KR101970885B1 - Pharmaceutical composition for treating or preventing filariasis and screening methods therefore - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating or preventing filariasis, and a screening method therefor. And more particularly to a pharmaceutical composition for treating or preventing onchocerciasis which specifically binds to calumenin of a willowworm, and a method for screening a ligand library through a computer program thereof.
The pharmaceutical composition for the treatment of filariasis of the present invention is capable of treating a selective and selective treatment for the pathogen, unlike the conventional treatment for river blindness, and can reduce adverse effects on the human body, and can effectively kill adults.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating or preventing onchocerciasis and a method for screening the same,

The present invention relates to a pharmaceutical composition for treating or preventing filariasis and a method for screening the composition, and more particularly, to a pharmaceutical composition comprising a drug for treating onchocerciasis which specifically binds to calumenin of a willowworm, Lt; / RTI >

Lymphatic filariasis and onchocerciasis are the major tropical diseases infected by the wasp nematode. Onchocerciasis is transmitted via mosquitoes (Culex, Anopheles and Aedes spp.), And 974 million people in 54 countries are threatened with this disease (Cobo, 2016). Wuchereria bancrofti, Brugia malayi and Brugia timori are the main causes of onchocerciasis.

Chlamydia inlithiasis can result in limb swelling of the extremities (edema of the tissue) or elephantiasis in which the skin tissue condenses. Onchocerciasis = river blindness caused by onchocerca volvulus is spread in 31 African sub-Saharan Africa, three Latin American countries, and Yemen (WHO, 2016) by Simulium spp. Chronic infections lead to itching and skin damage and eye diseases that can lead to permanent blindness.

Methods for treating such onchocerciasis include mediator control and massive drug administration (MDA). Despite numerous efforts, however, control and treatment of the disease has not been successful due to the absence of a vaccine or the limited effect of drugs used in group drug administration (MDA). Currently, diethylcarbamazine (DEC), albendazole (ALB), and Ivermectin (IVM) are used in group drug administration methods (Hoerauf, 2008). In addition, two drugs, diethylcarbamazine, albendazole and ivermectin, can be administered concurrently with onchocerciasis due to lymphadenopathy. However, ivermectin is the only treatment drug for onchocerciasis, and resistance to drugs is generated (Cobo, Cupp et al., 2011).

In order to solve the above problems, it is necessary to develop a new drug targeting a new molecule. New drugs should have a good insecticidal effect on adult insect pests, have a long-term effect on insect pests, and have no serious side effects from parasite insects. It should also be safe for children, pregnant women and breastfeeding women.

Since cuticle nematodes are deeply involved in drug delivery from the outside, mutation cuticle defects are highly relevant for drug development (Fetterer and Rhoads, 1993; Geary et al., 1995; Ho et al., 1990; Sheehy et al., 2000, Thompson et al., 1993). Furthermore, the cuticle of the nematode, an extracellular matrix composed of collagen, is important for nematode development and survival. Thus, many enzymes and chaperones associated with cuticle development have been proposed as potential drug targets for parasitic nematodes (Frand et al., 2005; Page et al., 2014).

Recently, computer algorithms for predicting protein structure have been developed, enabling the prediction of the three-dimensional (3D) structure of the target protein from amino acid sequences (Zhang, 2008b). These predictive protein models can be applied to virtual compound screening and ligand binding studies on a computer, and using these new indicators to regenerate FDA-approved drugs into new drugs is an efficient, (Chong and Sullivan, 2007).

The present inventors have selected calumenin, a Ca ²⁺ binding protein present in the ER as a target protein for a new drug.

Therefore, in the present invention, the structural modeling results of calumenin and human (H. sapiens) calumenin of nematodes (C. elegans, B. malayi and O. volvulus) were analyzed and a drug for treatment of river blindness was developed through virtual screening of the target drug .

Conventional treatments for onchocerciasis have problems in that (i) they do not kill adult pathogens that excrete microcephaly, (ii) they cause resistance to drugs, and (iii) they have low specificity and selectivity for pathogens.

In order to solve this problem, the present inventors have selected calumenin as a specific target for insect pests and have developed a novel treatment for river insect pestis that specifically binds to calumenin of the pathogens.

The present inventor has identified a novel use for the treatment of onchocerciasis of Itraconazole, Idarubicin, Paliperidone and Daunorubicin, leading to the present invention.

The present invention relates to a pharmaceutical composition for the treatment of filariasis comprising at least one compound selected from the group consisting of Itraconazole, Idarubicin, Paliperidone and Daunorubicin, A pharmaceutical composition for prevention is provided.

The present invention also provides a screening method for treating onchocerciasis, which specifically binds to calumenin of the pathogen.

The present invention is not limited thereto, and other objects not mentioned may be clearly understood by those skilled in the art from the following description.

The present invention relates to a pharmaceutical composition for the treatment of filariasis comprising at least one compound selected from the group consisting of Itraconazole, Idarubicin, Paliperidone and Daunorubicin, Gt;

In addition, the compound of the present invention or a salt thereof can specifically bind to calumenin of onion flukes causing onchocerciasis.

In addition, the compound or a salt thereof can inhibit the production of cuticle of onion flies causing onchocerciasis.

The present inventors have classified chemicals exhibiting high affinity only to the nematode calumenin in order to select drug candidates having a relatively low binding affinity to human calumenin and having specificity for calumenin of the nematode. As a result, Itraconazole, Idarubicin, Paliperidone, and Daunorubicin hydrochloride were selected as candidates for the active substance, but not limited thereto.

-demethylase 효소를 억제하여, 항진균 효과가 있는 것으로 알려져 있다(Carrillo-Munoz et al., 2006; Gachotte et al., 1997; Henry et. al., 2000).Itraconazole is involved in sterol biosynthesis. Itraconazole has been shown to reduce lanosterol to ergosterol, an essential component of the cell membrane,

-demethylase enzyme (Carrillo-Munoz et al., 2006; Gachotte et al., 1997; Henry et al., 2000).

Idarubicin is a 4-demethoxy analogue of daunorubicin hydrochloride and has the same pharmacological properties as these (Bigioni et al., 1994; Fukushima et al., 1993; Hollingshead and Faulds, 1991).

Paliperidone has a therapeutic effect on schizophrenia by acting as a dopamine D2 receptor antagonist and antagonist to the serotonin 5-HT2 receptor (Cohen, 1994; He and Richardson, 1995; Leysen et al., 1994).

Daunorubicin is known to be a potent anticancer drug that inhibits DNA and RNA synthesis by inserting double-stranded DNA (Aubel-Sadron and Londos-Gagliardi, 1984). It also acts as an inhibitor of DNA topoisomerase II, inhibiting DNA replication, repair, and RNA and protein synthesis (Zunino and Capranico, 1990).

Preferably, the present invention provides a pharmaceutical composition for treating or preventing filariasis comprising itraconazole or a salt thereof.

There are complex sterol biosynthetic pathways in yeast, plants, insects and mammals, but free living and parasitic nematodes can not synthesize sterols and thus need to supplement the exogenous sterols for survival (Chitwood, 1999; Hieb and Rothstein, 1968). Since the sterol is a precursor of free living nematode and parasitic nematode ecdysteroid, the present inventor has determined that itraconazole, which is an inhibitor of sterol biosynthesis, has a high possibility as a treatment for onchocerciasis, and confirmed this through the screening method of the present invention.

The pharmaceutical composition of the present invention may further comprise at least one compound selected from the group consisting of Diethylcarbamazine (DEC), Mectizan, Albendazole and Ivermectine can do.

The onchocerciasis of the present invention may be lymphatic filariasis or onchocerciasis. In addition, the causal organism causing the onchocerciasis of the present invention may be, but is not limited to, Brugia malayi or Onchocerca volvulus.

The present invention provides a screening method of a drug for treating onchocerciasis which specifically binds to calumenin of the pathogen.

The screening method for treating onchocerciasis may include the following steps:

(a) predicting the 3D structure of californicin and calumenin of the wasp strand;

(b) measuring the 3D structural homology between calmainin of californium and calumenin of human;

(c) docking one or more ligands selected from a candidate material ligand library with calmenin and calumenin of the wasp strand; And

(d) analyzing the binding affinity between the docked ligand and calumenin to select a ligand that specifically binds to calumenin of the pathogen.

The 3D structure of the step (a) can be predicted through the I-TASSER program, but is not limited thereto.

The homology of step (b) may be measured by RMSD value through the PyMOL program, but is not limited thereto.

The docking in step (c) may use a PyRx computer program including AutoDock Vina, but is not limited thereto.

The binding affinity of step (d) may be calculated through binding energy between the ligand and calumenin through a computer program, but is not limited thereto.

The pharmaceutical composition for the treatment or prevention of filariasis of the present invention is capable of treating a selective and selective treatment for a pathogen, unlike the conventional treatment for river blindness, capable of reducing adverse effects on the human body, have.

In addition, the screening method of the medicament for treating onchocerciasis of the present invention can predict the effect on the treatment of onionism in advance, which can save enormous cost and time for drug development.

Figure 1 shows the amino acid sequences of calumenin (Ce_calu) of C. elegans, calumenin (Bm_calu) of B. malayi, calumenin (Ov_calu) of O. volvulus and calumenin (Hs_calu) of H. sapiens.
Fig. 2 shows the 3D structure of calumenin (Ce_calu) of C. elegans, calumenin (Bm_calu) of B. malayi, calumenin (Ov_calu) of O. volvulus and calumenin (Hs_calu) of H. sapiens.
Figure 3 shows the results of calculating the RMSD values through structural alignment between the canine and human calumenin.
Figure 4 shows the Itraconazole formula, 3D structure, and Itraconazole docking to CeCALU-1.
Figure 5 shows the results of the assay for the sensitivity of calu-1 (tm1783) mutations to Albendazole or Ivermectin.
Figure 6 shows the results of an experiment measuring the resistance of calu-1 (tm1783) to Itraconazole.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods and materials similar or equivalent to those described herein can be used to practice the embodiments disclosed herein.

"Calumenin" is an enzyme that exists in the endoplasmic reticulum and binds to Ca ^2+, and is encoded by the CALU gene.

The term " ligand " as used herein is a molecule capable of binding to a biomolecule. Some ligands bind to the active site, but no catalytic conversion occurs. Examples include ligands that are evaluated in the field of drug design. The ligand may be a small molecule selected for its ability to non-covalently bind to a target biomolecule for pharmacological purposes. In some cases, the ligand is evaluated for its ability to enhance, activate, or inhibit the natural behavior of the biomolecule.

The term " library " as used herein refers to a nucleic acid sequence (e.g., a gene, oligonucleotide, etc.) or an expression product (e.g., an enzyme or other protein) therefrom of two or more different molecules, . Libraries generally contain a large number of different molecules. For example, a library typically comprises about ten or more different molecules. The macromolecule typically contains about 100 or more different molecules, more typically, about 1,000 or more different molecules. For some applications, the library comprises at least about 10,000 different molecules.

The term " docking " as used herein refers to a process by a computer that simulates or characterizes the binding of the active site of a molecule (e.g., a substrate or ligand) to a biomolecule (e.g., . Docking is typically performed on a computer system using a " doorker " computer program.

The term " screening " as used herein refers to a process of measuring one or more characteristics of one or more biomolecules. For example, a typical screening process involves measuring one or more characteristics of one or more members of one or more libraries. Screening can be performed computationally using computational models of biomolecules and virtual environments of biomolecules.

As used herein, the term " homology " refers to structural homology, and structural homology refers to the structural homology of two or more proteins that are folded in a three-dimensional (3D) Dimensional structure. Structural homology is determined within the range of 0% to 100%, and is determined using commercially available computer programs that can calculate homology using distance differences between two or more three-dimensional structures, RMSD and TM-score, etc. . In the present invention, calculation was performed using a " PyMOL " computer program.

In addition, " sequence homology " is interpreted to include homology with an amino acid sequence or a nucleotide sequence having one or several additions, deletions and / or substitutions compared to the subject sequence.

As used herein, the term " I-TASSER (Iterative Threading ASSEmbly Refinement) " refers to a bioinformatic program that predicts a stereostructure model of a protein molecule from an amino acid sequence.

As used herein, the term " PyRx " refers to Virtual Screening software that can be used to screen libraries of compounds for drug targets.

The term " AutoDock Vina " as used herein refers to molecular modeling simulation software effective for docking proteins and ligands.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Carriers included in the pharmaceutical composition of the present invention are those conventionally used and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, .

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like.

[Example]

subunit이 결실된 dpy-18(e364) 돌연변이 모델을 사용하여 시험하였다.In the present invention, the homology between the calumenin of the nematode and the human calumenin was evaluated, and the use of various compounds as a treatment for onchocerciasis was confirmed through virtual screening. To confirm the possibility of this, the calumenin mutant model of C. elegans and the prolyl 4-hydroxylase involved in the collagen biosynthesis of C. elegans wild type and C. elegans

were tested using the dpy-18 (e364) mutant model in which the subunit was deleted.

[Example 1] Structural modeling and homology evaluation of target protein

The amino acid sequence of the target protein

The amino acid sequences of CeCALU-1 (GenBank Accession No. AAF34189.1), BmCALU-1 (Bm5089), OvCALU-1 (OVOC5386), and HsCALU-1 (GenBank Accession No. AAB97725.1) Predicted structure.

Figure 1 shows the amino acid sequence alignment of C. elegans calumenin (Ce_calu), B. malayi calumenin (Bm_calu), O. volvulus calumenin (Ov_calu) and H. sapiens calumenin (Hs_calu) Five EF hands motifs were combined in magenta, orange, yellow, red, and cyan order. Additional EF hands motifs of human calumenin were shown in blue and green. Clustal X was used for multiple sequence alignment, and the alignment data was visualized using GeneDoc.

Through the amino acid sequence alignment of Figure 1, structural differences between human and nematode calumenin were identified.

Caluemnin (CeCALU-1) from C. elegans is 72% homologous to calumenin of the other weevil nematodes. On the other hand, human calumenin (HsCALU-1) showed 45% sequence homology with calumenin of C. elegans and 43% sequence homology with calumenin (BmCALU-1) of B. malayi with calumenin of O. volvulus (OvCALU-1) and 44% sequence homology.

 Calumenin has five EF hands motifs (calcium binding proteins), but human calumenin has seven EF hands motifs and PAEL is a C-terminal ER conserved signal only in nematodes. Respectively.

3D structure and homology evaluation of target protein

The predicted model of protein structure was analyzed using I-TASSER (Roy et al., 2010; Yang et al., 2015; Yang and Zhang, 2015; Zhang, 2008a) and PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrodinger , LLC) (Fig. 2).

FIG. 2C shows calumenin (OvCALU-1) of O. volvulus, FIG. 2D shows calumenin (OvCALU-1) of H. sapiens, (HsCALU-1). All of the structures in FIG. 2 are shown using PyMOL, and the EF hands motif to which Ca ²⁺ binds is represented by the chromatic color of the method shown in FIG.

2, it can be seen that the overall structure of the three nematode calumenins is similar, but different from the predicted structure of human calumenin. This structural difference implies that the nematode calumenin is the target protein of the selective and specific Filariasis drug.

The reliability of each protein model was quantitatively determined by the C-score, and the C-score was calculated based on the template alignment significance and the convergence parameter of the structural assembly simulation. The C-score is a confidence score, calculated based on the significance of the sequence with the template used for modeling and the convergence parameter in the structural assembly simulation. According to I-TASSER statistics, the C-score generally has a value between -5 and 2, and a C-score> -1.5 indicates a global topology.

The TM-score and root-mean-square deviation (RMSD) were also calculated to measure the quality of the modeling predictions. RMSD and TM-score (Zhang and Skolnick, 2004) measure the structural similarity between two structures by measuring the distance between the predicted model and the underlying structure.

The TM-score is a template modeling score, a parameter that measures the difference in three-dimensional structure between two proteins.

In the TM-score, the larger the distance error is calculated as the lower specific gravity than the smaller distance error, making the value more accurate (Zhang and Skolnick, 2004). By definition, TM-score ≤ 0.17 means any similarity, and TM-score> 0.5 indicates a global topology model (Table 1).

The RMSD analysis is calculated as the average deviation for the template using SuperPose. The SuperPose web server computes pairwise and multiple protein structure superposition using modified quaternion eigenvalue approaches. In the case of highly homogeneous templates, a model with a RMSD value of 1 to 2 Å is generated for traditional comparative modeling (Marti-Renom et al., 2000). In case of comparative modeling (CM) for a template with low homology to threading, there may be an error in the loop region and the RMSD value is in the range of 2 to 5 Å (Roy et al. 2010, Zhang, 2009).

The C-score, TM-score and RMSD for each model are shown in Table 1 below.

In order to calculate the RMSD between each calumenin of the present invention, each calumenin model was subjected to structural alignment (Fig. 3). FIG. 3A shows the structural alignment of C. elegans calumenin (CeCALU-1) and B. malayi calumenin (BmCALU-1), FIG. 3B shows C. elegans calumenin (CeCALU-1) and O. volvulus calumenin (OvCALU- 3C shows the structural alignment of B. malayi calumenin (BmCALU-1) and O. volvulus calumenin (OvCALU-1), FIG. 3D shows C. elegans calumenin (CeCALU-1) and H. sapiens calumenin HsCALU-1).

 1 was green, BmCALU-1 was gray (FIG. 3A) or purple (FIG. 3C), OvCALU-1 and HsCALU-1 were gray and EF hands motif was displayed in the same manner as in FIG. Respectively.

FIG. 3 shows that there are many overlapping structures between the calumenin of the nematode, but the overlapping structure between the human and the calumenin of the nematode is small.

The CeCALU-1 model showed high structural similarity with BmCALU-1 (RMSD = 1.038Å, Fig. 4A) and OvCALU-1 model (RMSD = 1.263Å, Fig. 4B) (20.627A). This implies that there is a significant difference between nematode and human calumenin structure. The BmCALU-1 model and the OvCALU-1 model showed structural similarities with each other due to the RMSD value difference of 1.260 Å (Fig. 3C), but showed different structural features from HsCALU-1 (RMSD = 19.877 Å and 19.918 Å) 4).

These results indicate that the calumenin structure is well preserved among the nematode even if the nematode is different, but it is clearly distinguished from the human calumenin structure. This means that the nematode calumenin can be a selective and specific target for the treatment of filariasis.

[Example 2] Virtual drug screening by molecular docking

Selection of ligand library

A molecular population (Zdd library) containing 1,701 commercially available drugs was obtained from the ZINC database (ZINC drug database Version 12, http://zinc.docking.org, Irwin and Shoichet, 2005). The Zdd library of 1,701 compounds was screened using PyRx, an open-source virtual screening software (Dallakyan and Olson, 2015).

Molecular docking analysis

We used the PyRx / AutoDock Vina (Version 0.8) program to select compounds that will molecular dock into the expected 3D model of calumenin.

The binding energy between each caluemnin model and the ligand was measured and screened. BcCALU-1, BcCALU-1, and OvCALU-1 to specifically bind to calumenin (CeCALU-1, BmCALU-1 and OvCALU-1) of nematode without binding to human calumenin (HsCALU- 1 and HsCALU-1 were used to select ligands.

The selected ligands are listed in Table 2 in the order of decreasing binding energy between the CeCALU-1 model and the ligand, that is, in the order of higher affinity between the ligand and the CeCALU-1 model, and BmCALU-1, OvCALU-1 or HsCALU- The order of binding affinity for the model is described.

High affinity only to the nematode calumenin (CeCALU-1, BmCALU-1, and OvCALU-1) was used to screen drugs for treatment or prevention of onchocerciasis with relatively low binding affinity to human calumenin and specificity to calumenin of the nematode The chemicals that they represent are classified. As a result, Itraconazole, Idarubicin, Paliperidone and Daunorubicin hydrochloride were selected as candidates of active substances.

As shown in Table 2, Itraconazole, Idarubicin, Paliperidone and Daunorubicin hydrochloride have high nematode specificity and itraconazole has the highest nematode specificity.

The docking state between Itraconazole and CeCALU-1 calumenin was visualized using PyMOL (Fig. 4). The chemical and 3D structures of Itraconazole are shown in Figure 4A. 4B to 4E, detailed docking schemes of Itraconazole and CeCALU-1 were visualized using PyMOL. Itraconazole is docked with the binding pocket of CeCALU-1 and connected to the third EF hand motif (marked yellow).

FIG. 4 shows that itraconazole is stably bound to calumenin of CeCALU-1.

[Example 3] Relationship between Itracozole and calumenin

Preparation of experiments

subunit이 결실된 dpy-18(e364) 돌연변이(비교예), C. elegans의 calumenin이 결실된 calu-1(tm1783) 돌연변이(실시예)를 준비하였다. 대조군, 비교예 및 실시예는 L4 단계의 유충에서 24시간 키운 성충(1 day-old adult)으로 준비하였다. In order to examine the effectiveness of itraconazole in the treatment of onchocerciasis, N2 (control), C. elegans, prolyl 4-hydroxylase involved in collagen biosynthesis of C. elegans

(e364) mutant in which the subunit was deleted (comparative example) and calu-1 (tm1783) mutant (example) in which C. elegans calumenin was deleted were prepared. The control, comparative, and example were prepared as adult (1 day-old adult) for 24 hours in L4 stage larvae.

The control and comparative examples were obtained from the Caenorhabditis Genetics Center (CGC), which was obtained from the National BioResource Project (Japan) and the parasite method was followed by standard methods (Brenner, 1974).

Albendazole (Experiment 1), Ivermectin (Experiment 2) and Itraconazole (Experiment 3) were treated with the control, comparative and example, respectively. Albendazole and Ivermectin are conventional treatments for onchocerciasis, and albendazole is a benzimidazole-based drug. Ivermectin is a class of Avermectin, an anti-parasitic antibiotic produced by Streptomyces avermitilis.

In Experiment 1 and 2, solid nematode growth media (NGM) containing OP50 nutrient was used. In Experiment 3, M9 buffer (22 mM KH2PO4, 42.3 mM Na2HPO4 and 80% 85.6 mM NaCl) and OP50 at a concentration of 20% (v / v) were used. Cholesterol was removed from the experimental nematode growth medium for Itraconazole for accurate experiments.

Experiment

Experiments 1 and 2 were performed using a solid growth plate and 10 to 15 control plates, comparative examples and examples per plate were incubated at 20 ° C. In Experiment 3, 100 μl of the liquid culture medium and 1 parasite were placed in each well of a 96-well culture plate (Cat # 32096, SPL, Republic of Korea) and cultured at 20 ° C.

In Experiment 1, the survival rate of the control group, the comparative example and the example was measured at 24 hours after exposure to Albendazole for 3 days, and the survival rate of the control group, the comparative example and the example was measured at 24 hour intervals by exposure to Ivermectin for 2 days , And experiment 3 was performed for 3 days and exposed to Itraconazole at 24 hour intervals to measure the survival rate of the control group, the comparative example and the example. The measurements were repeated at least three times.

The parasite was considered dead when the plate was touched and there was no response and no pharyngeal pumping of the parasite. When the parasite came out of the plate, it did not count the number.

In Experiment 1, the concentration of albendazole was adjusted to 0, 6.25, 12.5 and 25 μM. In Experiment 2, the concentration of Ivermectin was adjusted to 0, 1.2, 2.4, 4.8 and 9.6 nM, , 80 and 160 [mu] M, respectively.

result

The results of Experiment 1 are shown in FIG. 5A, the results of Experiment 2 are shown in FIG. 5B, and the results of Experiment 3 are shown in FIG.

In Experiment 1, the experimental group showed a more sensitive response to Albendazole than the control group (Fig. 5A). The control group was more sensitive to albendazole than the control group, but was relatively lower than the control group.

In Experiment 2, the comparative examples and the examples showed similar sensitivity to Ivermectin. Differences from the control group occurred 2 days after administration of Ivermectin.

These results indicate that deletion of cuticle can induce effective drug delivery to parasites and calumenin, an important factor in normal epidermal development, can be a new drug target.

In Experiment 3, the example showed a more resistant phenotype to Itraconazole than the control and comparative examples (Fig. 6). Survival rates of the Calumenin-deficient mutant nematodes were greater than 90% in 160 μM Itraconazole, but survival rates of the control and comparative examples were about 74% and 38% at the same concentrations. As a result, Itraconazole selectively and specifically inhibited calumenin of the nematode, indicating that it has an effect of treating and preventing onchocerciasis.

Claims (12)

delete A pharmaceutical composition for the treatment or prevention of filariasis comprising itraconazole or a salt thereof.
3. The pharmaceutical composition according to claim 2, wherein the itraconazole is combined with calmenin of onchocerciasis causing onchocerciasis.
3. The pharmaceutical composition according to claim 2, wherein the itraconazole inhibits the production of cuticle of onchocerciasis causing onchocerciasis.
3. The pharmaceutical composition according to claim 2, wherein the onchocerciasis is lymphatic filariasis or onchocerciasis.
[Claim 3] The pharmaceutical composition according to claim 2, wherein the causal organism causing onchocerciasis is Brugia malayi or Onchocerca volvulus.
The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition further comprises one or more compounds selected from the group consisting of Diethylcarbamazine (DEC), Mectizan, Albendazole, and Ivermectine Or a pharmaceutically acceptable salt, solvate or prodrug thereof. delete delete delete delete delete

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