MXPA99009998A - Methods for detecting and reversing resistance to macrocyclic lactone compounds - Google Patents

Abstract

Esta invención describe moléculas deácido nucleico o fragmentos de la misma, extraídos de plagas de nemátodos o artrópodos o recombinantes, las cuales codifican homólogos de la P-glicoproteína y regulan la resistencia a los compuestos de lactona macrocicliclas, y su producto de expresión. También se describen métodos para detectar el gen que codifica para la resistencia a los compuestos de lactona macrocíclicas en plagas de nemátodos o artrópodos los cuales comprende comparar losácidos nucleicos extraídos de un espécimen de plaga con aquellos codificados para la susceptibilidad y resistencia a los pesticidas. La invención además se dirige a métodos y composiciones para incrementar la eficacia de los compuestos de lactona macrocíclicas contra plagas de nemátodos o artrópodos resistentes, el cual comprende administrar a un mamífero o aplicar a las cosechas, una cantidad efectiva de un agente que invierte la resistencia a los múltiples fármacos.

Description

METHODS TO DETECT AND INVEST THE RESISTANCE TO MACROCICLIC LACTONE COMPOUNDS BACKGROUND OF THE INVENTION Field of the Invention This invention relates in general to new methods for diagnosing and overcoming resistance to macrocyclic lactone compounds. More specifically, the invention pertains to unique methods for detecting the development of resistance to macrocyclic lactones using nucleic acid probes and increasing the efficacy of macrocyclic lactones using agents that reverse the resistance to multiple drugs.

Description of Related Art Macrocyclic lactone compounds such as compounds LL-F28249, milbemycins and avermectins are widely used for the REF: 31801 treatment of nematode and arthropod parasites. The highly active LL-F28249 family of compounds are isolated endectocide agents from the fermentation broth of Streptomyomyces cyaneogri seus s ubsp. noncyanogenus. The North American Patent No. ,106,994 and its continuation, US Patent No. 5,169,956 describe the preparation of major and minor components, LL-F28249a- ?. The family of compounds LL-F28249 further includes, but is not limited to, the semi-synthetic 23-oxo derivatives and 23-imino derivatives of LL-F28249a-, which are shown in U.S. Patent No. 4,916,154. Moxidectin, chemically known as 23- (O-methyloxime) -LL-F28249a, is a particularly potent 23-imino derivative. Other examples of derivatives LL-F28249 include, but are not limited to, 23- (O-methyloxime) -5- (phenoxyacetoxy) -LL-F28249a, 23- (semicarbazone) -LL-F28249a and 23- (thiosemicarbazone) -LL -F28249a. Milbemycins, also known as the B-41 series of antibiotics, are macrocyclic lactones that originate naturally, isolated from microorganisms, Streptomyces hygroscopi cus subs. to ureol to crimosus. The North American Patent No. 3,950,360 shows the preparation of the macrolide antibiotics milbemycin? -aio / milbemycin? -p3, etc. These compounds are also commonly referred to as milbemycin A, milbemycin B, milbemycin D and the like, or antibiotic B-41A1, antibiotic B-41A3, etc. Avermectins, also known as the C-076 family of compounds, are macrocyclic lactones that originate naturally, produced by the actinomycete microorganisms of the soil, Streptomyces a vermi tilis. The patent US No. 4,310,519 describes the isolation and preparation of the major components Aia (e.g., avermectin Aia), A2a, B? A and B2a, and minor components A? B (e.g., avermectin ib), 2b. B? B and B2b- Family C-076 additionally encompasses semisynthetic derivatives such as 22, 23-dihydroavermectins described in US Patent No. 4,199,569. Semi-synthetic derivatives include, but are not limited to, ivermectin, abamectin, doramectin, eprinomectin, and the like. Resistance to all broad-spectrum macrocyclic lactone compounds has been found in many regions of the world, where compounds are routinely used in animal production. For example, drug resistance iver ectin (IVM), chemically known as 22, 23-dihydroavermectin Bi or 22, 23-dihydro C-076 Bi and a commonly used member of the avermectin family of drugs, has become a extended problem, particularly in nematodes of sheep, goats and cattle (Shoop, Parasitol. Today 9_: 154-159, 1993). In some parts of the world, the survival of commercial animal production is threatened by the development of antelminthic resistance. Additionally, there is conflicting evidence about whether resistance to ivermectin (avermectin) confers resistance to related milbemycins or other macrolides (Arena et al., J. Parasitol 81: 286-294, 1995, Oosthuizen and Erasmuns, J So. African Vet. Assoc. 6_4: 9-12, 1993; Po roy and helan, Vet. Rec. 132: 416, 1993; Shoop, 1993; Condora et al., Vet. Rec. 132: 651-652, 1993; Pomroy et al. , N.Z. Vet. J. 40:76, 1992; Pankavich et al. , Rec. 130: 241-242, 1992; Craig et al., Vet. Parasitol. 4_1: 329-333. 1992). The mechanisms of resistance to avermectins, milbemycins and other macrocyclic lactone compounds remain unknown. The P-glycoproteins (Pgp) were identified some years ago as proteins involved in multicarrenal resistance (MDR) of cells in mammals tumors (Julino and Ling, 1976, Gros and Buschman, 1993, Gotteesman and Pastan, 1993). MDR proteins may also be involved in drug resistance in the protozoan parasites Entamoeba histolytica (irth, Medical Research Archives 21 (Supp.1): 183-189, 1990; Samuelson et al., Mol. Biochem. Parasitol. : 281-290, 1990), Leishmania enrrietti (Chow, Mol.

Biochem. Parasitol 6_0: 195-208, 1993), L. dononani (Callahan et al., Mol. Biochem. Parasitol 68: 145-149, 1994); and Plasmodium falciparum (Volkman et al ..

Mol. Biochem. Parasitol. 57_: 203-211, 1993; Cowman et al., J. Cell Biol. 113: 1033-1042, 1991). While many seekers believe that the proposed mechanism for Pgp involved in drug resistance is that the Pgp behaves like a pump to increase the flow, Callahan et al. (1994) suggests that Pgp can work by decreasing the flow into the drug. However, the whole picture of how Pgp may be responsible for drug resistance is not clear yet. Only, homologs having Pgp in nematodes have recently been investigated (Sangster, Parasitol, Today 1_0: 319-322, 1994, Lincke et al., EMBO J. 12: 1615-1620, 1993, Lincke et al., J. Mol. Biol. 228: 701-711, 1992). Three full-length Pgp genes and one partial Pgp gene from the free-living nematodes, Caenorhabditis elegans, have been cloned, sequenced and mapped to chromosomes I, IV, and X (Lincke et al., 1992). Sangster et al. , J. Cell Biochem. 17 (Supp.): 1223, 1993, indicates the evidence for various partial genes for Pgp in the parasitic nematode Haemonchus contortus, even though the sequence information is lost. In vivo experiments have shown that the disruption of mouse mdrl, a P-glycoprotein gene, leads to a deterioration in the blood barrier in the brain and decreases the sensitivity to drugs in these mice (Schinkel et al., Cell 7_7: 491-502, 1994). The mice with the elimination of nidria were 50-100 times more sensitive to ivermectin than the normal mice. Drug resistance based on overexpression of P-glycoprotein has been shown to be reversed by verapamil and a number of other calcium channel blockers, calmodulin antagonists, steroids and hormone analogues, cyclosporins, dipyridamole and other agents who invest the MDR. (Ford, Hema t ol Oncol, Clin North Am. 9_ 337-361, 1995). However, the use of agents that invest MDR to combat resistance in nematodes and arthropods to pesticides has not been reported or suggested in the literature. There is a definite need to understand the mechanism of macrocyclic lactone resistance, for being able to detect incipient resistance before it becomes flagrant and is difficult to manage the health of animals. The ability to reverse resistance has a greater potential to maintain parasite control in the face of a conventional treatment failure. An important object of the present invention, thus, is to determine these resistance mechanisms to find viable, sensitive means to detect and overcome the problematic resistance, thereby improving the control of the parasite.

BRIEF DESCRIPTION OF THE INVENTION It is unknown until now that the mechanism of resistance to macrocyclic lactone compounds is due to the overexpression of the new homologues of P-glycoproteins. It has also recently been found that the nucleic acid molecules encoding the P-glycoprotein homologs or the fragments thereof regulating this resistance are used as unique probes in the methods for the diagnosis of resistance to macrocyclic lactones. For the first time, the reversal of resistance to macrocyclic lactone compounds using agents that reverse multidrug resistance is described.

BRIEF DESCRIPTION OF THE DRAWINGS The background of the invention and its departure from the art will be further described hereinafter, with reference to the accompanying drawings, wherein: Figure 1 shows the PCR product of 432 bp which is generated from a pBLUESCRIPT® library of Ha cDNA. emonch us with tortus as a template and degenerate primers based on the domains that bind the conserved ATP of P-glycoprotein genes from Caenorhabdi ti's the egans after electrophoresis on an agarose gel. Figures 2A and 2B represent, respectively, the nucleotide sequence of the 432 bp PCR product shown in Figure 1 and the predicted amino acid translation of the cDNA (corresponding to SEQ ID NO: 1 and SEQ ID NO: 2, respectively). Figure 3 shows the autoradiographies of Northern blots of RNAs extracted from eggs of nematode strains sensitive and resistant to ivermectin (MKIS and MKIR; ACIS and ACIR) respectively. The PRC product of [32P] -432 bp, with homology to Pgp, is used as a probe, and, a fragment of [3"P] -actin from pBAl is used as a second probe." Figures 4A to 4B represent the full-length cDNA sequence (4175 bp) of the clone PGP-A of the cDNA library of H. cont ortus with high homology to the known P-glycoproteins (corresponding to SEQ ID NO: 3).

Figure 5 depicts the partial cDNA sequence (1810 bp) of the 5 'end of clone PGP-A from the cDNA library of H. with t ort us (corresponding to SEQ ID NO: 4). Figure 6 depicts the partial cDNA sequence (2698 bp) from the 3 'end of clone PGP-A from the cDNA library of H. with t ortus (corresponding to SEQ ID NO: 5). Figure 7 depicts a translation of the putative amino acid (1275 a.) Of the ANDc of PGP-A (corresponding to SEQ ID NO: 6). Figure 8 to 8B represents the sequence Partial cDNA (3512 bp) from the 3 'end of the related but different PGP-0 clone of the H DNA library with tortus (corresponding to SEQ ID NO: 7). Figure 9 depicts the partial cDNA sequence (2681 bp) of the 3 'end of the related but different PGP-B clone from the H cDNA library with t ortus (corresponding to SEQ ID NO: 8). Figure 10 shows the autoradiographs of the Southern blotches of genomic DNA extracted from sensitive and resistant ivermectin eggs from strains of H. with t ortus (MKIS and MKIR) after digestion with PvuII, electrophoresis and subjected to probes with the probe Pgp of H. with t ort us of [32P] ~ 432bp. Figure 11 shows the restriction length polymorphism of the DNA PCR products of individual male adult worms of H. cont ort susceptible strains (lines 1-9) or resistant strains (lines 11-20) to ivermectin, generated with P-glycoprotein PGP2S and PGPAS primers followed by digestion with L > and separation in electrophoresis with non-denaturing polyacrylamide gel. The arrows point to the three digestion fragments that are associated with the resistance. Figures 12A and 12B depict the nucleic acid sequences comprising the sense primer PGP2S (Figure 12A, which corresponds to SEQ ID NO: 9) and the PGPAS antisense primer (Figures 12B, corresponding to SEQ ID NO: 10) ) which are constructed from the clone PGP-O-3 'of the homology cDNA of the P-glycoprotein of the nematode (intron region of 53 bp) and are used to generate PCR products that are diagnostic for the endectocida resistance to the macrocyclic lactone. Figures 13A and 13B illustrate the efficacy of moxidectin (MOX) against strains of H. with susceptible t (Figure 13A) or resistant to moxidectin (Fig. 13B) in jirds. Figures 14A and 14B illustrate the efficacy of ivermectin (IVM) against H. strains with susceptible turtles (Fig. 14A) and resistant to moxidectin (Fig. 14B) in jirds. Figures 15A and 15B illustrate the efficacy of verapamil (VRP) with or without ivermectin (IVM, LD50) against strains of H. with susceptible tortus (Fig. 15A) or resistant to moxidectin (Fig. 15B) in jirds. Figures 16A and 16B illustrate the efficacy of the combination of moxidectin (MOX; Figure 16A) or ivermectin (IVM; Figure 16B) with verapamil (VRP) against strains of H. with resistant to moxidectin in j irds. Figures 17A, 17B and 17C illustrate, respectively, Hinfl digestion of PRC fragments of P-glycoproteins from the DNA of individual H. worms with ivermectin-resistant t ores, susceptible and resistant to moxidectin, using PGP2S and PGPAS primers , followed by digestion and separation in electrophoresis with non-denaturing polyacrylamide gel. The arrows on the right side of Figures 17B and 17C point to the digestion fragments that are associated with the resistance, while the arrows on the left side point to the position and size of the standard markers. Figures 18A, 18B and 18C illustrate, respectively, the AluI digestion of PCR fragments of p-glycoproteins from the DNA of individual worms of H. with t ortus resistant to ivermectin, and resistant to moxidectin, using PGP2S primers and PGPAS, followed by digestion and separation in electrophoresis with non-denaturing acrylamide gel. The arrows on the right side of Figures 18B and 18C point to the digestion fragments that are associated with the resistance, while the arrows on the left side point to the position and size of the standard markers.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, novel isolated and purified nucleic acid molecules encoding novel P-glycoprotein homologs or fragments thereof, which regulate the acyclic lactone resistance, are provided. These nucleic acids find use as probes in innovation methods for the early diagnosis of a resistance developed by the endectocides. In the past, the detection methods of available macrocyclic lactone resistance were not based on DNA or RNA. At present, the present invention provides only the genetic basis of resistance and resistance diagnosis using nucleic acid probes. Early detection under the guidance of this invention allows to maintain adequate control of parasites and maintain the usefulness of the macrocyclic lactone compounds. Additionally, the mechanism of resistance to macrocyclic lactones can be used in the development of screens to identify new antiparasitic agents. The new methods of the present invention, which are employed to detect resistance to macrocyclic lactone compounds in nematode or arthropod pests, use the new nucleic acid probes, described herein. A variety of techniques well known to those skilled in the art can be employed for analysis. Desirably, the method detects changes in genomic DNA or RNA to provide a viable means for the diagnosis of macrocyclic lactone resistance. These methods include, for example, the analysis by Polymerase Chain Reaction (PCR), hybridization in Southern spotting, spotting Dot or Northern blotting or the use of an antibody to a sequence of peptides corresponding to the translation of the nucleotide sequences between the novel primers of the invention of an individual pest or combinations of the pests, such as, worms, which use primers or probes, for example, which correspond to the portion of the PGP-0 cDNA sequence between the sequences identified as PGP2S and PGPAS (see Figures 12A and 12B). Alternate primers or probes within this region that can be used in the methods of the invention include, but are not limited to, all combinations of PCR primers or probes within this region or those of the other PGP homologous sequences such as PGP. -A, PGP-B, PGP-0 and the like. Basically, the coding region of the homologous glycoprotein genes, corresponding to the cDNA sequences identified as PGP-A, PGP-A-3 ', PGP-B, PGP-B-3', PGP-O, PGP -O-3 'and the like, are detected by PCR, Southern spotting, Dot spotting, Northern spotting, Fragment Length Restriction Polymorphism (RFLP) and other standard means of analysis. Surprisingly, it has been found that the digestion model from the PCR fragment, the spotting data or the antibody-antigen reaction, are associated with the susceptible or resistant traits that are diagnostic for the development of lactone resistance. macrocyclic. The Polymerase Chain Reaction (PCR) can be used to detect resistance to macrocyclic lactone compounds by synthesizing a nucleic acid product which can be tested in conjunction with Southern blot analysis or initially digested with a restriction enzyme for the RFLP analysis as described herein. The primers are used to initiate a PCR reaction using the nucleic acids extracted from the pest specimen. They are used to synthesize a P-glycoprotein sequence or sequences. The PCR products can then be cut with restriction enzymes and the digested sequences run on electrophoresis gel. Examples of suitable restriction enzymes that can be used in the digestion of PCR products include, but are not limited to, Alul, Ddel, Hinfl, Rsa l and the like. Patterns or patterns of bands observed in a Southern stain or Northern spotting indicate that P-glycoprotein alleles are present in a pest specimen such as worm or worm groups. Some of the alleles may be associated with sensitivity to macrocyclic lactone and others with resistance to macrocyclic lactones. The PCR products, followed by the digested restriction enzyme, provide viable means for detecting resistance. The process for cutting PCR products or nucleic acids such as DNA for RFLP analysis greatly increases the sensitivity and specificity of the diagnosis. Reverse Transcriptase - Analysis of the Polymerase Chain Reaction (RT-PCR), can similarly be used for the detection or resistance to macrocyclic lactone compounds in nematode or arthropod pests. Typically, RNA from a nematode or arthropod specimen is extracted, and reverse transcriptase, followed by PCR, as described herein, is used to detect resistance.

By means of the illustration, the nucleic acids, typically DNA for the PCR procedure or mRNA for RT-PCR, are extracted from the pest specimen, a pest known to be resistant to macrocyclic lactone compounds and a pest known to be susceptible to macrocyclic lactones. Nucleic acids derived from resistant and susceptible pests are used as a reference point. The DNA, or cDNA produced by mRNA by Reverse Transcriptase, is denatured and the primers of the invention are added to form a mixture. The three mixtures are subjected to many cycles of PCR, usually digested by a restriction enzyme and subjected to gel electrophoresis.

Subsequently, the model or pattern and the intensity of the bands from the specimen or that of the reference nucleic acids, i.e. DNA or ANDc, of the resistant and susceptible extracts, are compared to detect the resistant population. Optionally, hybridization by a probe of the invention or by the use of a dye such as ethidium bromide, to assist in the visualization of the bands, is included in the process.

New probes are used in the diagnosis of macrocyclic lactone resistance by detecting the susceptibility or resistance to macrocyclic lactones in the PCR assay. The primers that are used in the PCR assay are constructed, for example, from the nucleic acid sequences for the parasite of the cDNA clones homologous to the P-glycoprotein. Examples of suitable PCR primers that can be used in the PCR analysis are the PGP2S and PGPAS primers used in the sense and antisense directions, respectively, which are constructed from PGP-O-3 'or PGP-0 (see figures 12A and 12B). The primers can also be prepared from the full or partial sequences of other P-glycoprotein nucleic acids such as PGP-A, PGP-A-3A PGP-B-3 ', PGP-O, etc. and the complementary strands of them that contain the region found to be diagnostic of the. resistance to macrocyclic lactone. The alternative employed sequences can be obtained by conventional methods such as hybridization techniques under standard or severe conditions. Southern staining, spotting Dot or Northern staining can be prepared with the nucleic acid molecules of the nematode specimen or the arthropod and, using a probe comprising one of the sequences of the nucleic acid molecule encoding the resistance or portion from it, the level of the nucleic acids extracted from the specimen can be compared to the level of the nucleic acids of the probe, for example, by measuring or detecting the level of DNA or mRNA. In general, three nucleic acids and extracts are mapped to make the comparison: of the plague specimen, of a plague known to be resistant and of a plague known to be susceptible. In the case of Southern spotting, the pattern or pattern of the bands is compared. With Northern staining, either the pattern or pattern, or the intensity of the bands, are compared. For spotting Dot, the intensity of the spots is compared. Another technique involves conducting a Fragment Length Restriction Polymorphism (RFLP) analysis, by extracting the nucleic acids from a nematode or arthropod specimen, digesting the nucleic acid with a restriction enzyme, using a probe comprising one of the sequences of the nucleic acid molecule that codes for the resistance or portion thereof and comparing the model or pattern of digestion of that of the model or pattern of digestion of nematodes or arthropods known to be either resistant or sensitive populations. the macrocyclic lactone endectocides. When the DNA is cut with the restriction enzyme, run in gel and probed under the RFLP technique, the probe hybridizes with similar sequences, but its length will vary depending on where the restriction sites for such enzymes occur. By repeating DNA analysis from individual worms, slightly different patterns or patterns are observed due to polymorphism. The specific models or patterns are diagnostic for the resistance of the gene. PvuII is an example of a preferred restriction enzyme that can be used for RFLP analysis. Other conventional restriction enzymes known to those skilled in the art can be substituted in the method. A further example of a method employed in the present invention to detect resistance refers to the preparation of an antibody that employs the new homologs to the PGP protein. For example, an antibody can be prepared to a peptide sequence corresponding to the translation amino acid of the nucleic acids or fragments thereof, which encode the homologues of the P-glycoprotein which regulates resistance. Then, a specimen of the pest of the nematode or arthropod, or the extract thereof, is prepared by reaction with the previous antibody. The specimen or extract is reacted with the antibody under suitable conditions that allow binding to the antibody-antigen to occur and, subsequently, the presence of the antibody-antigen binding is detected by conventional methods. The methods described above for the detection of resistance to macrocyclic lactone compounds, can optionally use a ligand or dye specific for the P-glycoprotein. Usually, the level of the P-glycoprotein in the specimen can be observed more easily, using the ligand or dye and comparing the levels obtained in the known macrocyclic lactone resistance and in susceptible arthropod or nematode populations. The ligand or dye is usually radiolabelled so that it can be easily detected. Examples of suitable ligands employed in this method include, but are not limited to, prazosin, azidoprazosin, iodoaryl-azidoprazosin and the like. A variety of conventional dyes can be employed such as, for example, rhodamine 123, ethidium bromide and others. For purposes of this invention, the nucleic acid molecule can be DNA, cDNA or RNA. However, in the most preferred embodiment of this invention, the nucleic acid probe is a cDNA molecule. Many of the aforementioned methods illustrate the nucleic acids extracted from Haemonch us cont ort us. It is contemplated that the present invention encompasses the use of recombinant nucleic acids encoding resistance or susceptibility to macrolides as well as nucleic acids isolated from other strains of worms or pest species. The plasmids containing the cDNA derived from Haemonchus with tortus are deposited in conjunction with the present patent application and maintained according to the Budapest Treaty in the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110 -2209, US A. The cDNA sequences described herein are contained within the plasmids (pBLUESCRIPT® II, commercially available from S tra tagene Inc., La Jolla, CA), transformed into bacterial strains Escheri chia coli XLI-blue. The plasmids identified as PGP-B-3 ', PGP-0-3' and PGP-A-5 'have been deposited with the ATCC on January 29, 1997, and have been assigned the numbers of ATCC Designation 98307, 98309 and 98310, respectively. Plasmid PGP-A-3 'has been deposited with the ATCC on February 26, 1997 and has been assigned with the designation number ATCC 98336. It should be appreciated that other plasmids, which can be easily constructed using site-directed mutagenesis, and the techniques described herein are also encompassed within the scope of the present invention. The present invention further relates to investing only the resistance in parasites to the macrocyclic lactone compounds by the administration or application of the agents that reverse the resistance to the multiple drugs. This reversal of an existing resistance problem that allows the control of the parasite to be renewed satisfactorily. The nematode or arthropod parasite or pests of this invention, refers to crop insects, mammalian nematodes or crops, ectoparasite arthropods and endoparasites of mammals including acarids and the like.

Desirably, the agent that reverses the resistance to multiple drugs, is a blocker of calcium channels, such as verapamil, nifedipine and the like; a calmodulin antagonist such as trifluoroperazine, perchlorozine and the like; an analogue of the vinca alkaloid such as vindoline, taliblastin, and the like; a spheroidal agent such as progesterone and the like; a hormonal agent such as tamoxifen, estradiol and the like; an immunosuppressive agent such as cyclosporin A, SDZ-PSC 833 and the like; an antibiotic such as erythromycin, cefoperazone, ceftriazone, tetracycline and the like; miscellaneous compounds such as dipyridamole, quinidine, reserpine, amiodarone, etc .; and other agents that reverse the resistance to the multiple drugs known to those skilled in the art. To increase the efficacy of the pesticidal macrolides, the compounds of the invention are administered to mammals orally, parenterally, topically (local activity) or trapsdermally (systemic activity), depending on the bioavailability of the selected medicine by the desired administration route. Parenteral administration of the drugs encompasses any other means than orally, such as, for example, intravenously, intramuscularly, subcutaneously, intratracheally, intra-rinally, etc. It is apparent that agents that reverse MDR are administered in conjunction with the administration of the macrocyclic lactone compound of the resistance found in nematodes or ectoparasite arthropods or endoparasites of mammals. However, the administration of the agents that invest the MDR can be elaborated either before or during the concurrent administration of the macrocyclic lactones. If the agent who invests the MDR will be given before the endectocide, the doctor or veterinarian can quickly determine, through the appropriate levels of the blood, with the rapidity in the advance, the agent that invests the MDR that can be given to increase the efficacy of the macrolide. Typically, the agent that invests the MDR will be administered within 24 hours of the initiation of the endectocide therapy and, preferably, within 4 hours before or concomitantly with the administration of the macrocyclic lactone. In terms of dosage, the quantity / The agent of the MDR reversing agent that is effective in increasing the efficacy of the macrocyclic lactone compound against resistant nematodes or ectoparasitic arthropods or resistant endoparasites typically will vary within a wide range of amounts at a variety of concentrations. The agent that invests the particular MDR, selected for use with the specific endectocide, will clearly affect the dose used of the agent that invests the MDR. It is contemplated that the selection of the appropriate dosages of each agent that invests the MDR and the macrocyclic lactone compound will reach the pesticide by increasing the effective amount which can be easily titrated by routine tests known to those of ordinary skill in the art. veterinary and medicine. For use in the parasiticidal treatment, the macrocyclic lactone compounds can be administered orally in a unit dosage form such as a capsule, a pill, or a tablet. Capsules and pills comprise the active ingredient mixed with a conventional carrier vehicle such as starch, talcum, magnesium stearate or dicalcium phosphate. The unit dosage form, solid, dry, is prepared by intimately and uniformly mixing the active ingredient with suitable finely divided diluents, fillers, disintegrating and / or binding agents such as starch, lactose, talc, magnesium stearate, vegetable gums and similar ones Such unit dosage formulations can be widely varied with respect to their total weight and content of the active ingredient depending on factors such as the type and weight of the mammal to be treated and the type and severity of the infection or infestation. In general, the amount of the macrocyclic compound given in oral administration is from about 0.001 mg to about 10 mg per kg of body weight and preferably, from about 1 mg to about 5 mg per kg of body weight. However, the amount will vary depending on the extent of resistance already developed in the parasite. For animals, the macrocyclic lactone compound and many of the agents that reverse MDR can also be administered via animal feed forage, by intimately dispersing the active ingredient in the feed or using as a top compost or in the form of pellets which can be added to the finished food or optionally separately from the food. Suitable compositions include premixes of foods or supplements in which the active compound is present in relatively large amounts, wherein said premixes of foods or supplements are suitable for direct feeding to the animal or by addition to the food either directly or after an intermediate dilution or mixing stage. Typical carriers or diluents suitable for such compositions include dry grain distillers, corn flour, citrus flour, fermentation residues, ground oyster shells, wheat products, molasses, corn cob meal, ground food of edible beans , soy en masse, crushed limestone and the like. The active compounds are intimately dispersed through the carrier by methods such as grinding, stirring, milling or tumbling. Compositions containing about 0.005% to about 2.0% by weight of the active compound are particularly suitable as food premixes.

The food supplements, which are feeding directly to the animal, contain approximately 0.0002% to 0.3% by weight, of the active compounds. Such supplements are added to the animal's food in an amount to give the finished food, the desired concentration of the active compound for the treatment or control of the resistant parasitic disease. Although the desired concentration of the active compound will vary, depending on a variety of factors such as the particular compound employed or the severity of the affliction, the macrocyclic compounds of this invention are usually foods of a concentration of about 0.00001% to about 0.02. % in the diet. Alternatively, the compounds of the present invention can be administered to parentally afflicted mammals, in which event the active ingredient is dissolved, dispersed or suspended in a sterile, isotonic, non-toxic liquid carrier vehicle. The active material is mixed with the pharmaceutically acceptable, non-toxic carrier, preferably a vegetable oil, such as peanut oil, cottonseed oil, or the like. Other parenteral vehicles such as propylene glycol, glycerol and the like can also be used for parenteral formulations. In parenteral formulations, the active macrolides are typically dissolved or suspended in the formulation in an amount sufficient to provide from about 0.005% to about 5.0% by weight of the active compound in said formulation. Conveniently, macrolides can also be administered to afflicted mammals, via the topical or transdermal route to achieve their systemic or local effects. When used in animals, the compounds can be applied as a liquid solution. The liquid solution is usually a solution, suspension or dispersion of the active compound, usually in water, together with a suspending agent, such as bentonite and a similar wetting agent or excipient. In general, the solutions also contain an antifoaming agent. Solution formulations typically contain about 0.001% to about 0.5% by weight of the active macrocyclic compound. Preferred solution formulations contain about 0.01% to about 0.1% by weight.

Additionally, the macrocyclic compounds can be administered by application as a gel, lotion, solution, cream or ointment to human skin or poured into the animal's skin or hide via a solution. Topical or transdermal formulations comprise the active ingredient in combination with conventional inactive excipients and carriers. The cream, for example, may use liquid petrolatum, white petrolatum, propylene glycol, stearyl alcohol, cetearyl alcohol, sodium lauryl sulfate, sodium phosphate buffer, polysorbates, parabens, emulsifying waxes, polyoxyethylene-polyoxypropylene block copolymers, purified water , and the like. Ointments, for example, can use petrolatum, mineral oil, mineral wax, glycerin and the like. Topical solutions can provide the active ingredient composed of propylene glycol, parabens, hydroxypropyl cellulose, preservatives. Pouring formulations may constitute the active ingredient dissolved or dispersed in an aromatic solvent, myristyl ether propionate PPG-2, polybutene, an antimicrobial agent, an antioxidant and a non-toxic pharmaceutically acceptable mineral or vegetable oil.

To increase the efficacy of macrolides as pesticide agents, agents that reverse multidrug resistance are applied to crops, crop seeds or to oil or water in which crops or seeds are growing or growing with an amount effective increasing the pesticide. The agents that invest the MDR can be applied either before or concurrently with the application of the macrocyclic lactone. Typically, the agent that invests the MDR will be applied within 4 hours before or, preferably, concomitantly with the application of the macrocyclic lactone. In terms of application rates, the proper amount of the agent that MDR invests, which is effective to increase the efficacy of the macrocyclic lactone compound against pests of resistant crops, will typically vary within a wide range of amounts to a variety. of concentrations and proportions. The agent that invests the particular MDR selected for use with the pesticide in the crops will clearly affect the rate of application of the agent that invests the MDR. It is contemplated that the selection of the appropriate amounts, concentrations, atomization rates and the like of each agent that invests the MDR and the macrocyclic lactone compound to achieve the effective amount increasing in pesticide, can be easily determined by known routine procedures by those who have ordinary skill in agricultural technique. As insecticidal, nematicidal or acaricidal agents used to protect crop seeds or growth or crops of crops from the attack of pests, the compounds of the present invention can be formulated in dry compacted granules, flowable compositions, wettable powders, powders. , concentrated powders, microemulsions and the like, of which all are also provided in application to soil, water or foliage and provide the protection required in the plant. Such compositions include compounds of the invention, mixed with agronomically acceptable solid or liquid carriers. In the agricultural composition, the active compounds are intimately mixed or comminuted with the excipients and carriers in amounts sufficient to typically provide from about 3% to about 20% by weight of the macrocyclic lactone compound in said composition. The compositions of this invention are employed in the combat of agricultural pests that inflict damage on crops, while they are growing or in storage. The compounds are applied using known techniques such as spraying, powders, emulsions, wettable, flowable powders, and the like, to growing or stored crops to provide protection against infestation by agricultural pests. Unexpectedly, it is found that the mechanism of resistance to macrocyclic lactone compounds is due to the overexpression of the new homologues of the P-glycoprotein which causes an outflow of the parasite's anthelmintic. The present invention illustrates the development of the homologous Pgp genes in the IVM resistance in H. con tortus. Overexpression of the Pgp protein in IVM resistant strains of H. contortus is shown to be regulated in both rearrangements of the genomic DNA encoding PGP homologs and by transcription of the gene. H. with t ortus in jirds (Meriones ungui cula t us) have been used for the evaluation of antelmnthic efficacy and have been shown to correlate well with studies of this parasite in sheep (Conder et al., J. Parasi tol. 7_8_ : 492-497, 1992). Using the jird model, the present invention determines that agents that reverse the multiple drugs can unexpectedly be used to increase the efficacy of macrocyclic lactones against resistant parasites. As a representative example, verapamil (VRP), an agent that reverses multidrug resistance (MDR), is shown to only increase the action of moxidectin and ivermectin against H. with susceptible or resistant to moxidectin. Parasites such as H. con tortus contain Pgp homologous genes which are expressed in different stages of the parasite's life cycle. This invention finds that the level of expression of P-glycoprotein is surprisingly high in different strains that are resistant to macrocyclic lactones., such as ivermectin, compared to the levels in the susceptible strains from which the resistant strains are derived. The higher level of Pgp expression in strains resistant to ivermectin is associated with an alteration in the genomic level. The P-glycoproteins can act as molecular pumps for the exit of hydrophobic xenobiotics from the cells. An elevation in the level of P-glycoproteins is the basis of multidrug resistance in cancer cells and also appear to be involved in some forms of drug resistance in some protozoa. A high level of Pgp has not been described so far as the mechanism of drug resistance in nematode parasites. This is a first evidence that shows that resistance to ivermectin may be due to an elevation in P-glycoproteins. Resistance to ivermectin is the beginning of a common problem in nematode parasites of animals and potentially in arthropod parasites. The continued use so far against arthropods probably leads to the selection of resistance similar to that in nematodes. There is evidence that ivermectin shares a common action with other avermectins, such as doramectin, milbemycins (Arena et al., 1995) and moxidectin. It can be predicted that the development of resistance against other macrocyclic lactone compounds will involve the hyper-expression of the P-glycoprotein leading to high drug release rates. This work is significant because it allows the sensitive detection of the. resistance to ivermectin and resistance to other macrocyclic lactone compounds in nematodes and arthropods using DNA and cDNA probes based on the demonstrated differences found in the delayed DNA PvuJI from susceptible and resistant organisms. It also allows the prediction of the degree of resistance from the level of P-glycoprotein expression based on the levels of Pgp protein or Pgp mRNA. This understanding of the mechanism of resistance to macrolides allows active analogues to be synthesized, which will remain effective in the presence of a mdr-based mechanism of resistance to other macrocyclic lactones. More specifically, the chemicals that act in the mode of action of the receptor, the glutamate-gated chloride channel (exit) (Arena et al., 1995), but which do not emanate efficiently, by the P-glycoprotein pump, that is to say . , are poor substrates for the Pgp emanation, can be selected to overcome the resistance. This will lead to improvements in parasite controls, especially in the prevention and treatment of ivermectin resistance and cross resistance to other avermectins, milbemycins and compounds LL-F28249. This invention provides new evidence that in the resistance to macrocyclic lactone endectocides, such as ivermectin, in nematode parasites or animal arthropods, the expression of the P-glycoprotein is raised compared to the level of expression in the parental susceptible strains or maternal parasites. It is also shown that the higher level of expression is associated with differences, at the genomic level of the P-glycoprotein genes. For example, using Southern blot analysis of DNA clustering and by PCR analysis (Polymerase Chain Reaction) in individual worms, differences in genomic DNA for Pgp in Haemonchus are determined with ivermectin-resistant tortus compared to the maternal or paternal susceptible strain, and the diversity of the allele for Pgp in resistant worms seems to be markedly reduced compared to the maternal or susceptible parental strains. New nucleic acid probes which can differentiate between resistant and susceptible parasites are now found and are thought to be employed in the early detection of the development of resistance to macrocyclic lactone compounds. For the first time, this invention demonstrates that resistance to macrolactone can be overcome by the use of an agent that reverses the MDR. For example, verapamil, a well-known, relatively weak reversal agent of MDR, significantly increases the efficacy of moxidectin against H. with moxidectin-resistant tyrosine. Moxidectin-resistant worms that show collateral resistance to ivermectin and resistance to ivermectin are also overcome with the use of a mild MDR reversal agent. The following examples demonstrate certain aspects of the present invention. However, it is understood that these examples are for illustration only and do not mean to be completely definitive in the conditions and scope of this invention. It should be appreciated that when the typical reaction conditions (eg, temperature, reaction times, etc.) have been given, the conditions to which both are above and below the specified ranges can also be used, in spite of which in general, are less convenient. The examples were carried out at room temperature (about 23 ° C to about 28 ° C) and at atmospheric pressure. All parts and percentages referred to herein are on a weight basis and all temperatures are expressed in degrees centigrade unless otherwise specified. A further understanding of the invention can be obtained from the following non-limiting examples.

EXAMPLE 1 Synthesis of PCR and cloning of a 432 bp DNA for a P-glycoprotein homolog from a H cDNA library.

Based on the highly conserved ATP binding domains of C. the egans Pgp, a pair of degenerate PCR primers is designated. The sense primer is 5'-ACNGTNGCNYTNGTNGG-3 '(which corresponds to SEQ ID NO: 11) and the antisense primer is 5'-GCNSWNGTNGCYTCRTC-3' (which corresponds to SEQ ID NO: 12). The PCR is performed for 40 cycles at a denaturing temperature of 94 ° C for 1 minute, a quench temperature of 37 ° C for 1 minute, and an extension temperature of 72 ° C for 3 minutes using a cDNA library H. contort us (Geary et al., Mol. Bi ochem. Parasi tol., 50: 295-306, 1992) as a template. A 432 bp product is purified by agarose gel electrophoresis and the purified product is used as a template for a second round of PCR amplification with the same primers. An enriched 432 bp product is subsequently cloned into a TA vector (invitrogen) in accordance with standard protocols. The plasmids that are inserted are transformed into Escheri chi a coli and then laminated on Ampicillin LB plates containing a chromogenic substrate, X-GAL® (5-bromo-4-chloro-3-indol i1-β-D-galactopyranoside, commercially available from Gibco BRL, Bethesda, MD) (Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989). Ten clones are identified as ATP binding domain sequences of the P-glycoprotein.

EXAMPLE 2 Selection of the cDNA library of H. with t ort us The 432 bp fragment is cut by EcoRl, labeled by random priming with [32P] d-CTP and used as a probe to select the cDNA library (Sambrook et al., 1989). Approximately one million clones are selected and nine putative clones are identified. Positive clones are digested with PvuI I and three of them containing inserts in the predicted size are subsequently sequenced.

EXAMPLE 3 Strains of Parasites Two pairs of H. strains are used with susceptible and resistant to ivermectin. The first pair is a strain resistant to ivermectin (MFR) developed in the Research Laboratory Merck (for its acronym in English the Merck Research Laboratories), Rahway, NJ (Rohrer et al., J.

Parasi t ol. 8_0: 493-497, 1994) and the mother strain susceptible to ivermectin (MFS) from which the resistant strain is selected over the seventeen generations of ivermectin selection. The second for is a strain resistant to ivemectin (ACR) developed in the Cyanamid American Company (for its acronym in English American Cyanamid Company, Princeton, NJ and the mother strain susceptible to ivermectin (ACS) from which the strain is selected resistant over fourteen generations of ivermectin selection.The MFR strain is reported to be 10X resistant to ED95 compared to MFS, and ACR, after twelve generations of selection, is found to be 6.3X resistant to ED9J compared to ACS.

EXAMPLE 4 RNA Extraction and Northern Hybridization Adult worms of H. with susceptible and ivermectin-resistant tortus were collected from the abomasum (or fourth stomach in ruminants) of sheep (Lubega and Prichard, Bi ochem.Pharma col. 41: 93-101, 1991). The eggs of each strain were collected and isolated from sheep droppings (eston et al., J. Parasi Tol 14: 159-164, 1984) which have previously been free of worms and inoculated with one of the four strains of H. con tortus Total RNA is extracted from tissues of susceptible and ivermectin-resistant strains, respectively, using TRIzoL® reagent (protocol from Gibco BRL Life Technologies, Inc., Gaithersburg, MD). The total RNA is run on electrophoresis with denaturing formaldehyde agarose gel and transferred to nylon N-linkage membranes. The membranes are prehybridized at 6 ° C in 10% dextran disulphate, SDS 1% (sodium dodecyl sulfate), 1.0 M NaCl for 4 hours. The Pgp_ fragment of 432 bp 32P-tagged H. with tortus and an actin probe consisting of a 1.25 kb PstI fragment from pBAl (Degen et al., J. Biol. Chem. 258: 12153, 1983 ) are mixed and incubated overnight with the membranes at 65 ° C in the same hybridization buffer. The membranes are washed with 2X SSC (1: 2 mixture of trisodium citrate and sodium chloride), 0.1% SDS at 65 ° C for 30 minutes and 0.5X SSC at 35 ° C for 1 hour and then autoradiographed. Image analysis of gel autoradiographies is done by quantitative determination of mRNA expression, using the IMAGE program (O'Neil et al., Appl. Theor., Ectrophor., 1: 163-167, 1989). Actin DNA probes from a mouse source, labeled and hybridized with the same spotting, using the same method as the previous one, are used as an internal control for the mRNA loaded. The results are shown in Figure 3 (S = unselected strains; R = selected IVM strains; MKI = strains developed at the Merck Research Laboratories (Merck Research Laboratories); Rahway, NJ; ACI = strains developed in the American Cyanamid Company (American Cyanamid Company), Princeton, NJ).

EXAMPLE 5 Stained Southern Genomic DNA was extracted from both strains susceptible and resistant to ivermectin (Sambrook et al., 1989). Four restriction enzymes EcoRI, Cl a l, Pvu I I and Pst I are used to digest the genomic DNA after the directions of the suppliers. Each reaction is carried out with both strains, resistant and susceptible to ivermectin. After a digestion of the restriction enzyme overnight, the samples are run on 1% agarose gels and then stained on H-binding membranes (Sambrook et al., 1989). The membranes with DNA are exposed under UV light to fix the DNA to the membranes. The membranes are prehybridized at 65 ° C in buffer (10% dextran sulphide, 10% SDS and 1M NaCl) by al. minus 4 hours The 432 bp fragment, labeled with [32P], is added as a probe and hybridizes with the genomic DNA in the prehybridization buffer, overnight. The membranes are subsequently washed twice with 2X SCC for 10 minutes, twice with SCC IX for 15 minutes and then autoradiographed.

RESULTS Amplification of the PCR Two rounds of PCR amplification generate a 432 bp product (Figure 1) which is highly homologous to the highly conserved ATP binding domain of the P-glycoprotein (Figure 2A). The putative amino acid sequence (Figure 2B) shows that this fragment is highly homologous to the P-glycoprotein or multidrug-resistant proteins from C. egan s, mouse or other species. These data indicate that the 432 bp fragment represents the ATP binding sequence of a Pgp homolog of H. with tortus.

Expression of P-glycoprotein mRNA in Strains of H. with t ortus Susceptible or Resistant to Ivermectin _ A single animal species may have different Pgp, which may vary in size. Northern hybridization, with the PRC product homologous to H. with t ortus Pgp of 432 bp, shows that the molecular size of the mRNA for H. with t ortus Pgp is approximately 4 kb. However, it is found that the levels of Pgp mRNA in strains of H. with t ortus susceptible and resistant to ivermectin are different. For illustration, the results of a number of representative Northern blots in RNA from eggs of H. with t ortus are shown in Figure 3. The RNA is also subjected to probes with an actin probe to allow correction for different amounts of RNA loaded in the gels.

The intensity of the Pgp mRNA band varies with the strain of the parasite. After correction for the intensity of the actin band, it is found that the amount of the 4bk mRNA band recognized by the Pgp probe of 432 bp is much higher in both strains resistant to ivermectin, compared to their susceptible precursor strains. the ivermectin. The increase varies from 250% to 670% after standardization for the expression of actin mRNA in the susceptible and drug resistant strains (Table 1). Similar results are also obtained in comparison of the expression of Pgp using RNA extracted from H. with adult tortus. Table 1 shows the relative intensity of mRNA for P-glycoprotein and actin in strains of It has emonchus with t susceptible and resistant to ivermectin. RNA is extracted from eggs of the Merck (MKI) and American Cyanamid paired strains (ACI). Each susceptible and resistant pair is processed at the same time. The RNA is separated on agarose gel and subjected to probes with both H. - with t ortus Pgp of 432 bg and with the radiolabeled probes pBAl of actin. The relative intensity of each band is determined, after gel autoradiography, by gel densitometry. The intensity of each Pgp band is corrected for the intensity of its corresponding actin band to adjust to different amounts of RNA that have been loaded on the gels. All comparisons are made in pairs (resistant (R) with corresponding susceptible tra (S)).

TABLE 1 Comparison of strains Corrected R / S ratio MKIS / MKIR 6.77 MKIS / MKIR 6.08 MKIS / MKIR 2.57 ACIS / ACIR 4.19 Sequencing of P-glycoprotein homologs from a cDNA library of H. with tortus Large clones (4.2 kb, 3.5 kb and 2.7 kb) identified using a 432 bp probe, which are shown to be homologous to the P-glycoprotein, are completely or partially sequenced. Figures 4A to 4B show the complete cDNA sequence for clone PGP-A (4175 bp) which have high homology to known P-glycoprotein people such as the Xenpus putative protein resistant to multiple drugs (Xemdr) and C. egans cepgpA gene for P-glycoprotein A. Figures 5 and 6 show the partial sequence in the direction sense (Figure 5; PGP-A-5 ') and the antisense direction (Figure 6; A-3 ') of the DNA fragment which is also highly homologous to the P-glycoprotein. Figures 7-9 illustrate, respectively, the translation of the putative amino acid of the PGP-A cDNA, the partial cDNA sequence of the 3 'end of the PGP-0 clone (3.5 kb), antisense direction, and the partial DNA sequence of the 3-terminus. 'of clone PGP-B (2.7 k), antisense direction.

Differences of Genomic DNA between Strains of H. with t ortus Resistant and Susceptible to the Ivermectin and the Determination of an Acid Probe Nucleic for the Detection of Susceptibility or Resistance to Macrolactone Hybridizations of genomic DNA show that at least two bands are recognized by the 432 bp probe in the Clal and PstI digestion maps of both strains susceptible and resistant to ivermectin. The EcoRl digestion maps show three strongly hybridizing bands and one light band for both susceptible and resistant strains. However, PvuII models or patterns of digestion are clearly different between susceptible and ivermectin-resistant strains (Figure 10). The PCR products are generated using pairs of primers which are specific to the Pgp genes of parasites. In one example, the reverse primer is specific for a region of 53 base pairs in length present in one of the Pgp (PGP-O) clones. The forward primers temper a region common to multiple Pgp clones. Genomic DNA extracted from individuals of H. with adult male tt from populations of IVM-sensitive (24 worms) and IVM-resistant (29 worms) (M IS and MKIR), are used as temples for amplification by PCR The pgp PCR products, approximately 900 bp in length, are deferred with the restriction enzyme Ddel and the digestion products are separated by non-denaturing polyacrylamide gel electrophoresis (figure 11, see also figures 17A-18C illustrating the diagnosis of models or restriction patterns for resistance, after selection with either ivermectin or moxidectin, using different strains of worms and different restriction enzymes). The model or pattern of digestion for worms from susceptible populations is variable, while for worms of resistant populations it is more homogeneous. An identical model or pattern of digestion of the three bands (arrows) is found in 28 of the 29 worms of the resistant population (figure 11, lines 11-18 and 20, for example), while only 4 or 5 worms the susceptible population has this model or pattern (figures 11, lines 6 and 9, for example). The examples of the probes are shown in Figures 12A and 12B. These results are repeated several times. The PCR data and the Southern blot data clearly indicate that the selection for endectocide resistance of the macrocyclic lactone causes a reduction in the genetic diversity of the Pgp alleles and that the differences in Pgp at the DNA level can be detected by genetic techniques. specific probes such as PCT (Polymerase Chain Reaction), Southern Spotting analysis and RFLP (Fragment Length Restriction Polymorphism).Additional Methods EXAMPLE 6 Establishment of LD $ Q for Moxidectin and Ivermectin Against H. with Susceptible and Resistant Moxidectin in the Jird The Jirds, which are fed a standard commercial proportion to which 0.02% hydrocortisone has been added 5 days prior to infection, are inoculated with H. contortus L3 exenvainado 1000. On day 10 after inoculation, the Jirds are treated with either water or several doses of moxidectin or ivermectin orally.

Each treatment group contains 6 jirds. The proportions of parasite strains and the antelimitic doses are shown in Table 2. The results of these dose titrations are shown in Figures 13A-14B. Probit analyzes are used to estimate LD50 levels for each anthelmintic against each strain. The estimated LD50 of moxidectin against susceptible and moxidectin-resistant strains are 0.010 and 0.017 mg / kg, respectively, and for ivermectin, the estimated LD50 levels are 0.024 and 0.046 mg / kg, respectively.

The results indicate that moxidectin (i) is more potent than ivermectin against both susceptible and moxidectin-resistant strains, and (ii) the H. with t ortus resistant to moxidectin is resistant collateral to ivermectin.

Table 2. PROPORTION OF DOSES (mg / kg) of MOXIDECTIN (MOX) OR IVERMECTIN (IVM) AGAINST STRAINS OF H. contort us RESISTANT AND SUSCEPTIBLE TO MOXIDECTIN IN JIRDS (n = 6) EXAMPLE 7 Determination of the Toxicity and Efficacy of Verapamil alone and in Combination with Ivermectin This experiment was carried out to determine the toxicity of verapamil, a weak agent that invests MDR, alone and in combination with ivermectin, the efficacy of verapamil alone against H. with t ort us and the effect of verapamil 20 mg / kg on the efficacy of ivermectin against susceptible and moxidectin-resistant worms. The dose ratio of verapamil between 20 and 80 mg / kg was used alone or in combination with ivermectin at 0.024 and 0.046 mg / kg in H. infected jirds with t susceptible or resistant to moxidectin. Verapamil is given concomitantly with ivermectin by the oral route. The results are shown in Table 3.

Table 3. DEMONSTRATION OF TOXICITY AND EFFECTIVENESS OF THE VERAPAMIL (VRP) WITH OR WITHOUT IVERMECTIN AGAINST STRAINS OF H. with t ort us SUSCEPTIBLE OR RESISTANT TO MOXIDECTIN IN JIRDS (n = # per group) ** The dose of ivermectin is 0.024 mg / kg against strain PF14 and 0.046 mg / kg against strain MOF14.

As no deaths or other signs of toxicity were observed at a verapamil dose rate of 20 mg / kg, in the absence or presence of ivermectin, this dose ratio was used for subsequent experiments that reverse resistance. The varapamil alone, was found to have no significant effect on the count of the worm at any of the dose proportions used.

The toxicity of verapamil is summarized in the Table 4 Table 4. VERAPAMIL TOXICITY IN JIRDS * Ivermectin was used at 0.24 mg / kg or 0.046 mg / kg in accordance with Table 3.

Due to the toxicity of verapamil, at doses of 40 mg / kg above, only the effects of verapamil at 20 mg / kg on the efficacy of ivermectin against susceptible worms and resistant to moxidectin were considered. These results, shown in Figures 15A and 15B, are summarized in Table 5. Verapamil at 20 mg / kg significantly increases the efficacy of ivermectin against moxidectin-resistant worms.

Table 5. EFFECT OF VERAPAMIL (20 mg / kg) ON THE EFFICACY (%) OF IVERMECTIN # Ivermectin is administered at 0.024 mg / kg in jirds infected with strains PF14 and at 0.046 mg / kg in jirds infected with the strain MOF14"n.s" indicates that the worms counted are not significantly different from the controls; "A" suggests significantly different from the controls, but not from other counted worms, from the same strain, with the same letter; lB 'suggests significantly different from the worms counted from WA "by the same strain and dose ratio of ivermectin.

EXAMPLE 8 The Effects of Verapamil on the Efficacy of Moxidectin and Ivermectin Against H. contortus Susceptible and Resistant to Moxidectin.

This experiment was conducted in jirds to determine the effects of verapamil at 20 mg / kg on the efficacy of moxidectin and ivermectin against H. contortus susceptible and resistant to moxidectin. All treatments have 7 jirds / group. The dose ratios of moxidectin and ivermectin are selected to give approximately 50% efficacy in the absence of verapamil. Verapamil at 20 mg / kg significantly increases the efficacy of moxidectin against resistant worms. The increase is observed when verapamil coadministered with ivermectin is not significant in this experiment as the efficacy obtained with ivermectin alone which is already relatively high. The results are shown in Table 6 (see graphic representation of the results in Figures 16A and 16B).

Table 6. EFFECT OF VERAPAMIL ON THE (%) EFFICACY OF MOXIDECTIN AND IVERMECTINE AGAINST JET CEPAS. with t ort us SUSCEPTIBLE AND RESISTANT TO MOXIDECTIN (MOF14) * Verapamil is administered at 20 mg / kg. All treatments are by oral route. # of different letters indicate that the counted male worms are statistically different. However, the results of ivermectin (+ _ verapamil) are not compared with the results of moxidectin (+ _ verapamil). The verapamil itself increases the count of worms, but the male counting is not statistically different from the control.

This experiment confirms that the weak verapamil agent that invests the MDR overcomes the resistance in nematodes to acrolactones. These results are completely consistent with previous molecular evidence that resistance to macrolactone is associated with over-expression of the P-glycoprotein homologue due to a change in the P-glycoprotein DNA in resistant parasites. More potent agents that reverse MDR, such as cyclosporin A, SDZ-PSC 833 or other potent reversal agents can, at low dose rates, markedly increase the efficacy of macrocyclic lactone endectocides against resistant parasites. In the aforesaid, a detailed description of particular embodiments of the present invention has been provided for the purposes of illustration and not limitation. It is understood that all other modifications, ramifications and equivalents obvious to those skilled in the art, based on this description, are intended to be included within the scope of the invention as claimed.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT; (A) NAME: McGill University (B) ADDRESS: 845 Sherbrooke Street W. (C) CITY: Montreal (D) PROVINCE: Quebec (E) COUNTRY: Canada (F) POSTAL CODE: H3A 2T5 (ii) TITLE OF THE INVENTION; METHODS TO DETECT AND INVEST THE RESISTANCE TO MACROCICLIC LACTONE COMPOUNDS (iii) SEQUENCE NUMBER; 12 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: hard disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (v) CURRENT APPLICATION DATE: (A) APPLICATION NUMBER: PCT / IB98 / 00735 (B) SUBMISSION DATE: APRIL 29, 1998 (C) CLASSIFICATION: (vi) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 60 / 045,160 (B) DATE OF SUBMISSION: APRIL 30, 1997 (A) APPLICATION NUMBER: US 09 / 067,676 (B) DATE OF SUBMISSION: 28 -ABRIL-1998 (vii) PERMITTED INFORMATION / AGENT: (A) NAME: SMART & BIGGAR (B) ADDRESS: POBOX 2999, STATION D (C) CITY: OTTAWA (D) PROVINCE: ONTARIO (E) COUNTRY: CA ADÁ (F) POSTAL CODE: K1P 5Y6 (G) REFERENCE NUMBER / DOCUMENT: 73836- 3 (viii) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (613) -232-2486 (B) TELEFAX: (613) -232-8440 (2) INFORMATION FOR SEQ ID NO: 1:ERISTICS OF THE SEQUENCE: (A) LENGTH: 432 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: ACGGTGGCGT TTGTTGGGCA GTCTGGTTGT GGAAAAAGCA CTGTGAAGGC GTTGTTGGAC 60 GGTTTTACAA TCAAAACAAG GGCGTGATTA CGGACGCCGA AAACATCAGA AACATGAACA 120 TACGCAATCT TCGTGAGCAA GTGTGTATTG TAAGCCAGGA ACCAACGCTG TTCGACTGTA 180 CCATCATGGA AAACATCTGT TACGGTCTCG ATCGACCCCA AGCTCCTACG AACAGGTTGT 240 TGCTGCAGCA AAATCGGTCG AGTCGAAATG GCGAACATTC ACAATTTTGT GCTGGGACTA 300 CCAGAGGGTT ACGATACGCG TGTTGGTGAG AAAGGCACTC AGCTGTCAGG CGGACAGAAG 360 AAACGAATAG CCATAGCCAG AGCGCTGATT CGAGATCCGC CTATACTTCT GCTGGATGAG 420 GCTACGACGG CC 432 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 144 amino acids (B) TYPE: amino acids (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protelna (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Thr Val Wing Phe Val Gly Gln Ser Gly Cys Gly Lys Ser Thr Val Lys 1 5 10 15 Wing Leu Leu Glu Arg Phe Tyr Asn Gln Asn Lys Gly Val lie Thr Asp 20 25 30 Wing Glu Asn lie Arg Asn Met Asn lie Arg Asn Leu Arg Glu Gln Val 35 40 45 Cys lie Val Ser Gln Glu Pro Thr Leu Phe Asp Cys Thr lie Met Glu 50 55 60 Asn lie Cys Tyr Gly Leu Asp Asp Pro Lys Leu Leu Arg Thr Gly Cys 65 70 75 80 Cys Cys Ser Lys lie Gly Arg Val Glu Met Wing Asn lie His Asn Phe 85 90 95 Val Leu Gly Leu Pro Glu Gly Tyr Asp Thr Arg Val Gly Glu Lys Gly 100 105 110 Thr Gln Leu Ser Gly Gly Gln Lys Lys Arg lie Ala lie Wing Arg Ala 115 120 125 Leu lie Arg Asp Pro Pro lie Leu Leu Leu Asp Glu Ala Thr Thr Ala 130 135 140 (2) INFORMATION FOR SEQ ID NO: 3: Ei) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4175 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: GGTTTAATTA CCCAAGTTTG AGAGATCGTT CTCAAGCTGG TAAAATGTTC GAAAAAGGCC 60 AAGATGATGA ACGTATACCA TTACTCGGTT CATCCAAGAA AAGTTCAATC GGCGAAGTCA 120 GTAAAAAAGA AGAACCGCCT ACAATAACAA ACCGTGGAAT TCTCTCCTTA GCCACTACAT 180 TGGATTATGT GCTTCTTGCG GCTGGTACGC TGGCGCCGTG TGTTCATGGC GCTGGATTCT 240 CAGTACTCGG TATTGTACTC GGTGGTATGA CGACAGTCTT TCTCAGAGCT CAGAACTCAG 300 AATTCGTTCT GGGCACTGTT AGTCGGGATC CTGAAGGGCT ACCAGCTCTT ACTAAGGAAG 360 AATTTGACAC ACTAGTACGT AGGTATTGCT TATACTACCT TGGATTAGGC TTTGCTATGT 420 TTGCAACATC TTATATACAG ATTGTGTGTT GGGAGACGTT CGCCGAACGA ATTACCCATA 480 AATTACGAAA AATTTATCTA AAAGCCATAC TTCGGCAGCA GATCTCATGG TTTGACATTC 540 AACAAACAGG AAATCTCACA GCTCGTCTAA CCGATGATCT CGAACGTGTT CGTGAAGGAC 600 TTGGTGATAA ACTGTCGCTT TTTATACAAA TGGTGTCTGC TTTTGTGGCT GGTTTCTGTG 660 TAGGATTCGC GTATAGCTGG TCAATGACGC TCGTGATGAT GGTCGTGGCG CCGTTTATAG_720_TTATTTCTGC TAATTGGATG TCAAAAATCG TTGCTACTAG GACCCAAGTT GAACAGGAAA 780 CCTACGCTGT TGCCGGTGCT ATAGCGGAGG AGACTTTCTC ATCGATACGA ACCGTACACT 840 CGATATGTGG CCATAAAAGA GAGCTAACAA GATTTGAGGC AGCGTTGGAG AAAGGACGTC 900 AGACAGGCCT TGTCAAATAT TTCTATATGG GTGTTGGTGT GGGATTTGGT CAGATGTGTA 960 CCTATGTGTC CTACGCCTTG GCTTTTTGGT ATGGCAGTGT ACTGATCATC AACGACCCTG 1020 CATTGGATCG TGGCCGAATT TTCACAGTCT TTTTTGCTGT GATGTCCGGC TCAGCAGCTC 1080 TCGGCACATG TCTGCCACAT CTTAACACCA TATCCATCGC TCGAGGAGCG GTACGAAGTG 1140 TACTGTCAGT GATTAATAGT CGTCCAAAAA TCGATCCCTA TTCGTTAGAT GGCATTGTGC 1200 TCAACAATAT GAGAGGATCT ATCCGCTTCA AGAACGTGCA TTTCTCCTAT CCTTCCCGAA 1260 GAACATTGCA GATATTGAAA GGTGTGTCAC TGCAAGTGTC GGCTGGCCAA AAAATTGCTT 1320 TGGTGGGTTC AAGCGGTTGT GGAAAGTCAA CGAACGTCAA TTTATTATTG AGATTTTATG 1380 ATCCGACAAG GGGAAAGGTA ACCATAGATG ATATTGATGT GTGTGATCTC AACGTGCAAA 1440 AACTTCGTGA ACAAATCGGT GTTGTTAGTC AGGAACCAGT GCTTTTCGAT GGCACACTAT 1500 TCGAAAATAT CAAGATGGGT TATGAACAGG CCACAATGGA GGAGGTCCAA GAAGCGTGCC 1560 GTGTGGCGAA TGCTGCCGAC TTCACCAAAC GACTTCCAGA AGGTTACGGC ACCCGAGTTG 1620 GTGAACGTGG TGTGCAGTTA AGTGGCGGAC AAAAGCAGCG AATTGCCATA GCTCGTGCGA 1680 TCATCAAGAA CCCTCGCATA CTGCTGCTCG ATGAAGCCAC CAGTGCTCTA GACACAGAAG 1740 CGGAATCAAT CGTGCAAGAG GCTCTGGAGA AGGCTCAAAA AGGGAGAACA ACCGTCATTG 1800 TAGCGCATCG TCTGTCTACT ATCAGAAACG TGGATCAGAT TTTCGTTTTC AAGAACGGAA 1860 CGATCGTTGA GCAGGGCACT CATGCCGAGT TGATGAACAA ACGTGGAGTA TTCTTTGAAA 1920 TGACTCAAGC ACAAGTCCTC CGACAAGAGA AGGAAGAGGA AGTTTTAGAT AGCGATGCGG 1980 AATCCGATGT CGTGTCACCG GATATTGCAT TACCCCATCT TAGTTCACTT CGATCCCGTA 2040 AAGAATCCAC AAGAAGTGCT ATCTCCGCGG TCCCCAGCGT TCGAAGTATG CAAATCGAAA 2100 TGGAGGACCT TCGTGCCAAA CCAACTCCAA TGTCGAAAAT TTTCTATTTT AACCGTGACA 2160 AATGGGGATA TTTCATTTTG GGACTCATCG CCTGTATTAT TACTGGAACT GTTACACCGA 2220 CATTTGCAGT TTTATATGCG CAGATCATAC AGGTATACTC GGAACCTGTT GATCAAATGA 2280 AAGGCCATGT GCTGTTCTGG TGTGGAGCTT TCATCGTCAT TGGTCTCGTA CACGCTTTTG 2340 CGTTCTTTTT CTCGGCTATT TGTTTGGGAC GTTGCGGCGA AGCGTTAACG AAAAAATTAC 2400 GTTTCGAGGC GTTCAAGAAC CTTCTGCGAC AGAATGTGGG ATTCTACGAC GATATCCGAC 2460 ACGGTACCGG TAAACTCTGT ACGCGATTTG CTACAGATGC ACCCAATGTC CGATATGTGT 2520 TCACTCGACT TCCGGGTGTG CTTTCATCGG TGGTGACCAT AATTGGAGCT TTGGTTATTG 2580 GATTCATCTT CGGGTGGCAG CTGGCTTTGA TTCTTATGGT GATGGTACCG TTGATCATCG 2640 GTAGTGGATA CTTCGAGATG CGCATGCAGT TTGGTAAGAA GATGCGTGAC ACAGAGCTTC 2700 TTGAAGAGGC TGGGAAAGTT GCCTCTCAAG CCGTGGAGAA CATTCGTACC GTGCATGCCC 2760 TGAATAGGCA AGAGCAGTTC CATTTCATGT ATTGCGAGTA TTTGAAGGAA CCCTATCGAG 2820 AAAATCTTTG CCAGGCGCAC ACCTACGGGG GTGTATTCGC GTTCTCACAA TCGTTGTTAT 2880 TCTTTATGTA TGCTGTAGCA TTTTGGATTG GTGCAATCTT CGTGGACAAC CACAGCATGC 2940 AACCGATTGA CGTTTACCGA GTATTTTTCG CGTTCATGTT TTGTGGACAA ATGGTCGGCA 3000 ACATTTCTTC TTTTATTCCT GACGTTGTGA AAGCTCGCCT GGCTGCATCG CTCCTTTTCT 3060 ACCTTATCGA ACACCCATCA GAAATTGATA ATTTGTCCGA GGATGGTGTC ACGAAGAAAA 3120 TCTCTGGTCA TATCTCGTTC CGCAATGTCT ATTTCAATTA TCCGACAAGA AGACAGATCA 3180 GAGTACTCCG TGGACTTAAC CTAGAGATAA ATCCTGGCAC GACGGTAGCG CTTGTTGGGC 3240 AGTCTGGTTG TGGAAAAAGC ACTGTGATGG CGTTGTTGGA ACGGTTTTAC AATCAAAACA 3300 AGGGCGTGAT TACGGTGGAC GGCGAAAACA TCAGAAACAT GAACATACGC AATCTTCGTG 3360 AGCAAGTGTG TATTGTTAGC CAGGAACCAA CGCTGTTCGA CTGTACCATC ATGGAAAACA 3420 TCTGTTACGG TCTCGATGAC CCCAAGCCGT CCTACGAACA GGTTGTTGCT GCAGCAAAAA 3480 TGGCGAACAT TCACAATTTT GTGCTGGGAC TACCAGAGGG TTACGATACG CGTGTTGGTG 3540 ARAAAGGCAC TCAGCTGTCA GGCGGACAGA AGCAACGAAT AGCCATAGCC AGAGCGCTGA 3600 TTCGAGATCC GCCTATACTT CTGCTGGATG AGGCGACAAG CGCGCTGGAT ACCGAGAGTG 3660 AAAAGATCGT GCAAGACGCC CTAGAGGTTG CTCGCCAAGG TAGAACGTGC CTTGTAATTG 3720 CCCATCGCCT TTCT CAATT CAAGACAGTG ACGTCATAGT GATGATCCAG GAGGGGAAAG 3780 CTACAGACAG AGGCACTCAT GAACATTTAC TGATGAAGAA CGATCTATAC AAACGGCTAT 3840 GCGAAACACA ACGACTCGTT GAATCACAAT GAGTTTTTAG TGCCAATCGA TAGTGATCGA 3900 TAAGCTATGG ATTAGTCTTT AACACTTACT GATCATATGA CTCTATCTCG TGCTTTATTA 3960 TAATGTACAT ATGTAATGGT TTTGATCTTA CATATCTTGT AATTGGTCCT CACTATCATA 4020 ATGCCTTTAG TAGTATATTA ACAGTTTTAT TAATACAACT TAAGTAACAT ATTAACAATT 4080 TTATTAATAT AACTTAAGTA AGATATTGAC AGTTTTATTA ATTTGGAGGA TTTATAATAA 4140 AACCTCGTGC CGCTCGTGCC GAAACGATAT CAAGC 4175 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1810 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GGTTTAATTA CCCAAGTTTG AGAGATCGTT CTCAAGCTGG TAAAATGTTC GAAAAAGGCC 60 AAGATGATGA ACGTATACCA TTACTCGGTT CATCCAAGAA AAGTTCAATC GGCGAAGTCA 120 GTAAAAAAGA AGAACCGCCT ACAATAACAA ACCGTGGAAT TCTCTCCTTA GCCACTACAT 180 TGGATTATGT GCTTCTTGCG GCTGGTACGC TGGCGCCGTG TGTTCATGGC GCTGGATTCT 240 CAGTACTCGG TATTGTACTC GGTGGTATGA CGACAGTCTT TCTCAGAGCT CAGAACTCAG 300 AATTCGTTCT GGGCACTGTT AGTCGGGATC CTGAAGGGCT ACCAGCTCTT ACTAAGGAAG 360 AATTTGACAC ACTAGTACGT AGGTATTGCT TATACTACCT TGGATTAGGC TTTGCTATGT 420 TTGCAACATC TTATATACAG ATTGTGTGTT GGGAGACGTT CGCCGAACGA ATTACCCATA 480 AATTACGAAA AATTTATCTA AAAGCCATAC TTCGGCAGCA GATCTCATGG TTTGACATTC 540 AACAAACAGG AAATCTCACA GCTCGTCTAA CCGATGATCT CGAACGTGTT CGTGAAGGAC 600 TTGGTGATAA ACTGTCGCTT TTTATACAAA TGGTGTCTGC TTTTGTGGCT GGTTTCTGTG 660GTATAGCTGG TCAATGACGC TCGTGATGAT GGTCGTGGCG CCGTTTATAG_720_TTATTTCTGC TAATTGGATG TCAAAAATCG TTGCTACTAG GACCCAAGTT GAACAGGAAA 780 CCTACGCTGT TGCCGGTGCT ATAGCGGAGG AGACTTTCTC ATCGATACGA ACCGTACACT 840 CGATATGTGG CCATAAAAGA GAGCTAACAA GATTTGAGGC AGCGTTGGAG AAAGGACGTC 900 AGACAGGCCT TGTCAAATAT TTCTATATGG GTGTTGGTGT GGGATTTGGT CAGATGTGTA 960 CCTATGTGTC CTACGCCTTG GCTTTTTGGT ATGGCAGTGT ACTGATCATC AACGACCCTG 1020 CATTGGATCG TGGCCGAATT TTCACAGTCT TTTTTGCTGT GATGTCCGGC TCAGCAGCTC 1080 TCGGCACATG TCTGCCACAT CTTAACACCA TATCCATCGC TCGAGGAGCG GTACGAAGTG 1140 TACTGTCAGT GATTAATAGT CGTCCAAAAA TCGATCCCTA TTCGTTAGAT GGCATTGTGC 1200 TCAACAATAT GAGAGGATCT ATCCGCTTCA AGAACGTGCA TTTCTCCTAT CCTTCCCGAA 1260 GAACATTGCA GATATTGAAA GGTGTGTCAC TGCAAGTGTC GGCTGGCCAA AAAATTGCTT 1320 TGGTGGGTTC AAGCGGTTGT GGAAAGTCAA CGAACGTCAA TTTATTATTG AGATTTTATG 1380 ATCCGACAAG GGGAAAGGTA ACCATAGATG ATATTGATGT GTGTGATCTC AACGTGCAAA 1440 AACTTCGTGA ACAAATCGGT GTTGTTAGTC AGGAACCAGT GCTTTTCGAT GGCACACTAT 1500 TCGAAAATAT CAAGATGGGT TATGAACAGG CCACAATGGA GGAGGTCCAA GAAGCGTGCC 1560 GTGTGGCGAA TGCTGCCGAC TTCACCAAAC GACTTCCAGA AGGTTACGGC ACCCGAGTTG 1620 GTGAACGTGG TGTGCAGTTA AGTGGCGGAC AAAAGCAGCG AATTGCCATA GCTCGTGCGA 1680 TCATCAAGAA CCCTCGCATA CTGCTGCTCG ATGAAGCCAC CAGTGCTCTA GACACAGAAG 1740 CGGAATCAAT CGTGCAAGAG GCTCTGGAGA AGGCTCAAAA AGGGAGAACA ACCGTCATTG 1800 TAGCGCATCG 1810 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2698 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AGTGCTTTTC GATGGCACAC TATTCGAAAA TATCAAGATG GGTTATGAAC AGGCCACAAT 60 GGAGGAGGTC CAAGAAGCGT GCCGTGTGGC GAATGCTGCC GACTTCACCA AACGACTTCC 120 AGAAGGTTAC GGCACCCGAG TTGGTGAACG TGGTGTGCAG TTAAGTGGCG GACAAAAGCA 180 GCGAATTGCC ATAGCTCGTG CGATCATCAA GAACCCTCGC ATACTGCTGC TCGATGAAGC 240 CACCAGTGCT CTAGACACAG AAGCGGAATC AATCGTGCAA GAGGCTCTGG AGAAGGCTCA 300 AAAAGGGAGA ACAACCGTCA TTGTAGCGCA TCGTCTGTCT ACTATCAGAA ACGTGGATCA 360 GATTTTCGTT TTCAAGAACG GAACGATCGT TGAGCAGGGC ACTCATGCCG AGTTGATGAA 420 CAAACGTGGA GTATTCTTTG AAATGACTCA AGCACAAGTC CTCCGACAAG AGAAGGAAGA 480 GGAAGTTTTA GATAGCGATG CGGAATCCGA TGTCGTGTCA CCGGATATTG CATTACCCCA 540 TCTTAGTTCA CTTCGATCCC GTAAAGAATC CACAAGAAGT GCTATCTCCG CGGTCCCCAG 600 CGTTCGAAGT ATGCAAATCG AAATGGAGGA CCTTCGTGCC AAACCAACTC CAATGTCGAA 660 AATTTTCTAT TTTAACCGTG ACAAATGGGG ATATTTCATT TTGGGACTCA TCGCCTGTAT 720 TATTACTGGA ACTGTTACAC CGACATTTGC AGTTTTATAT GCGCAGATCA TACAGGTATA 780 CTCGGAACCT GTTGATCAAA TGAAAGGCCA TGTGCTGTTC TGGTGTGGAG CTTTCATCGT 840 CATTGGTCTC GTACACGCTT TTGCGTTCTT TTTCTCGGCT ATTTGTTTGG GACGTTGCGG 900 CGAAGCGTTA ACGAAAAAAT TACGTTTCGA GGCGTTCAAG AACCTTCTGC GACAGAATGT 960 GGGATTCTAC GACGATATCC GACACGGTAC CGGTAAACTC TGTACGCGAT TTGCTACAGA 1020 TGCACCCAAT GTCCGATATG TGTTCACTCG ACTTCCGGGT GTGCTTTCAT CGGTGGTGAC 1080 CATAATTGGA GCTTTGGTTA TTGGATTCAT CTTCGGGTGG CAGCTGGCTT TGATTCTTAT 1140 GGTGATGGTA CCGTTGATCA TCGGTAGTGG ATACTTCGAG ATGCGCATGC AGTTTGGTAA 1200 GAAGATGCGT GACACAGAGC TTCTTGAAGA GGCTGGGAAA GTTGCCTCTC AAGCCGTGGA 1260 GAACATTCGT ACCGTGCATG CCCTGAATAG GCAAGAGCAG TTCCATTTCA TGTATTGCGA 1320 GTATTTGAAG GAACCCTATC GAGAAAATCT TTGCCAGGCG CACACCTACG GGGGTGTATT 1380 CGCGTTCTCA CAATCGTTGT TATTCTTTAT GTATGCTGTA GCATTTTGGA TTGGTGCAAT 1440 CTTCGTGGAC AACCACAGCA TGCAACCGAT TGACGTTTAC CGAGTATTTT TCGCGTTCAT 1500 GTTTTGTGGA CAAATGGTCG GCAACATTTC TTCTTTTATT CCTGACGTTG TGAAAGCTCG 1560 CCTGGCTGCA TCGCTCCTTT TCTACCTTAT CGAACACCCA TCAGAAATTG ATAATTTGTC 1620 CGAGGATGGT GTCACGAAGA AAATCTCTGG TCATATCTCG TTCCGCAATG TCTATTTCAA 1680 TTATCCGACA AGAAGACAGA TCAGAGTACT CCGTGGACTT AACCTAGAGA TAAATCCTGG 1740 CACGACGGTA GCGCTTGTTG GGCAGTCTGG TTGTGGAAAA AGCACTGTGA TGGCGTTGTT 1800 GGAACGGTTT TACAATCAAA ACAAGGGCGT GATTACGGTG GACGGCGAAA ACATCAGAAA 1860 CATGAACATA CGCAATCTTC GTGAGCAAGT GTGTATTGTT AGCCAGGAAC CAACGCTGTT 1920 CGACTGTACC ATCATGGAAA ACATCTGTTA CGGTCTCGAT GACCCCAAGC CGTCCTACGA 1980 ACAGGTTGTT GCTGCAGCAA AAATGGCGAA CATTCACAAT TTTGTGCTGG GACTACCAGA 2040 GGGTTACGAT ACGCGTGTTG GTGARAAAGG CACTCAGCTG TCAGGCGGAC AGAAGCAMCG 2100 AATAGCCATA GCCAGAGCGC TGATTCGAGA TCCGCCTATA CTTCTGCTGG ATGAGGCGAC 2160 AAGCGCGCTG GATACCGAGA GTGAAAAGAT CGTGCAAGAC GCCCTAGAGG TTGCTCGCCA 2220 AGGTAGAACG TGCCTTGTAA TTGCCCATCG CCTTTCTACA ATTCAAGACA GTGACGTCAT 2280 AGTGATGATC CAGGAGGGGA AAGCTACAGA CAGAGGCACT CATGAACATT TACTGATGAA 2340 GAACGATCTA TACAAACGGC TATGCGAAAC ACAACGACTC GTTGAATCAC AATGAGTTTT 2400 TAGTGCCAAT CGATAGTGAT CGATAAGCTA TGGATTAGTC TTTAACACTT ACTGATCATA 2460 TGACTCTATC TCGTGCTTTA TTATAATGTA CATATGTAAT GGTTTTGATC TTACATATCT 2520 TGTAATTGGT CCTCACTATC ATAATGCCTT TAGTAGTATA TTAACAGTTT TATTAATACA 2580 ACTTAAGTAA CATATTAACA ATTTTATTAA TATAACTTAA GTAAGATATT GACAGTTTTA 2640 TTAATTTGGA GGATTTATAA TAAAACCTCG TGCCGCTCGT GCCGAAACGA TATCAAGC 2698 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1275 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Met Phe Glu Lys Gly Gln Asp Asp Glu Arg lie Pro Leu Leu Gly Ser 1 5 10 15 Ser Lys Lys Ser Ser lie Gly Glu Val Ser Lys Lys Glu Glu Pro Pro 20 25 30 Thr lie Thr Asn Arg Gly lie Leu Ser Leu Wing Thr Thr Leu Asp Tyr 35 40 45 Val Leu Leu Wing Wing Gly Thr Leu Wing Pro Cys Val His Gly Wing Gly 50 55 60 Phe Ser Val Leu Gly He Val Leu Gly Gly Met Thr Thr Val Phe Leu 65 70 75 80 Arg Wing Gln Asn Ser Glu Phe Val Leu Gly Thr Val Ser Arg Asp Pro 85 90 95 Glu Gly Leu Pro Wing Leu Thr Lys Glu Glu Phe Asp Thr Leu Val Arg 100 105 110 Arg Tyr Cys Leu Tyr Tyr Leu Gly Leu Gly Phe Wing Met Phe Wing Thr 115 120 125 Ser Tyr He Gln He Val Cys Trp Glu Thr Phe Wing Glu Arg He Thr 130 135 140 His Lys Leu Arg Lys He Tyr Leu Lys Wing He Leu Arg Gln Gln He 145 150 155 160 Being Trp Phe Asp He Gln Gln Thr Gly Asn Leu Thr Wing Arg Leu Thr 165 170 175 Asp Asp Leu Glu Arg Val Arg Glu Gly Leu Gly Asp Lys Leu Ser Leu 180 185 190 Phe He Gln Met Val Ser Wing Phe Val Wing Gly Phe Cys Val Gly Phe 195 200 205 Wing Tyr Ser Trp Ser Met Thr Leu Val Met Met Val Val Ala Pro Phe 210 215 220 He Val He Ser Wing Asn Trp Met Ser Lys He Val Wing Thr Arg Thr 225 230 235 240 Gln Val Glu Gln Glu Thr Tyr Ala Val Ala Gly Ala He Ala Glu Glu 245 250 255 Thr Phe Ser Ser He Arg Thr Val His Ser He Cys Gly His Lys Arg 260 265 270 Glu Leu Thr Arg Phe Glu Ala Ala Leu Glu Lys Gly Arg Gln Thr Gly 275 280 285 Leu Val Lys Tyr Phe Tyr Met Gly Val Gly Val Gly Phe Gly Gln Met 290 295 300 Cys Thr Tyr Val Ser Tyr Ala Leu Ala Phe Trp Tyr Gly Ser Val Leu 305 310 315 320 He He Asn Asp Pro Wing Leu Asp Arg Gly Arg He Phe Thr Val Phe 325 330 335 Phe Wing Val Met Ser Gly Wing Wing Wing Leu Gly Thr Cys Leu Pro His 340 345 350 Leu Asn Thr He Ser Wing Wing Arg Gly Wing Val Arg Ser Val Leu Ser 355 360 365 Val He Asn Ser Arg Pro Lys He Asp Pro Tyr Ser Leu Asp Gly He 370 375 380 Val Leu Asn Asn Met Arg Gly Ser He Arg Phe Lys A = n Val His Phe 385 390 395 400 Ser Tyr Pro Ser Arg Arg Thr Leu Gln He Leu Lys Gly Val Ser Leu 405 410 415 Gln Val Ser Wing Gly Gln Lys Wing Wing Leu Val Gly Being Ser Gly Cys 420 425 430 Gly Lys Ser Thr Asn Val Asn Leu Leu Arg Phe Tyr Asp Pro Thr 435 440 445 Arg Gly Lys Val Thr He Asp Asp He Asp Val Cys Asp Leu Asn Val 450 455 460 Gln Lys Leu Arg Glu Gln He Gly Val Val Ser Gln Glu Pro Val Leu 465 470 475 480 Phe Asp Gly Thr Leu Phe Glu Asn He Lys Met Gly Tyr Glu Gln Ala 485 490 495 Thr Met Glu Glu Val Gln Glu Wing Cys Arg Val Wing Asn Wing Wing Asp 500 505 510 Phe Thr Lys Arg Leu Pro Glu Gly Tyr Gly Thr Arg Val Gly Glu Arg 515 520 525 Gly Val Gln Leu Ser Gly Gly Gln Lys Gln Arg He Wing He Wing Arg 530 535 540 Wing He He Lys Asn Pro Arg He Leu Leu Leu Asp Glu Wing Thr Ser 545 550 555 560 Ala Leu Asp Thr Glu Ala Glu Ser He Val Gln Glu Ala Leu Glu Lys 565 570 575 Wing Gln Lys Gly Arg Thr Thr Val He Val Wing His Leu Arg Ser Thr 580 585 590 He Arg Asn Val Asp Gln He Phe Val Phe Lys Asn Gly Thr He Val 595 600 605 Glu Gln Gly Thr His Wing Glu Leu Met Asn Lys Arg Gly Val Phe Phe 610 615 620 Glu Met Thr Gln Ala Gln Val Leu Arg Gln Glu Lys Glu Glu Glu Val 625 630 635 640 Leu Asp Ser Asp Ala Glu Be Asp Val Val Ser Pro Asp He Ala Leu 645 650 655 Pro His Leu Be Ser Leu Arg Be Arg Lys Glu Be Thr Arg Be Wing 660 665 670 Be Ser Wing Val Pro Ser Val Arg Ser Met Gln He Glu Met Glu Asp 675 680 685 Leu Arg Ala Lys Pro Thr Pro Met Ser Lys He Phe Tyr Phe Asn Arg 690 695 700 Asp Lys Trp Gly Tyr Phe He Leu Gly Leu He Wing Cys He He Thr 705 710 715 720 Gly Thr Val Thr Pro Thr Phe Ala Val Leu Tyr Ala Gln He He Gln 725 730 735 Val Tyr Ser Glu Pro Val Asp Gln Met Lys Gly His Val Leu Phe Trp 740 745 750 Cys Gly Wing Phe He Val He Gly Leu Val His Wing Phe Wing Phe Phe 755 760 765 Phe Ser Wing Cys Leu Gly Arg Cys Gly Wing Glu Leu Thr Lys Lys 770 775 780 Leu Arg Phe Glu Wing Phe Lys Asn Leu Leu Arg Gln Asn Val Gly Phe 785 790 795 800 Tyr Asp Asp He Arg His Gly Thr Gly Lys Leu Cys Thr Arg Phe Wing 805 810 815 Thr Asp Ala Pro Asn Val Arg Tyr Val Phe Thr Arg Leu Pro Gly Val 820 825 830 Leu Ser Val Val Thr He He Gly Ala Leu Val He Gly Phe He 835 840 845 Phe Gly Trp Gln Leu Ala Leu He Leu Met Val Met Val Pro Leu He 850 855 860 He Gly Ser Gly Tyr Phe Glu Met Arg Met Gln Phe Gly Lys Met 865 870 875 880 Arg Asp Thr Glu Leu Leu Glu Glu Wing Gly Lys Val Wing Ser Gln Wing 885 890 895 Val Glu Asn He Arg Thr Val His Wing Leu Asn Arg Gln Glu Gln Phe 900 905 910 His Phe Met Tyr Cys Glu Tyr Leu Lys Glu Pro Tyr Arg Glu Asn Leu 915 920 925 Cys Gln Wing His Thr Tyr Gly Gly Val Phe Wing Phe Ser Gln Ser Leu 930 935 940 Leu Phe Phe Met Tyr Wing Val Wing Phe Trp He Gly Wing He Phe Val 945 950 955 960 Asp Asn His Ser Met Gln Pro He Asp Val Tyr Arg Val Phe Phe Wing 965 970 975 Phe Met Phe Cys Gly Gln Met Val Gly Asn He Ser Ser Phe He Pro 980 985 990 Asp Val Val Lys Ala Arg Leu Ala Wing Ser Leu Leu Phe Tyr Leu He 995 1000 1005 Glu His Pro Ser Glu He Asp Asn Leu Ser Glu Asp Gly Val Thr Lys 1010 1015 1020 Lys He Ser Gly His He Ser Phe Arg Asn Val Tyr Phe Asn Tyr Pro 1025 1030 1035 1040 Thr Arg Arg Gln He Arg Val Leu Arg Gly Leu Asn Leu Glu He Asn 1045 1050 1055 Pro Gly Thr Thr Val Wing Leu Val Gly Gln Ser Gly Cys Gly Lys Ser 1060 1065 1070 Thr Val Met Wing Leu Leu Glu Arg Phe Tyr Asn Gln Asn Lys Gly Val 1075 1080 1085 He Thr Val Asp Gly Glu Asn He Arg Asn Met Asn He Arg Asn Leu 1090 1095 1100 Arg Glu Gln Val Cys He Val Ser Gln Glu Pro Thr Leu Phe Asp Cys 1105 1110 1115 1120 Thr He Met Glu Asn He Cys Tyr Gly Leu Asp Asp Pro Lys Pro Ser 1125 1130 1135 Tyr Glu Gln Val Val Wing Wing Wing Lys Met Wing Asn He His Asn Phe 1140 1145 1150 Val Leu Gly Leu Pro Glu Gly Tyr Asp Thr Arg Val Gly Glu Lys Gly 1155 1160 1165 Thr Gln Leu Ser Gly Gly Gln Lys Gln Arg He Ala He Wing Arg Wing 1170 1175 1180 Leu He Arg Asp Pro Pro He Leu Leu Leu Asp Glu Wing Thr Ser Wing 1185 1190 1195 1200 Leu Asp Thr Glu Ser Glu Lys He Val Gln Asp Ala Leu Glu Val Ala 1205 1210 1215 Arg Gln Gly Arg Thr Cys Leu Val He Wing His Arg Leu Ser Thr He 1220 1225 1230 Gln Asp Ser Asp Val He Val Met He Gln Glu Gly Lys Wing Thr Asp 1235 1240 1245 Arg Gly Thr His Glu His Leu Leu Met Lys Asn Asp Leu Tyr Lys Arg 1250 1255 1260 Leu Cys Glu Thr Gln Arg Leu Val Glu Ser Gln 1265 1270 1275 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3512 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCCGTTGTTG CCGGTGCTAT AGCGGAGGAG ACTTTCTCAT CGATACGAAC CGTACACTCG 60 TTATGTGGCC ATAAAAGAGA GCTAACAAGG CAGCGTTGGA GAAAGGACGT CAGACAGGCC 120 TTGTCAAATA TTTCTATATG GGTGTTGGTG TGAGATTTGG TCAGATGTGT ACCTATGTGT 180 CCTACGCCTT GGCTTTTTGG TATGGCAGTG TACTGATCAT CAACGACCCT GCATTGGATC 240 GTGGCCGAAT TTTCACAGTC TTTTTGCTGT GATGTCCGGC TCAGCAGCTC TCGGCACATG 300 TCTGCCACAT CTTAACACCA TATCCATCGC TCGAGGAGCG GTACGAAGTG TACTGTCAGT 360 GATTAATAGT CGTCCAAAAA TCGATCCCTA TTCGTTAGAT GGCATTGTGC TCAACAATAT 420 GAGAGGATCT ATTCGCTTCA AGAACGTGCA TTTCTCCTAT CCTTCCCGAA GAACATTGCA 480 GATATTGAAA GGTGTGTCAC TGCAAGTGTC GGCTGGCCAA AAAATTGCTT TGGTGGGTTC 540 AAGCGGTTGT GGAAAGTCAA CGATCGTCAA TTTATTATTG AGATTTTATG ATCCGACAAG 600 GGGAAAGGTA ACCATAGATG ATATTGATGT GTGTGATCTC AACGTGCAAA AACTTCGTGA 660 ACAAATCGGT GTTGTTAGTC AGGAACCAGT GCTTTTCGAT GGCACACTAT TCGAAAATAT 720 CAAGATGGGT TATGAACAGG CCACAATGGA GGAGGTCCAA GAAGCGTGCC GTGTGGCGAA 780 0 TGCTGCCGAC TTCATCAAAC GACTTCCAGA AGGTTACGGC ACCCGAGTTG GTGAACGTGG 840 TGTGCAGTTA AGTGGCGGAC AAAAGCAGCG AATTGCCATA GCTCGTGCGA TCATCAAGAA 900 CCCTCGCATA CTGCTGCTCG ATGAAGCCAC CAGTGCTCTA GACACAGAAG CGGAATCAAT 960 CGTGCAAGAG GCTCTGGAGA AGGCTCAAAA AGGGAGAACA ACCGTCATTG TAGCGCATCG 1020 TCTGTCTACT ATCAGAAACG TGGATCAGAT 'TTTCGTTTTC AAGAACGGAA CGATCGTTGA 1080 GCAGGGCACT CATGCCGAGT TGATGAACAA ACGTGGAGTA TTCTTTGAAA TGACTCAAGC 1140 ACAAGTCCTC CGACAAGAGA AGGAAGAGGA AGTTTTAGAA AATACGGAAC CAGTAGCGAA 1200 GTGTCAAGAG GTATCCCTCC CTGCTCCTGA TGTCACTATT TTGGCTCCCC ATGAGGAACA 1260 ACCCGAGCTA CCTAGCCCGC CGGGTCGGTT AGAAAATACA AAGCAACATG AGCATCTCTG 1320 AATGTCTTTG TCTGAGATAG CGATGCGGAA TCCGATGTCG TGTCACCGGA TATTGCATTA 1380 CCCCATCTTA GTTCACTTCG ATCCCGTAAA GAATCCACAA GAAGTGCTAT CTCCGCGGTC 1440 CCCAGCGTTC GAAGTATGCA AATCGAAATG GAGGACCTTC GTGCCAAACC AACTCCAATG 1500 TCGAAAATTT TCTATTTTAA CCGTGACAAA TGGGCATATT TCATTTTGGG ACTCATCGCC 1560 TGTATTATTA CTGGAACTGT TACACCGACA TTTGCAGTTT TATATGCGCA GATCATACAG 1620 GTATACTCGG AACCTGTTGA TCAAATGAAA GGCCATGTGC TGTTCTGGTG TGGAGCTTTC 1680 ATCGTCATTG GTCTCGTACA CGCTTTTGCG TTCTTTTTCT CGGCTATTTG TTTGGGACGT 1740 TGCGGCGAAG CGTTAACGAA AAAATTACGT TTCGAGGCGT TCAAGAACCT TCTGCGACAG 1800 GATGTGGGAT TCTACGACGA TATCCGACAC GGTACCGGTA AACTCTGTAC GCGATTTGCT 1860 ACAGATGCAC CCAATGTCCG ATATGTGTTC ACTCGACTTC CGGGTGTGCT TTCATCGGTG 1920 GTGACCATAA TTGGAGCTTT GGTTATTGGA TTCATCTTCG GGTGGCAGCT GGCTTTGATT 1980 CTTATGGTGA TGGTACCGTT GATCATCGGT AGTGGATACT TCGAGATGCG CATGCAGTTT 2040 GGTAAGGAGA TGCGTGACAC AGAGCTTCTT GAAGAGGCTG GGAAAGTTGC CTCTCAAGCC 2100 GTGGAGAACA TTCGTACCGT GCATGCCCTG AATAGGCAAG AGCAGTTCCA TTTCATGTAT 2160 TGCGAGTATT TGAAGGAACC CTATCGAGAA AATCTTTGCC AGGCGCACAC CTACGGGGGT 2220 GTATTCGCGT TCTCACAATC GTTGTTATTC TTTATGTATG CTGTAGCATT TTGGATTGGT 2280 GCAATCTTCG TGGACAACCA CAGCATGCAA CCGATTGACG TTTACCGAGT ATTTTTCGCG 2340 TTCATGTTTT GTGGACAAAT GGTCGGCAAC ATTTCTTCTT TTATTCCTGA CGTTGTGAAA 2400 GCTCGCCTGG CTGCATCGCT CCTTTTCTAC CTTATCGAAC ACCCATCAGA AATTGATAAT 2460 TTGTCCGAGG ATGGTGTCAC GAAGAAAATC TCTGGTCATA TCTCGTTCCG CAATGTCTAT 2520 TTCAATTATC CGACAAGAAG ACAGATCAGA GTACTCCGTG GACTTAACCT AGAGATAAAT 2580 CCTGGCACGA AGGTAGCGCT TGTTGGGCAG TCTGGTTGTG GAAAAAGCAC TGTGATGGCG 2640 TTGTTGGAAC GGTTTTACAA TCAAAACAAG GGCGTGATTA CGGTGGACGG CGAAAACATC 2700 AGAAACATGA ACATACGCAA TCTTCGTGAG CAAGTGTGTA TTGTAAGCCA GGAACCAACG 2760 CTGTTCGACT GTACCATCAT GGAAAACATC TGTTACGGTC TCGATGACCC CAAGCCGTCC 2820 TACGAACAGG TTGTTGCTGC AGCAAAAATG GCGAACATTC ACAATTTTGT GCTGGGACTA 2880 CCAGAGGGTT ACGATACGCG TGTTGGTGAG AAAGGCACTC AGCTGTCAGG CGGACAGAAG 2940 CAACGAATAG CCATAGCCAG AGCGCTGATT CGAGATCCGC CTATACTTCT GCTGGATGAG 3000 GCGACAAGCG CGCTGGATAC CGAGAGTGAA AAGATCGTGC AAGACGCCCT AGAGGTTGCT 3060 CGCCAAGGTA GAACGTGCCT TGTAATTGCC CATCGCCTTT CTACAATTCA AGACAGTGAC 3120 GTCATAGTGA TGATCCAGGA GGGGAAAGCT ACAGACAGAG GCACTCATGA ACATTTACTG 3180 ATGAAGAACG ATCTATACAA ACGGCTATGC GAAACACAAC GACTCGTTGA ATCACAATGA 3240 GTTTTTAGTG CCAATCGATA GTGATCGATA AGCTATGGAT TAGTCTTTAA CACTTACTGA 3300 TCACAAATTT TATCTCGTGC TTTATTCTAA TGTACATATG TAACGGTTTT GATCTTACAT 3360 ATCTTGTAAT TGGTCCTCAC TATCATAATG CCTTTAGTAG TACATTAACA GTTTTATTAA 3420 TACAACTTAA GTAACATATT AACAATTTTA TTAATATAAC TTAAGTAAGA TATTGACAGT 3480 TTTATTAATT TGGAGGATTT ATAATAAAAC T 3512 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2681 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear ( ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CCCGACTTCC GGAAGGTTAC GGCACCCGAG TAGGTGAACG TGGTGTACAA CTAAGTGGCG 60 GACAAAAGCA GCGCATCGCT ATTGCTCGCG CCATCATTAA AAACCCTCGT ATACTTCTGC 120 TTGACGAAGC CACCAGTGCT CTGGACACAG AGGCGGAATC AATTGTGCAA GAAGCTCTCG 180 AGAAAGCTCA AAAAGGACGA ACGACCGTCA TTGTAGCGCA TCGCCTATCT ACCATCAGAA 240 ATGTCGATCA AATTTTCGTC TTCAAGAATG AAACGATTGT TGAGCAGGGT ACACATGCAG 300 AGTTGATGAA CAAACGAGGA GTGTTCTTTG AAATGACTCA AGCACAGGTC CTTCGACAAG 360 AAAAGGAAGA GGAGGTCTTAAACCAGTAGC GAAGTGTCAA GAGGCATCCT 420 TTCCTGCTCC TGATGTCACT ATTTTGACTC CCCATGACGA ACAACCCGAG CTACTTAGCC 480 CGCCGGATAG CGATGCGGAA TCCGACGTCA TGTCACCGGA TCTTGGCTTA CCCCATCTTA 540 GTTCACTTCG ATCACGTAAA GAGTCCACAA GAAGTGCTAT TTCCGCAGTC CCCAGCGTTC 600 GGAGTATGCA GATCGAAATG GAGGACCTTC GTGCCAAACC GACTCCGATG TCGAAAATTT 660 TCTATTTCAA CCGTGACAAA TGGGGATTTT TCATTTTGGG ACTCATCGCC TGTATTATAA 720 CTGGAACTGT TACACCGACA TTTGCAGTTT TATATGCGCA GATCATACAG GTATACTCGG 780 AACCTGTTGA TCAAATGAAA GGCCATGTGC TGTTTTGGTG TGGAGCTTTC ATCGTCATTG 840 GTCTCGTACA CGCATTTGCG TTCTTTTTCT CGGCCATTTG TCTGGGACGT TGCGGCGAAG 900 CTTTAACGAA GAAGTTACGT TTCGAGGCGT TCAAGAACCT TCTCCGACAA GATGTGGGAT 960 TCTACGACGA TATCCGACAC GGTACCGGTA AACTCTGTAC GCGATTTGCT ACAGATGCAC 1020 CCAATGTTCG ATATGTGTTC ACTCGACTTC CGGGTGTACT TTCATCGGTG GTGACCATAA 1080 TCGGAGCTTT GGTTATTGGA TTTATTTTCG GGTGGCAGCT GGCCTTGATT CTTATGGTCA 1140 TGGTACCGTT GATCATTGGC AGTGGATACT TCGAGATGCG CATGCAGTTT GGTAAAAAGA 1200 TGCGTGACAC AGAGCTTCTT GAAGAGGCTG GGAAA GTTGC CTCACAAGCC GTAGAGAATA 1260 TTCGTACCGT ACATGCCCTG AATCGGCAAG AGCAGTTCCA TTTCATGTAC TGCGAGTATT 1320 TGAAGGAACC CTATCGAGAG AATCTTTGCC AGGCGCACAC TTACGGGGGT GTATTCGCGT 1380 TTTCACAGTC GTTGTTATTC TTTATGTATG CTGTAGCATT TTGGATTGGT GCAATCTTCG 1440 TGGACAACCA CAGCATGCAA CCGATTGATG TTTACCGAGT ATTTTTCGCG TTCATGTTTT 1500 GTGGACAAAT GGTTGGCAAC ATTTCGTCCT TCATCCCTGA TGTTGTGAAA GCTCGCCTGG 1560 CTGCATCGCT CCTTTTCTAC CTCATCGAAC ACCCATCAGA AATTGATAAC TTGTCCGAGG 1620 ATGGTGTCAA GAAGAAAATC TCTGGTCACA TCTCGTTCCG CAATGTCTAT TTCAATTACC 1680 CGACGAGAAG GCAGATCAGA GTACTCCGTG GACTTAACCT AGAGATAAAT CCTGGCACGA 1740 CGGTAGCGCT TGTTGGACAA TCTGGTTGTG GAAAAAGCAC TGTGATGGCG TTGTTGGAAC 1800 GCTTTTACAA TCAAAACAAG GGCGTGATTA CGGTTGACGG CGAAAACATC AGAAACATGA 1860 ACATACGCAA TCTCCGTGAG CAAGTATGTA TAGTCAGCCA GGAACCAACA CTGTTCGACT 1920 GTACCATCAT GGAAAACATC TGTTACGGAC TCGATGACCC CAAACCGTCC TACGAACAGG 1980 TTGTTGCGGC AGCAAAAATG GCGAACATCC ACAATTTTGT GCTGGGACTG CCAGAGGGTT 2040 ATGACACGCG TGTTGGCGAG AAAGGCACTC AGCTGTCAGG CGGACAAAAG CAAAGAATAG_2100_CCATAGCCCG AGCGCTGATC CGAGATCCGC CTATACTTCT GCTGGATGAG GCGACAAGCG 2160 CTCTGGACAC GGAGAGTGAG AAGATCGTGC AAGACGCCCT AGAGGTTGCT CGCCAAGGTA 2220 GAACGTGCCT TGTAATTGCC CACCGCCTTT CTACAATTCA AGACAGTGAC GTCATAGTGA 2280 TGATCCAGGA GGGAAAAGCT ACAGACAGAG GCACTCATGA ACATTTACTG ATGAAGAACG 2340 ATCTATACAA ACGGCTATGC GAAACACAAC GACTCGTTGA ATCACAATGA GTTTTTAGTG 2400 CCGATCGATA GTGATCGATA AGCTATGGAT TAGTCTTCAA CACTTACTGA TCATATGACT 2460 ATCTCGTGCT TTATTATAAT GTACATATGT AATGGTTTTG ATGTAAGTTA AGTTATAATT 2520 GGTCTTCACT ATCATAATGC CTTTAGTAAT GCATTAACAC TTTTATAATA TAACTTGAAT 2580 AACATATTGA CAGTTTTATT AATATAACTT AAATAAGATA TTGACAGTTT TATTAATTTG 2640 GAGAATTTAT AATGAAACTT CTGGATTCCT GCAGCCCGGG G 2681 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GAAATGACTC AAGCACAAG 19 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: AGACAAAGAC ATTCAGAG 18 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11; ACNGTNGCNY TNGTNGG 17 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12; GCNSWNGTNG CYTCRTC 17 It is noted that in relation to this date, the best method known to the applicant, to carry out the aforementioned invention, is that which is clear from the present invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (17)

1. A recombinant nucleic acid molecule or a fragment thereof, characterized in that said recombinant nucleic acid or the fragment encoding a P-glycoprotein homologue which regulates the resistance to macrocyclic lactone compounds are selected from the group consisting of of LL-F28249a- ?, a 23-oxo derivative of LL-F28249a- ?, a 23-imino derivative of LL-F28249a- ?, a semicarbazone derivative of LL-F28249a- ?, a thiosemicarbazone derivative of LL-F28249a-? , an avermectin and a milbemycin.

2. The isolated and purified nucleic acid molecule or fragment according to claim 1, characterized in that said nucleic acid molecule or fragment is extracted from a nematode or arthropod pest and encodes a P-glycoprotein homologue which regulates the resistance to the macrocyclic lactone compound.

3. A biologically functional plasmid or viral vector, characterized in that it contains the nucleic acid molecule or fragment according to claim 2.

4. A suitable stable host cell transformed or transfected by a vector, characterized in that it comprises the nucleic acid molecule or a fragment according to claim 2.

5. A process for the production of a polypeptide product having part or all of the primary structural conformations and the biological activity of a homolog product of the P-glycoprotein, said process is characterized in that it comprises: growth, under suitable nutrient conditions, prokaryotic host cells or eukaryotes transformed or transfected with the nucleic acid molecule or the fragment according to claim 2 in a manner that allows the expression of said polypeptide product, and the isolation of the desired polypeptide product from the expression of said nucleic acid molecule or said fragment .

6. A homologous product of the P-glycoprotein of the expression of a prokaryotic or eukaryotic host cell, characterized in that the product of the protein is encoded by the nucleic acid molecule or the fragment according to claim 2.

7. The nucleic acid molecule or fragment according to claim 1 or 2, characterized in that it has a nucleotide sequence encoding the PGP-A set out in the SEC ID NO: 3, PGP-A-3 'set forth in SEQ ID NO: 5 (Accession number ATCC 98336), PGP-B, PGP-B-3 'set forth in SEQ ID NO: 8 (accession number ATCC 98307), PGP-0 or PGP-O-3' set forth in SEQ ID NO. : 7 (accession number ATCC 98309); complementary strands thereof or a nucleotide sequence which hybridizes at about 65 ° C in the presence of a dextran buffer for a period of at least about 4 hours to the nucleotide sequence coding for PGP-A, PGP-A-3 ' , PGP-B, PGP-B-3 ', PGP-0 or PGP-O-3'.

8. A method for detecting resistance to a macrocyclic lactone compound in a nematode or arthropod pest, characterized in that it comprises: comparing a nucleic acid molecule encoding a homolog of the P-glycoprotein which is extracted from a pest specimen to a nucleic acid molecule encoding a P-glycoprotein homologue that regulates resistance to the macrocyclic lactone compound and a nucleic acid molecule encoding a glycoprotein homolog associated with the macrocyclic lactone compound susceptibility.

9. The method according to claim 8, characterized in that it hybridizes to the nucleic acid molecule extracted from the pest specimen with a nucleic acid probe having a nucleotide sequence encoding the PGP-A set forth in SEQ ID NO: 3, PGP -A-3 'set forth in SEQ ID NO: 5 (accession number ATCC 98336), PGP-B, PGP-B-3' set forth in SEQ ID NO: 8 (Accession number ATCC 98307), PGP-0 or PGP-O-3 'set forth in SEQ ID NO: 7 (accession number ATCC 98309); complementary strands thereof or a nucleotide sequence which hybridizes at about 65 ° C in the presence of a dextran buffer for at least about 4 hours to the nucleotide sequence encoding PGP-A, PGP-A-3 ', PGP -B, PGP-B-3 ', PGP-0 or PGP-0-3A

10. The method according to claim 8, characterized in that one of the three nucleic acid molecules is mixed with a Polymerase Chain Reaction (PCR) or a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) primer. ); and the PCR primer or RT-PCR comprises a nucleotide sequence between PGP2S and PGPAS in the sense and antisense directions, respectively or a nucleotide sequence encoding 2PGP-A exposed in the SEQ ID NO: 3, PGP-A-3 'set forth in SEQ ID NO: 5 (Accession number ATCC 98336), PGP-B, PGP-B-3 'set forth in SEQ ID NO: 8 (accession number ATCC 98307), PGP-0 or PGP-0-3' set forth in SEQ ID NO: 7 (accession number ATCC 98309); complementary strands thereof or a nucleotide sequence which hybridizes at about 65 ° C in the presence of a dextran buffer for a period of at least about 4 hours to the nucleotide sequence coding for PGP-A, PGP-A-3 ', PGP-B, PGP-B-3', PGP-0 or PGP-O-3 '.

11. A method for detecting resistance to a macrocyclic lactone compound in a nematode or arthropod pest characterized in that it comprises: preparing an antibody to a sequence of a peptide corresponding to the translation of the amino acid of a nucleic acid molecule or a fragment thereof which encodes a homologue of the P-glycoprotein which regulates resistance to a macrocyclic lactone compound; preparing a specimen of the nematode or arthropod pest, or an extract thereof, for reaction with the antibody; reacting the specimen or extract with the antibody under suitable conditions that allow the antibody-antigen binding to occur; and rehearse for the presence of an antigene-antigen binding.

12. A method for increasing the efficacy of a macrocyclic lactone compound against a resistant pest in crops, characterized by the application to the crop, to the seed of the crop or to the oil or water in which the crop or seed is growing or is allowed to grow, of an increased effective amount of a pesticide from an agent that reverses resistance to multiple drugs.

13. A method for increasing the efficacy of a macrocyclic lactone compound against a resistant nematode or resistant ectoparasite or endoparasite arthropod of a mammal, characterized in that the mammal is administered an increased effective amount of a pesticide from an agent that reverses resistance to multiple drugs in conjunction with the administration of the macrocyclic lactone compound.

14. An improved composition for controlling or combating a crop pest, characterized in that the improvement comprises a pesticidally effective amount of an agent that reverses multidrug resistance in combination with a macrocyclic lactone compound and an agronomically acceptable carrier.

15. An improved composition for controlling or treating infestation or infection of helminth insects or endo or ectoparasitic arthropods of a mammal, characterized in that the breeding comprises an antelimitically effective amount or an arthropod endo or ectoparasiticly of an agent that reverses resistance to multiple drugs, in combination with a macrocyclic lactone compound and a pharmaceutically acceptable non-toxic carrier.

16. An improved method for controlling or combating a crop pest, characterized in that the improvement comprises applying to the crop, to the seed of the crop or to the soil or water in which the crop or seed is growing or growing, of a pesticidally effective amount of the composition of claim 14.

17. An improved method for controlling or treating an infection or infestation of an endo or ectoparasitic helminth arthropod or insect of a mammal, characterized in that the improvement comprises the administration to the mammal to be treated, of a quantity antelmntically, or an endo or ectoparasiticidally effective arthropod of the composition of claim 15.