisin peptide derivative CopA3 (HL) that reduces mice mortality caused by Cd bacteria infection, while recovering weight loss; and

FIG. 8 illustrates effects of a coprisin peptide derivative CopA3 (HL) that inhibits Interleukin-6 (inflammatory cytokine) as well as damage of brush border membranes in the small intestine/colon of each mouse suffering from acute pseudomembranous colitis derived from Cd bacteria infection.

Hereinafter, exemplary embodiments of the present invention will be described in detail in conjunction with the accompanying drawings. Those skilled in the art will appreciate that such embodiments are proposed for illustrative purposes and the scope and spirit of the present invention disclosed in the appended claims are not particularly limited to the foregoing embodiments. Although preferred embodiments are not enclosed, it would be apparent to those skilled in the art that an enteritis remedy composition containing an antimicrobial peptide according to the present invention, as an active ingredient, may be manufactured.

EXAMPLE 1

Coprisin peptide derivative CopA3 (HL) expresses strong antibiotic effects upon Cd bacteria as pathogenic bacteria causing acute pseudomembranous colitis

It is well known that intestinal pathogenic bacteria inducing acute pseudomembranous colitis, that is, C. difficile (Cd bacteria) are gram-positive bacteria and, when the number of normal bacterial flora is decreased by antibiotic treatment, colonized to ultimately secrete toxin, thus causing acute intestinal inflammation such as enteritis. After inducing an immune response, in order to verify antibiotic effects specific to Cd bacteria, coprisin peptide derivative CopA3 (HL) based peptides isolated from Copris tripartitus Waterhouse were used.

For this purpose, after placing 6.5g of meat broth in 50ml of distilled water in a vial, the vial was tightly sealed to prevent air from permeating into the vial and subjected to sterilization, thereby preparing a Cd bacteria culture medium. Anaerobic Cd bacteria were inoculated in a predetermined amount on a culture broth without air permeation and incubated therein at 37℃ for 5 days. In this case, D-type and L-type coprisin peptide derivatives CopA3 (HL) in a concentration of 1㎍ were respectively used to treat the cultured bacteria, followed by further culturing. Using an absorption meter, variation in growth rate of the bacteria was traced at 600nm and results thereof were compared. As shown in FIG. 1, for bacteria cultured under anaerobic conditions, it was confirmed that the growth rate exhibits an S-shaped pattern and increases exponentially.

However, as the foregoing coprisin peptide derivative CopA3 (HL) was simultaneously added to the bacteria during culture, it was found that growth rate is significantly decreased. In the case where the peptide derivative CopA3 (HL) is orally administered, digestive enzymes actively degrade the same, thus limiting application thereof as a cure remedy. Therefore, after further synthesizing D-type peptides, antibiotic effects thereof to Cd bacteria were compared to those of L-type peptides. As shown in FIG. 1, it was found that both L-type and D-type coprisin peptide derivatives CopA3 (HL) sufficiently inhibit growth of Cd bacteria. The figures show that the growth rate of Cd bacteria begins to rapidly increase from Day 3 and/or 4. On the other hand, for a group of Cd bacteria treated using the coprisin peptide derivative CopA3 (HL), increase in growth of Cd bacteria was not observed (FIG. 1). These results demonstrate that Cd bacteria antibiotic effects are expressed earlier at the beginning of treatment using the coprisin peptide derivative CopA3 (HL). Furthermore, it was confirmed that the D-type coprisin peptide derivative CopA3 exhibits stronger antibiotic effects than the L-type (FIG. 1). Such results demonstrate that D-type coprisin peptide derivative CopA3 possesses not only principal chemical properties of L-type coprisin peptide derivative, but also exhibits improved antibiotic effects, thereby having enhanced applicability as a cure remedy. In addition, FIG. 1 is a photograph of a culture test tube. For the group of Cd bacteria treated using the coprisin peptide derivative CopA3, turbidity was significantly reduced.

EXAMPLE 2

Determination of minimum antibiotic concentration of coprisin peptide derivative CopA3 to express antibiotic effects upon Cd bacteria

In order to determine a minimum concentration of the coprisin peptide derivative CopA3 to express antibiotic effects upon Cd bacteria, coprisin peptide derivatives at different concentrations were used to treat Cd bacteria, then, variation in growth rates of Cd bacteria was measured. Since the D-type coprisin peptide derivative CopA3 was determined to have stronger antibiotic effects than the L-type derivative from the previous results, subsequent experiments were implemented using the D-type coprisin peptide derivative CopA3. From results of such experiments, it was confirmed that Cd bacteria antibiotic effects observed when using the coprisin peptide derivative CopA3 at a concentration of 1㎍/ml are substantially similar to those obtained by treatment using the coprisin peptide derivative CopA3 at 5㎍/ml (FIG. 2). In other experiments conducted using the coprisin peptide derivative CopA3 at different concentrations of less than the above levels, it was found that a minimum concentration of the coprisin peptide derivative CopA3 to express Cd bacteria antibiotic effects is 1㎍/ml (FIG. 2). Further, a control group (con) refers to the initial number of bacteria measured during Cd bacteria inoculation. The right image is a photograph of the culture test tube (FIG. 2).

EXAMPLE 3

Antibiotic effects of coprisin peptide derivative CopA3 (HL) upon C. difficile bacteria

Clindamycin and kanamycin clinically used to treat a wide range of bacterial infections, and vancomycin known as a remedy for treating Cd infection-induced pseudomembranous colitis were subjected to comparison of antibiotic effects with CopA3 (HL) of the present invention. Compared to the coprisin peptide derivative CopA3 (HL) providing 60% decrease in growth rate, clindamycin showed 62% decrease in growth rate which is substantially similar to that of CopA3 (HL). On the contrary, antibiotic effects of vancomycin well known as the most effective remedy for Cd bacteria treatment were relatively low (30%). For kanamycin, Cd bacteria antibiotic effects were substantially not observed.

These results demonstrate that the coprisin peptide derivative CopA3 (HL) of the present invention has much stronger antibiotic effects specific upon Co bacteria than existing antibiotics. Specifically, it is notable that the coprisin peptide derivative CopA3 (HL) exhibits superior efficacy, compared to vancomycin.

EXAMPLE 4

Antibiotic effects of coprisin peptide derivative CopA3 (HL) to effective microorganisms including lactic-acid bacteria

Although drugs having high antibiotic effects upon intestinal lactic-acid bacteria, which are known to prevent proliferation of pathogenic microorganisms or inhibit toxin secretion, express favorable antibiotic effects upon pathogenic microorganisms, there are difficulties in practical utilization thereof. Therefore, for a coprisin peptide derivative CopA3 (HL) with strong Cd bacteria antibiotic effects, it is necessary to determine whether this drug has selective non-toxic efficacy upon effective intestinal microorganisms including lactic-acid bacteria. For this purpose, research workers attempted to examine effects of the coprisin peptide derivative CopA3 (HL) in relation to growth rate of colon microorganisms of a mouse.

Firstly, each of CD1 mice at 4 weeks of age was subjected to abdominal incision and stools were randomly extracted from different sites in the colon. While retaining anaerobic conditions, the extracted stool samples were homogenized in an LB broth medium and the obtained samples were cultured in equal amounts under anaerobic and aerobic conditions, respectively. Simultaneously, each sample was treated using 1㎍/ml of D-type coprisin peptide derivative CopA3 (HL), followed by comparing variation in growth rates of the above material with a control group. When the stool of the mouse was cultured for 24 hours, as shown in FIG. 4, it was found that bacteria grow to 3 times or more the initial number of bacteria to be cultured. However, it was confirmed that, even if the bacteria are treated using the coprisin peptide derivative CopA3 (HL), the growth rate of bacteria is not decreased. Meanwhile, it can be seen that both D-type and L-type coprisin peptide derivatives CopA3 (HL) do not influence growth of intestinal microorganisms.

EXAMPLE 5

Toxicity test of coprisin peptide derivative CopA3 (HL) upon effective microorganisms (EM bacteria)

Subsequently, after mixing EM bacteria including lactic-acid bacteria and culturing the same, the mixture was subjected to re-verification of effects of CopA3 (HL). For this purpose, mixed EM bacteria purchased from the Center for EM Research and Development, Jeonju University, were incubated in an LB broth medium, followed by comparison of effects of CopA3 (HL) on growth rate. The mixed EM bacteria may include, for example, yeast, lactic-acid bacteria, Aspergillus oryzae called ‘Koji bacteria,’ photosynthetic bacteria, and about 80 species of other microorganisms used for food fermentation or the like since ancient times. On the other hand, a control group was treated by simultaneously inoculating the mixed EM bacteria and adding 1㎍/ml of coprisin peptide derivative CopA3 (HL) thereto, then, after 24 hours, subjected to comparison of variation in growth rates through measurement of absorbance. By separating the coprisin peptide derivatives CopA3 into D-type and L-type derivatives, each was used at a concentration of 1㎍/ml, to treat the bacteria. Then, according to the procedures described above, the growth rate of the bacteria was measured. As shown in FIG. 5, it was confirmed after 3 days that the mixed EM bacteria inoculated on the LB broth medium are proliferated to 5 times or more the initial number of bacteria to be cultured. Like the results described above, treatment using the coprisin peptide derivative CopA3 (HL) did not influence growth of the mixed EM bacteria. It can be seen that neither D-type nor L-type coprisin peptide derivative CopA3 (HL) expressed antibiotic effects upon EM bacteria (FIG. 5). The foregoing results demonstrate that the coprisin peptide derivative CopA3 (HL) does not have cytotoxicity upon effective intestinal microorganisms and also suggest that the coprisin peptide derivative CopA3 (HL) expresses very unique antibiotic effects specific to Cd bacteria.

EXAMPLE 6

Coprisin peptide derivative CopA3 (HL) does not cause decrease in growth rate of Bifidobacterium, Lactobacillus delbrueckii subsp. Lactis, and Lactobacillus bulgaricus .

Since non-toxicity of the coprisin peptide derivative CopA3 (HL) was sufficiently verified from a liquid culture experiment using colon microorganisms of a mouse and a culture experiment using EM mixed bacteria, the present researchers attempted to trace variation in growth rate caused by stimulation of a coprisin peptide derivative CopA3 (HL) after culturing individual lactic-acid bacteria. For this purpose, Bifidobacterium, Lactobacillus delbrueckii subsp. Lactis and Lactobacillus bulgaricus were respectively distributed by the Rural Development Administration, separately inoculated on suitable media and incubated for 24 hours. Then, these bacteria were treated using a high concentration (5㎍/ml) of coprisin peptide derivative CopA3 (HL), followed by tracing variation in growth rates of the bacteria over time. For vancomycin (5㎍/ml), clindamycin (5㎍/ml) and kanamycin (5㎍/ml), respectively, the above procedures were repeated to treat the bacteria and effects thereof were compared to effects of the coprisin peptide derivative CopA3 (HL). As shown in FIG. 6, it was confirmed that Bifidobacterium, Lactobacillus delbrueckii subsp. Lactis and Lactobacillus bulgaricus were proliferated, respectively, to at least 8 times, 3 times and 2 times the initial number of bacteria to be cultured. As identified from the previous results, treatment using the coprisin peptide derivative CopA3 (HL) had absolutely no influence upon growth rate of bacteria. On the other hand, it was found that, although vancomycin did not decrease growth rates of Lactobacillus delbrueckii subsp. Lactis and Lactobacillus bulgaricus, this drug exhibited strong antibiotic effects upon Bifidobacterium. For clindamycin, strong antibiotic effects to the foregoing three kinds of bacteria were verified. Furthermore, kanamycin showed strong antibiotic effects upon Lactobacillus delbrueckii subsp. Lactis and Lactobacillus bulgaricus, except for Bifidobacterium. Such results demonstrate that the coprisin peptide derivative CopA3 (HL) expresses antibiotic effects specific to C. difficile only as a pathogenic bacterium while seldom influencing normal intestinal bacteria flora, thereby suggesting suitability of the foregoing derivative as a remedy for treatment of acute intestinal inflammation such as enteritis. By comparing the coprisin peptide derivative CopA3 (HL) with vancomycin, positive results from which therapeutic applicability of CopA3 (HL) higher than that of vancomycin may be assumed were obtained.

EXAMPLE 7

Influence of coprisin peptide derivative CopA3 (HL) upon acute enteritis of mouse derived from C. difficile infection

Since the coprisin peptide derivative CopA3 (HL) exhibits relatively selective non-toxicity upon effective intestinal microorganisms, whereas having strong Cd bacteria antibiotic effects, the present inventors attempted to verify whether treatment using the coprisin peptide derivative CopA3 (HL) leads to expression of anti-inflammatory efficacy, by directly infecting a mouse with Cd bacteria to cause acute enteritis then using this as an animal model of acute enteritis. A composite antibiotic (including 0.4mg/ml of kanamycin, 0.035mg/ml of gentamicin, 850U/ml of colistin, 0.215mg/ml of metronidazole and 0.045mg/ml of vancomycin) was mixed with water and fed to CD1 mice (n=12) at 4 weeks of age for 3 days, followed by providing water alone for 1 day. The next day, clindamycin was administered i.p. in an amount of 10mg/kg to the mice to thus induce additional antibiotic stimulation. Following this, Cd bacteria (1×10⁵ cells) were directly fed into the stomach of each mouse using a sonde. Treatment using the coprisin peptide derivative CopA3 (HL) was performed by diluting CopA3 (HL) in water to 1㎍/ml and providing the diluted drug to the mice with drinking water, 1 day before Cd bacteria injection, that is, the day when clindamycin was administered i.p. In this regard, in order to prevent colonic degradation, 1㎍/ml of D-type coprisin peptide derivative CopA3 (HL) was dissolved in drinking water and provided. Weight loss and mortality were compared and traced between the treated group and a control group given drinking water alone after Cd infection. From the next day after Cd bacteria injection, weight change, fecal consistency and lethal dose were investigated. As shown in FIG. 7, Cd bacteria injection induced rapid weight loss. 3 days after Cd bacteria injection, weight loss was significant and an average 2 to 3 g of weight loss was observed each day. On the contrary, for the treated group that received the coprisin peptide derivative CopA3 (HL) dissolved in drinking water, it was found that weight loss was noticeably reduced, thus beginning to recover. For the control group, weight loss was not considerable until 3 days after Cd bacteria injection. However, it was observed that mice given the corprisin peptide derivative CopA3 show noticeable weight recovery 4 days after Cd bacteria injection, in turn returning to original weight after 6 days. Meanwhile, for a control group of mice that received drinking water only after Cd bacteria infection, mortality was about 76%. However, mice given the coprisin peptide derivative CopA3 (HL) exhibited mortality of 16%. Feeding the coprisin peptide derivative CopA3 (HL) mixed with drinking water to mice remarkably inhibited villous necrosis in the small intestine (FIG. 8). It was also confirmed that expression of an inflammatory cytokine, Interleukin-6 (IL-6), is significantly decreased in the group fed with the coprisin peptide derivative CopA3 (HL) in drinking water after Cd bacterial infection, whereas the control group that received drinking water only exhibited relatively high IL-6 levels (FIG. 8).

The foregoing description suggests that a coprisin peptide derivative CopA3 (HL) having non-toxicity to intestinal bacteria flora may exhibit strong antibiotic effects upon Cd bacteria, to thereby efficiently relieve Cd bacteria-derived acute pseudomembranous colitis.

Claims

A composition for treatment of pseudomembranous colitis, including a coprisin peptide derivative CopA3 (HL) having the following amino acid sequence, as an active ingredient:

L-L-C-I-A-L-R-K-K
The composition according to claim 1, wherein the coprisin peptide derivative CopA3 (HL) is a regulator to inhibit pathogenic bacteria and selectively increases growth rate of normal bacterial flora in the small intestine and the colon.
The composition according to claim 1 or 2, wherein the coprisin peptide derivative CopA3 (HL) is D-type.