MXPA97001217A - Compositions and methods to control insectosdañi - Google Patents

Abstract

Compositions of a purine, a xanthine oxidase inhibitor and / or a dihydrofolate reductase inhibitor and methods for using same, to control the growth of harmful insects that recover, store or excrete their nitrogenous residues through a metabolic pathway of puri

Description

"COMPOSITIONS AND METHODS TO CONTROL HARMFUL INSECTS" BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION The present invention is directed to the regulation of the growth of harmful insects that use the metabolic access route of purine to save, store or excrete their nitrogenous waste. It comprises contacting the harmful insecticides, formulations containing growth control amounts of compositions comprising purines, purine metabolic enzyme inhibitors and enzyme inhibitors that regulate the production of specific co-factors of this access pathway. 2. DESCRIPTION OF THE PREVIOUS TECHNIQUE Despite recent development and great promise and advanced insect control techniques such as chemical sterilizers, pheromones and ecologically based control strategies, the use of chemical insecticides still has a predominant role. However, increasing public awareness of environmental issues, stricter government regulations, and increased resistance of insects to conventional modes are driving the insect control industry to seek safer alternatives to these conventional chemical insecticides. 5 Others have attempted to identify and evaluate the efficacy of insect growth inhibitors. However, given the continuing need for increased selectivity and efficacy of insect control agents, it has become desirable to pursue a rational formulation of control agents based on the understanding of key nutritional and metabolic access routes of insects.

COMPENDIUM OF THE INVENTION 5 It is widely recognized that most insects are uricotelic and that they excrete their excessive nitrogen, such as uric acid and uricolytic derivatives thereof (Cochran (1975), "Excretion in Insects" in Insect 0 -Bioche-iis ry and Function pp. 171-281). Uric acid is synthesized through a catabolic purine access pathway shown in Figure 1, and either excreted outward or in some cases stored by the insect as a metabolic reserve.

Cockroaches are a good model of the essential nature of storage-excretion of uric acid. For example, in German cockroaches, a thick slurry of uric acid is passed to the female during mating as a paternal investment. The female, in turn, invests the developing eggs with a supply of uric acid that is used during embryogenesis (Mullins &Keil (1980), Nature 283: 567-569).

The interruption of this life cycle appears highly detrimental to the growth of the cockroach population, which depends heavily on these stores of uric acid (Engebretson &Mullins (1986), Comp.Chemchem.Physiol.83B: 93-97; Suiter and others (1992), J. Econ. Entomol. 85 (1): 117-122). In the fatty body of the cockroach, de novo synthesis of uric acid is effected, largely through purine recovery, in trophocytes and uric acid is stored in specialized urocytes for recycling (Cochran (1985), Ann. Rev. Entomol 30: 29-49). This is achieved through uricolitic digestion of the urates stored by endosimbiont bacteria that are sequestered in bacteriocite cells adjacent to the urocytes (Wren &Cochran (1987), Comp. Biochem. Physiol. 88B: 1023-1026). In this part of the uric acid cycle, the endosimbiont bacteria use xanthine dehydrogenase to reduce the urates in xanthine, and the interruption of any part of this system also inhibits the growth of the population. Another essential aspect of insect physiology is the molting cycle, when the cuticular epithelial cells multiply and synthesize a new larger exoskeleton just before desquamation (Chapman (1982), The Insects Structure and Function, Cambridge, MA: Harvard University Press; Hepburn (1985), "The Integument" in Fundamentals of Insect Physiology. Editors M.S. Blum, pages 139-183. New York: John Wiley & Sons, Inc.). At the same time, many of the internal tissues are growing, as in cockroaches where, for example, the development of the internal and external reproductive organs advances with each stage, culminating in the final molt towards the sexually mature adult. (Chapman (1982) The Insects Structure and Function, Cambridge MA: Harvard University Press). During this process, the insects intensely extract their metabolic reserves to achieve the rapid growth of the cells that is made. The purine metabolic pathway is central to all of these processes, and, therefore, to homeostasis of the insects. As in any of the biochemical access routes, the hydrolytic enzymes and their co-factors are essential for the functioning of the purine degradative access pathway. This gateway also serves to recover free purine bases for reuse in nucleotide and nucleic acid biosynthesis (Lehninger (1970) biochemistry: The Molecular Basis of Cell 5 Structure and Function, Second Edition, pages 740-742). Two of the enzymes involved in this access pathway are xanthine oxidase and dihydrofolate reductase (also known as * - tetrahydrofolate dehydrogenase). The xanthine oxidase (E.C. 1.2.3.2), a molybdenum iron sulfur flavo-enzyme, works late in the recovery pathway of purine catabolism recovery of guanosine monophosphate and inosine monophosphate in xanthine, and finally, in uric acid. In this access road, the oxidized is xanthine catalyzes both the conversion of hypoxanthine to xanthine, and the conversion of xanthine to uric acid (Coughlan (1980) Moly-b enu-O and Molybdenum-Containing Enzymes. New York: Pergamon Press). Functioning as xanthine dehydrogenase, the same enzyme reduces uric acid in xanthine in the uricolitic access pathway of the endosimbiont bacteria in the fatty body of the cockroach (Wren &Cochran (1987), Comp.biochem.physiol.88B: 1023-1026). Dihydrofolate reductase catalyzes the synthesis of tetrahydrofolate, which is an essential co-factor in the pathways access of uric acid and purine synthesis (Kucers &Bennett (1979), "Trimethoprim and Cotrimoxazole" in The Use of Antibiotics, Third Edition London: William Heinemann Medical Boocks, Ltd.). An understanding of these insect systems, which depend on the recycling and excretion of their purines, leads to the present invention, which provides novel compositions and methods for disrupting insect homeostasis and inhibiting the growth of the insect population. Thus, in one embodiment, these compositions comprise (1) a purine such as guanine (2-amino-1, 7-dihydro-6H-purin-6-one); hypoxanthine (1, 7-dihydro-6H-purin-6-one); or xanthine (3,7-dihydro-lH-purine-2,6-dione), and mixtures thereof, and (2) a xanthine oxidase inhibitor, preferably one from the pyrazolo group [3, 4-d] ] 6-unsubstituted pyridimidine, such as oxipurinol (4,6-dihydroxypyrazolo [3,4-d] pyrimidine); 4-mercapto-6-hydroxypyrazolo [3,4-d] pyrimidine; 4,6-dimercaptopyrazolo [3,4-d] pyrimidine, 4-amino-6-hydroxypyrazolo [3,4-d] pyrimidine; 4-hydroxy-6-mercapto [3,4-d] pyrimidine; or allopurinol (4-hydroxypyrazolo [3,4-d] pyrimidine), and mixtures thereof. In another embodiment, these compositions comprise (1) a purine; (2) a xanthine oxidase inhibitor; and (3) a dihydrofolate reductase inhibitor such as trimethoprim (2,4-diamino-5- (3,4,5-trimethoxybenzyl) -pyrimidine), methotrexate (N- [4- [[(2,4-diamino) -6-pteridinyl) methyl] methylamino] benzoyl] -L-glutamic acid), or pyrimethamine (5- (4-chlorophenyl) -6-ethyl-2, -pyrimidinediamine), and mixtures thereof. Even when the specific purines in combination with specific enzyme inhibitors are used to illustrate the present invention, it will be understood that any of the purines and inhibitors of any of the enzymes of the access path of Figure 1, can be applied in accordance with the present invention. In addition, even when the cockroach is used to illustrate the present invention, it will be understood that the compositions and methods of the present invention can be applied to regulate the growth of any harmful insect utilizing the purine metabolic pathway. to save, store or excrete to the outside, its nitrogen waste. -. A further embodiment of the invention comprises an insect bait or an attractant formulation containing an effective growth regulating amount of insects of the compositions.

DESCRIPTION OF THE FIGURES Figure 1 shows the access path for purine catabolism.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery that the ingestion of formulations containing amounts of growth control of certain novel compositions by harmful insects, particularly cockroaches, disrupts homeostatic and inhibits the growth of the population. The compositions of the present invention can be the sole active ingredients of the formulation or can be mixed with one or more additional active ingredients such as other conventional insecticides. The compositions of the present invention can be formulated with a "bait" or "attractant". For purposes of describing the present invention, these terms refer to any formulation to which harmful insects are attracted and which they will ingest. These compositions are well known to those skilled in the art and it will be understood that any material that is inert with respect to the compositions of the present invention may be employed in the practice of the invention. During use, the formulations can be applied to harmful insects, to the site of harmful insects, and / or to the habitat of harmful insects.

The following examples are included for illustration purposes only and are not intended to be limiting unless otherwise specified.

Example 1 - General Procedure The German cockroaches (German philately L.) of the "VPI" strain of laboratory material were used to form experimental colonies of mixed life stages. Unless otherwise specified, each insect colony of 42 insects contained five of each of newly emerging adult males and females, eight of each of male and female pupae in the fifth stage of pupae and eight of each one of male and female pupae in the third pupal stage. Care was taken to select insects from the same colonies of material for each experimental block and each colony was allowed to acclimate for twenty-four (24 hours) before treatment. The colonies were housed in glass battery jars of 3,785 liters equipped with wooden pulp board platforms, with clean tap water that was continuously offered in small glass jars covered with cotton. The jars were covered with a thin coating of petrolatum, and they were closely covered with three layers of cheesecloth that are held in place with strong elastic bands. These measures prevented the escape of test insects, as well as contamination by other insects. Each test included "control" colonies in which the food was untreated and "test" colonies where the food was mixed with the compositions being tested to form the weight percentage of the concentrations (in weight / weight). Unless otherwise specified, the feed was Agway Lab Rat Food and was prepared by grinding the food pellets to a fine powder, and for the test colonies, incorporating the test compounds by grinding and mixing them with the food, using a mortar and pestle. The food, whether treated or untreated, was previously weighed in stainless steel plates and offered with the plates placed in plastic cups to avoid loss through spillage. During the tests, the plates were weighed weekly and the food was replenished when necessary. The duplicated colonies were started on consecutive days, with all the colonies housed in the laboratory of the material under the same conditions of ambient temperature (25 ° C), and humidity as during rearing. A control "blank colony" that was identical to the control colony except that insects were not included, was monitored for loss or increase of moisture in the feed due to changes in ambient humidity. Any of these changes became calculations of food consumption. A record was kept of all dead insects that were counted and selected weekly, when the food was weighed. The dead insects were frozen and stored at -4 ° C before undergoing a whole-body uric acid test. Unless otherwise specified, the total population of each colony was counted every three (3) weeks. When all the insects, or all the females were dead or dying, the colony was determined to be non-viable and the experiment was terminated. The remaining insects were killed by freezing and stored frozen, as previously, waiting to be tested for uric acid. The change in mean percentage (?%) In the population number for each colony was calculated, with the initial number (42) representing 100 percent. Feed consumption, in milligrams per individual cockroach (ICmg), was calculated during the first three (3) weeks of the experiment before the chrysalis matured. These measures determined whether the test compositions were ingested, and whether these compositions were effective in inhibiting population growth.

Example 2 - Uric Acid Test The determination of the whole-body uric acid content of dead cockroaches was carried out essentially in accordance with a normal uricase assay (Cochran (1973) Comp.biochem. Physiol.A46: 409-418). The individual cockroaches, with wings and legs trimmed, were dried for 24 to 48 hours at 60 ° C, weighed and ground until they formed a fine powder. Uric acid was extracted from the dead tissue with 0.6 percent aqueous lithium carbonate for three (3) hours at 60 ° C with continuous agitation. The extracts were centrifuged to remove the garbage from the tissue. After mixing with the uricase, the maximum absorption at 292 nm was determined spectrophotometrically, and the uric acid concentration was calculated in micrograms of uric acid / milligram of dead tissue.

Example 3 - Evaluation of Xanthine Food Compositions In two experiments (3a) and (3b), the effects of adding 1 percent xanthine [Sigma Chemical Co.] to the basic diet of the rat food cockroach were studied milled. The colonies in each experiment were established as described in Example I, with the diets being either rat food alone (RC), or rat food + 1 percent xanthine (RCX). Each experiment included three duplicate colonies for each condition (n = 3). Populations were counted at 6 and 9 weeks (3a) or 10 and 12 weeks (3b) and the percentage change in the average population numbers (?%) Was calculated. The individual consumption (ICmg) of the diets during the first three weeks of treatment was calculated from the weight data of the food. 15 The results are shown in Table 1. The addition of xanthine appeared to not inhibit the '' - * food did not detrimentally affect the growth of the population. In fact, xanthine had the appearance of improving reproduction, since the number of the The population was higher in the colonies treated with xanthine than in those fed the rat food alone.

Table 1 EXPERIMENT 3a TIME ICmg?% (*) (week) (± SEM) RC RCX RC RCX 3 55.8 55.3 (± 0.9) (± 2.7) 6 + 224% + 278% 9 + 707% + 921% 12 Table 1 (continued) EXPERIMENT 3b TIME ICmg (week) (± SEM) RC RCX RC RCX • vO 58.0 57.9 (± 0.4) (± 0.8) 6 9 + 1405% + 1433% 12 + 1774% + 1869% (*) + = increase Table 1: Average individual consumption (ICmg) and percentage change (?%) In the average number of population over the course of time (weeks), in the colonies of German cockroaches administered with the food offered without (RC) or with 1 percent xanthine (RCX). n = 3 Example 4 - Evaluation of Xanthine-Oxypurinol Compositions Colonies of German cockroaches were prepared as described. The diets administered were the rat food alone (CR); Rat food with oxipurinol [Sigma Chemical Co. ] (RC + OXY%); and rat feed with 1 percent xanthine (RCX) and oxipurinol (+ OXY%) at five concentrations (in weight / weight). Individual consumption (IC g), control of population growth and uric acid concentrations throughout the body were determined. Individual consumption (ICmg) in the first three weeks was calculated, and the results are shown in Table 2a presented below. The addition of oxipurinol alone caused a decrease in feed intake through the controls fed untreated feed. The addition of xanthine to the diet caused the consumption of food treated with oxipurinol to increase the concentration of oxypurinol by 35 percent to 0.1 percent and oxipurinol concentration by 56 percent to 1.0 percent.

Table 2a XANTINE TIME 0% XANTHIN 1% (week) RC RC + 0X1% RC + 0XY% 0. 1 1.0 0.1 0.5 1.0 2.0 3.0 3 53.7 36 32 48.5 58.3 49.9 52.6 45.6 (± 2.0) (± 1.4) (± 0.8) (± 1.9) (± 1.5) n = 9 * n = ln = ln = 6 n = 3 n = 6 n = 6 n = l * n = number of colonies Table 2a: Average individual consumption (ICmg) of rat food over three weeks, with or without 1 percent xanthine, and with different concentrations (in weight / weight) of oxipurinol (OXY%) The percent change (?%) In the population numbers of the average colony at 5.5, 6, 7, 9, 10 and 12 weeks of treatment was determined as described, with the results shown in Table 2b presented then. The addition of oxipurinol alone to the diet did not inhibit the growth of the population. The addition of / xanthine plus oxipurinol inhibited population growth to the point of extinction.

Table 2b 5 XANTINE TIME 0% XANTINE 1% - RC RC + OXY% RC + OXY% (weeks) CONTROL 0.1 1.0 0.1 0.5 1.0 2.0 3.0 . 5 + 690% + 460% +1060 n = l n = l n = l 6 + 126% -31% -50% - 5% -11% -55% n = 5 9 + 812% -92% -92% -64% -77% -88% n = 5 20 7 +719 -64% -75% -69% + 1405% -91% -100% 14 + 1774% -94% -100% 100% Table 2b: Percentage of changes (+ or -?%) In the number of the average population, in colonies of German cockroaches that were offered food with or without 1 per of xanthine, and with different concentrations (in weight / weight) of oxipurinol (OXY%), through the course of time (mermen). Except when noted, n = 3.) Whole body uric acid concentrations were calculated from normal uricase tests for cockroaches that followed the diet for 5 to 9 weeks of treatment. Samples from the VPI laboratory strain of German cockroaches were also tested to show the "baseline" levels of urates before treatment. As shown in Table 2c presented below, females of the IPV strain typically exhibit a slightly higher level of uric acid than males, regardless of the stage. However, as shown in Tables 2d-2f below, after several weeks of feeding on xanthine and oxypurinol in the diet, there was a marked decrease in the concentration of whole body urate in all groups independently of age or sex.

Table 2c STAGE SEX URE ACID AGE (weeks) μg / mg ± SEM adult males 6-7 1.80 n = 9 ± 0.12 females 2.41 n = 10 ± 0.06 male chrysalis 5-6 2.34 n = 10 ± 0.10 females 2.44 n = 10 ± 0.22 chrysalis males 3-4 0.77 n = 10 ± 0.10 females 1.51 n = 10 ± 0.10 Table 2c: Whole body, average uric acid concentrations (microgram / milligram dry tissue weight, ± SEM), in different age groups and sex of the VPI laboratory strain of the German cockroaches that were typical of those used in feeding experiments.

Table 2d TIME RC RCX + OXY% (weeks) 0.1 1.0 2.0 2.42 0.54 0.32 0.31 ± 0.12 ± 0.05 ± 0.06 ± 0.05 n = 5 n = 25 n = 17 n = 17 6 2.79 0.43 0.30 0.27 ± 0.21 ± 0.04 ± 0.04 ± 0.03 7 2.78 0.54 0.25 0.21 ± 0.25 ± 0.10 ± 0.04 ± 0.04 9 3.16 0.51 0.14 0.32 ± 0.06 ± 0.04 ± 0.10 n = 10 n = ln = 7 n = 3 Table 2d: Mean whole-body uric acid concentrations (microgram / milligram dry tissue weight ± SEM) in cockroaches German males with food without (RC), or with 1 percent xanthine (RCX) and concentrations of different percentages (in weight / weight) of oxipurinol (OXY%).

Table 2e TIME RC RCX + OXY% (weeks) 0.1 1.0 2.0 2. 63 0.31 0.31 0.28 ± 0.14 ± 0.13 ± 0.04 ± 0.08 n = 3 n = 6 n = 8 n = 7 3. 13 0.31 0.34 0.35 D ± 0.04 ± 0.03 ± 0.06 ± 0.06 n = 4 n = 27 n = 27 n = 18 7 2.95 0.43 0.22 0.26 ± 0.18 ± 0.04 ± 0.04 ± 0.06 n = 14 n = 24 n = 23 n = 14 9 3.14 0.21 0.29 0.34 ± 0. 03 ± 0. 04 ± 0. 05 n = ln = 21 n = 14 n = 13 Table 2e: Mean whole body uric acid concentrations (microgram / milligram dry tissue weight + SEM) in female German cockroaches in food without (RC), or with 1 percent xanthine (RCX) and concentrations at different percentages (in weight / weight) of oxipurinol (OXY).

Table 2f r. "TIME RC RCX + OXY% 10 (weeks) 0.1 1.0 2.0 1.95 0.53 0.32 +0.36 +0.04 +0.18 15 n = 4 n = 3 n = 2 6 2.95 0.08 +0.09 +0.06 r 'n = 5 n = 2 20 7 3.14 0.13 +0.03 +0.08 n = 4 n = 2 3.26 0.14 n = ln = l Table 2f: Mean whole body uric acid concentrations (microgram / milligram dry tissue weight + SEM) in German cockroach pupae to which food was offered without (RC) or with 1 per percent xatin (RCX) and concentrations of different percentage (in weight / weight) of oxipurinol (OXY).

Example 5 - Evaluation of the Compositions of Xanthine-Oxipurinol that was offered through Different Durations Colonies were prepared as described. The food was treated with 1 percent xanthine and various concentrations of oxipurinol and offered through durations of either 24 hours, or 1, 2 or 3 weeks. At the end of the treatment time, the treated food was removed, and the insects were offered untreated rat food for the remainder of the test time. As shown in Table 3, which is presented below, the data indicates that a minimum dose of oxipurinol should be ingested over time to achieve population inhibition. For example, the 24-hour treatment affected population numbers when compared to the control but did not control population numbers at any oxipurinol concentration. The calculation reveals that the individual intake of oxipurinol ingested during this time period ranged from 6 to 104 micrograms. Table 3 TREATMENT TIME RC RCX + OXY% DURATION (weeks) 0.1 1.0 + 109% 24 hours 6 + 500% + 250% + 114% + 109% 1 week 6 + 887% + 137% -455 -49% 9 + 1157% + 320% -63% -57% 12 + 1580% + 853% -5% -31% 2 weeks 9 + 591% + 36% -65% -90% 12 + 750% + 213% -66 -94% 15 > + 750% + 561% -45% -96% 3 weeks 6 + 391% -58% -71% -92% 9 + 1050% -71% -92% -97% 12 + 1604% -79% -96% -98% Table 3: Percentage of change (+ o 1) in the average population numbers in colonies fed a diet with rat food alone (RC), or rat food combined with 1 percent xanthine (RCX), and with different concentrations (in weight / weight) of oxipurinol (OXY%). The duration of the treatments was 24 hours, or 1, 2, or 3 weeks after which rat food was offered alone. n = 3 ^ Treatment with 0.1 percent oxipurinol for one or two weeks also resulted in lower population numbers when compared to controls and delayed incubation for 1 to 2 weeks, but treated colonies recovered when they were terminated at 12 weeks. However, three (3) weeks r-J ~. treatment of 0.1 percent oxypurinol caused a considerable reduction in the numbers of the population in the weeks after treatment, without being observed recovery after 12 weeks, and with only one viable egg box, which were incubated six weeks later than normal. Colonies treated for two (2) weeks with 2 percent oxipurinol, or for three (3) weeks with 1 percent or 2 percent oxipurinol did not recover, even after the "recovery" time was extended to fifteen (15) weeks. The average individual consumption of oxipurinol was 734 micrograms, 579 micrograms and 1,140 micrograms, respectively.

Example 6 - Evaluation of Food Selection Colonies were prepared as described with three duplicates of each condition. Plates containing either treated food (RC) or food treated with xanthine + oxypurinol (RCX + 0 percent) were offered together in each colony. The weights of the food for each plate were calculated to determine how much of each one had been consumed. The treatments consisted of rat food with 1 percent xanthine and oxipurinol at a concentration of either 0.1 percent, 0.5 percent, or 1.0 percent (w / w). The control colony was provided with two untreated rat food plates. The results as shown in Table 4 below show that the insects consumed either the same amount of the treated and untreated food (at 0.5 percent oxipurinol), or ate more of the treated food than the untreated food. (at 0.1 percent and 2.0 percent oxipurinol). The oxipurinol scale ingested was calculated as being 29 micrograms and 265 micrograms per individual through the first three weeks, and a high level of control of population growth was achieved especially at oxipurinol concentration of 1.0 percent. Table 4 TEST TIME REC RC RCX + 0% RC RCX + 0% RC RCS + 0% (se- CONTROL 0.1 0.5 1.0 manas) 3 ICmg 58.9 23.1 29.4 25.7 25.6 24.7 26.5 + SEM +1.7 +3.1 +0.3 +1.0 +1.3 +0.9 +2.0 ICμg 0 0 29.4 0 128 0 265 OXY % 100% 43% 57% 50% 50% 48% 52% TOTAL 7?% + 422% -64% -72% -83% 9?% + 1378% -71% -80% -94% 12?% + 20007% -76% .71% -96% Table 4: Individual consumption (ICmg) and percentage of change in the average population numbers (?%) Through the course of time (weeks), in colonies in where the treated (RCX + 0%) and untreated (RC) food was offered together with a selection of the diet. The amount of oxipurinol that was ingested through the first three weeks is shown as microgram / individual (ICμg OXY), and the ratio of treated and untreated food consumed is given as a percentage of the total amount eaten (% TOTAL ).

Example 7 - Effects of the Life Stage of Xanthine-Oxipurinol Compositions The German cockroach colonies were housed as described above with the usually mixed stages separated into three different colonies. The colonies consisted of either freshly moved adults (five males and five females, 6 to 7 weeks old); large pupae (eight males and eight females, 5 to 6 weeks old); or small chrysalises (eight males and eight females, from 3 to 4 weeks old) The colonies of older adults (five males and five females, from 7 to 8 weeks old) were also tested.

The colonies were fed untreated rat (RC) feed, or rat feed treated with 1 percent xanthine (RCX) plus different levels (w / w) of oxipurinol (OXY%). The individual consumption (ICmg) and the percentage of change in the average population number (?%) Were determined for each stage, and are shown in Tables 5a to 5d that are presented below for adults, large chrysalis, small chrysalis and older adults, respectively. The data in these tables confirms that the primary impact of treatment with xanthine plus oxypurinol occurs as cockroaches attempt to reproduce. The effect is probably caused by depletion of the metabolic reserves of the insects, including the storage of uric acid that can not be replaced due to irreversible inhibition of the enzyme. However, the very small chrysalids that were incubated in the colony that was dying are also affected since they are usually too weak to survive and hardly reach their second stage. It is likely that they are not invested with metabolic reserves that normally pass to them before they are born. Their continuous feeding of treated food also prevents young pupae from developing their own metabolic storage, especially storage of uric acid.

The adult males were observed to be the first to die. During mating, adult males use much of their reserves to pass the urates as well as the mature sperm to the females. The females that have just produced a box of eggs, which need a large investment of nutritional reserves, die shortly thereafter, usually with the non-viable egg box protruding from the ovipositor. Cochran observed that cyclic feeding occurs in adult females in relation to egg reproduction (Cochran (1983) Entomol, Exp. Appl. 34: 51-57). In this breeding cycle, the females feed vigorously while the oocytes mature, and only as long as they carry a box of eggs. These phenomena would be responsible for the high feeding regimes and early mortality of the newly departed adults (Table 5a), as well as the low feeding regimes of the elderly (Table 5d). These last females would tend to have already matured the eggs that fill the ovarialcaja shortly after the colony was assembled and therefore were part of the low diet of their cycle. Their first incubation of chrysalises would be responsible for the precipitous rise in population numbers in these colonies (Table 5d), followed by the gradual weakening of the colonies as the adults tried to reproduce further and the newly incubated pupae died. The pupae followed the same mortality pattern as adults, and were not affected by the diet after moulting to the adult stage, when they were normally vigorously fed in preparation to mature their first oocytes. The delay in the regime to which the population in the large chrysalis colony decreased (Table 5b) and the small chrysalis colony (Table 5c), is further evidence that a predominant impact occurs during reproduction. This would have happened between weeks 9 to 11 of the experiment for these age groups. The effective dosing scale for oxipurinol with xanthine is very broad in these experiments causing high mortality at 99.5 micrograms / individual that is measured over three weeks in newly molted adults (Table 5a), and slower control at consumption regimes higher elevations when the colonies were started as chrysalises. However, it is evident that, even when there is a different effect on cockroaches depending on their age when the treatment is started, all are affected when they try to reproduce.

Table 5a COLONY INITIATED AS ADULTS (n = l! TEST TIME RC RCX + OXY% weeks 0.1 1.0 2.0 IC g 87.0 99.5 76.8 1696 3 IcμgOXY 0 99.5 768 1696 6?% + 1430% -94% + 75% -88% 9?% -1310% -100% -90% -100% 12?% + 1810% -100% -100% -100% Table 5a: Individual consumption (ICmg) and percentage of change in the average population number (?%) In colonies of freshly molted adult German cockroaches were fed with the untreated rat (RC) feed or rat feed treated with 1 percent xanthine (RCX) and different concentrations (in weight / weight) of oxipurinol OXY%).

/ "Table 5b COLONY INITIATED AS LARGE CHRISTMAS (n = l) TEST TIME RC RCX + OXY% weeks 0.1 1.0 2.0 ICmg 82.8 76.9 65.3 79.3 3 ICμg OXY 0 76.9 653 1586 6?% -6% -50% -31% -6% 9?% + 1613% -69% -81% -63% 12?% -1800% -88% -100% -100% Table 5b: Individual consumption (ICmg) and percentage of change in the average number of population (?%) In colonies of large cockroaches of German cockroach (5-6 weeks of birth on the date of departure) fed with rat food not treated (RC) or food for rat treated with 1 percent xanthine (RCX) and different concentrations (in 25 weight / weight) of oxipurinol (0XY%).

Table 5c COLONY INITIATED AS SMALL CHILDREN (n = l) TEST TIME RC RCX + OXY% weeks 0.1 1.0 2.0 ICmg 54.9 53.9 52.4 40.4 3 ICμg OXY 0 53.9 524 808 6?% -50% -31% -19% -81% 9?% + 719% -69% -81% -88% 12?% + 775% -88% -100% -100% f.

Table 5c: individual consumption (ICmg) and percentage of change 20 in the average number of the population (?%) Of small pupae of the German cockroach (3 to 4 weeks of birth on the date of departure) fed with rat food untreated (RC) or rat food treated with 1 percent xanthine (RCX) and different concentrations (in 25 weight / weight) of oxipurinol (0XY%).

Table 5d COLONY INITIATED AS ADULTS MORE MATURE (N = 3) TEST TIME RC RCX + OXY% weeks 0.1 1.0 2.0 ICmg 38.7 37.2 35.0 35.2 + SEM +1.9 +0.6 +1.8 ICμg OXY 37.2 350 704 ?% + 1150% + 557Í + 403% + 823% ?% + 1030% + 33% + 40% + 197% 12?% + 1820% -73% -67% -30% Table 5d: Average individual consumption (ICmg) and percentage of change in the average number of population (?%) In colonies of more mature adults of the German cockroach (from 8 to 9 weeks of birth on the date of departure) fed with food for untreated rat (RC) or rat food treated with 1 percent xanthine (RCX) and different concentrations (in weight / weight) of oxipurinol (OXY%).

Example 8 - Evaluation of Compositions Containing Trimethoprim Duplicate colonies of German cockroaches were prepared as described. The diets administered were either rat food alone (CR); Rat food with different concentrations of trimethoprim (RC + T%) (in weight / weight), or food for rat with 1 percent xanthine (RCX) and different concentrations (in weight / weight) of trimethoprim (T%). As shown in Table 6a presented below, the addition of trimethoprim alone did not inhibit population growth even when there was some eventual weakening of the treated colonies. As shown in Table 6b below, however, the combination of xanthine and trimethoprim caused rapid inhibition of population growth. Whole body uric acid concentrations were calculated from normal uricase assays as described above. As shown in Table 6c below, the metabolism of uric acid was not affected by treatment with a combination of xanthine and trimethoprim. During the first three weeks, there was an average of -82 percent of the populations in the treated colonies, with 65 percent of these pupae still when they died. This represents 72 percent of the pupae used for the experiment and confirms that the effects are most pronounced during chrysalis moult.

Table 6a TEST TIME RC RC + T% Weeks 0.5 1.0 2.0 ICmg 62 61 58 54 + SEM +2.2 +3.5 +3.4 +1.7 12?% + 1398% + 1246% + 1013% + 384% - "* Cuaero 6a: Average individual consumption (IC g) of food for rat (RC) or with different concentrations (in weight / weight) of trimethoprim (RC + T%), through the course of time 20 (weeks), which is shown together with the percentage change in the average population number (?%), in German cockroach colonies where the starting number (42) = 100 percent, n = 5.

Table 6b TEST TIME RC RCX + Tí weeks 1.0 2.0 3.0 n = 6 n = 3 n = 12 n = 3 ICmg 17.3 12.0 8.8 5.8 + SEM +2.4 +0.9 +0.7 +0.1 ?% -1% -4% -28% -41% ICmg 44.7 33.9 22.6 13.4 + SEM + 2.1 + 1.1 + 2.8 + 1.3 ?% -16% -23% -77% -98% 6?% + 36% -44% -67% -98% Table 6b: Individual average consumption (ICmg), and percentage of change in the average number of population (?%), Through the course of time (weeks), in colonies of German cockroaches who were offered food without (RC) and with (RCX) 1 percent xanthine and different concentrations (in weight / weight) of trimethoprim (T%), where the starting number of the colony (42) = 100%.

Table 6c WEEK GRUPO REC RCX + 2% T 3-4 males 2.04 2.61 +0.12 +0.05 n = 19 n = 9 females 2.54 2.64 +0.06 +0.03 n = 17 n = 3 pupa 2.76 2.62 n = l n = 9 Table 6c: Mean whole body uric acid concentrations (microgram / milligram dry tissue weight + SEM), in three groups of German cockroaches to which untreated food (CR) was offered, or food treated with 1 per xanthine percent (RCX) and 2 percent trimethoprim (weight / weight) Example 9 - Treatment of resistant cockroaches with Xanthine-Oxipurinol compositions Cockroach colonies were prepared as described above with the exception that the insects were taken from laboratory materials of two strains of German cockroaches known to be resistant to insecticides that are commonly used to control cockroaches. The two strains were: (A) the Ha thorne strain, and (B) the La Palms strain. The profiles of the resistance relationships exhibited by these two strains are shown in Table 7a, which is presented below Table 7a INSECTICIDE HAWTHORNE LAS PALMS ORGANOGROPHOSPHATES RR Diazinon 2.0 > 75 Chlorpyrifos 10.8 > fifty Acefato 2.0 1.2 Malation 5.5 > 50 CARBAMATES Propoxur 1.7 > 60 Bendiocarb 2.2 > 70 PIRETROIDES f- • Pyrethrins > 140 > 140 Aletrina > 140 > 140 Permetrin 0.5 3.2 Fenotrin 0.6 > 120 - Fenvalerate 0.9 > 60 Esfenvalerate 0.8 7.0 Ciflutrin 1.8 2.5 Cipermetrin 1.6 > 80 BIO-CHEMISTRY Avermectin 2.4 1.5 Table 7a: Resistance ratio (RR) profiles for the Hawthorne and Las Palms resistant strains, where in a continuous high resistance mode RR > 2.0 indicates that resistance is developing, and RR > 3.0 indicates that the frequency of the gene for resistance has increased. RR is calculated as (Test strain LT50) + (susceptible strain LT50), where LT50 is the time required for the toxic product to achieve 50 percent mortality in a tratrated population.

Individual consumption (ICmg) during the first three weeks was calculated as described above. As shown in ls Tables 7b and 7c are presented below, the ICmg for both strains was compatible across all concentrations of the food mixtures. The Hawthorne strain exhibited a maximum decrease in consumption of 22 percent for a diet containing 3 percent oxipurinol. This represents a dose of 1,260 micrograms of oxipurinol through the first three weeks.

Table 7b CEPA HAWTHORNE TIME RC RCX + OXY% (weeks) 0.1 1.0 2.0 3.0 3 53.6 47.1 48.0 47.1 42.0 (+3.5) (+0.6) (+1.3) (+0.8) (+0.4) n = 4 n = 3 n = 3 n = 3 n = 4 CEPA LAS PALMS 45. 2 39.5 40.0 40.0 40.3 (+1.3) (+1.0) (+0.4) (+2.3) (+ 0.5) n = 4 n = 3 n = 3 n = 3 n = 4 Table 7b: Individual average consumption (ICmg), through the course of time (weeks), of the rodent meal that was offered without (RC), or with 1 percent xanthine (RCX), and with different concentrations (in weight / weight) of oxipurinol (OXY% ), by German cockroaches of resistant strains Hawthorne and Las Palms The effect of xanthine-oxipurinol combinations on population growth was determined as described above. As shown in Tables 7c and 7d presented below, the combination controlled the population growth of both resistant strains. Table 7c TIME RC RCX + OXY% weeks 0.1 1.0 2.0 3.0 6 + 438% -325 -22% + 12% -21% 9 + 997% -55% -59% -38% -67% 12 + 1.601% -77% -78% -76% -98% Table 7c: Percentage of changes (+ or -) in the average number of the population in colonies of German cockroaches of the resistant strain Hawthorne, to whom food was offered without (RC) and with 1 percent xanthine (RCX), and with different concentrations (in weight / weight) of oxipurinol (OXY%), through the course of time (weeks). n = 3 Table 7d TIME RC RCX + OXY% weeks 0.1 1.0 2.0 3.0 + 146% + 50% + 68% + 31% -25% 9 + 1.074% -50% -8% -60% -70% 12 + 1.624% -78% -67% -88% -95% Table 7d: Percentage of changes (+ or -) in the average population number in colonies of German cockroaches of the resistant strain Las Palms, to whom food was offered without (RC) or with 1 percent xanthine (RCX), and with different concentrations (in weight / weight) of oxipurinol, through the course of time (weeks), n = 3.

Example 10 - Treatment of Resistant Cockroaches with Xanthine-Trimethoprim Compositions Cockroach colonies were prepared as described using the resistant Hawthorne and Las Palms strains. As shown in Table 8a presented below, for the Hawthorne strain, the feeding was inhibited in relation to the control, in direct relation to the concentration of trimethoprim in the diet. The maximum decrease of 62 percent occurred at a concentration of 4.05% T, which represents a dose of 639 micrograms of trimethoprim per individual over the first three weeks. The population growth of the Hawthorne strain was controlled at higher concentrations. Table 8a TEST TIME REC RCX + T% Weeks 0.5 1.0 2.0 3.0 4.0 3 ICmG 42.5 37.6 37.1 30.4 17.2 15.9 (+ SEM) (+0.7) (+2.1) (+1.7) (+2.0) (+1.2) (+1.4) n = 7 n = 3 n = 3 n = 6 n = 6 n = 3 3?% -7% -2% -6% -27% -75% -79% n = 7 n = 3 n = 3 n = 6 n = 4 n = 3 6?% + 368% -70% -79% -89% n = 4 n = 3 n = 4 n = 3 9?% + 606% + 369% + 298% -17% -95% -94% n = 7 n = 3 n = 3 n = 6 n = 4 n = 3 12?% + 913% -51% -93% -97% n = 3 n = 3 n = 3 n = 3 Table 8a: Individual average consumption (ICmg), and percentage of change (?%) In the average numbers of population, in colonies of German cockroaches of the Hawthorne resistant strain to which food was offered without (RC), or with 1 percent xanthine (RCX), and different concentrations (in weight / weight) of trimethoprim (T%) through the course of time (weeks). r " For the Las Palms strain, as shown in Table 8b presented below, there was a uniform decrease in ICmg of the treated food in direct relation to the increase in the trimethoprim concentration. The maximum inhibition, compared to The control was 38 percent at a concentration of 6 percent T, which constitutes an ingested dose of 1,758 micrograms of trimethoprim per individual over the course of three weeks. The population numbers were reduced by two thirds to six weeks of treatment. twenty Table 8b TEST TIME RC RCX + T% (weeks) 3.0 4.0 5.0 6.0 3 IC g 47.0 43.0 41.3 37.0 29.3 (+ SEM) (+3.8) (+3.5) (+2.2) (+2.3) (+1.8) 3?% -12% -24% -26% -43% -57% 6?% + 336% + 100% -37% -37% -67% Table 8b: Individual average consumption (ICmg) and percentage of change (?%) In the average population number in colonies of German cockroaches of the Las Palms resistant strain to which food was offered without (RC), or without 1 percent of xanthine (RCX), and with different concentrations (in weight / weight) of trimethoprim (T%) through the course of time (weeks). n = 3 Example 11 - Treatment of Cockroaches with Compositions of Xanthine-Oxipurinol-Trimethoprim-so ¬ German cockroach colonies of the VPI susceptible strain and Hawthorne resistant strain colonies were offered either untreated rat (RC) feed, or treated rat feed (weight / weight) with 1 percent xanthine (RCX) , combined with 2 percent oxipurinol (OXY) and 2 percent trimethoprim (T). Individual consumption and changes in the populations of the colony are shown in Tables 9a (strain VPI) and 9b (strain Hawthorne). In both colonies they had virtually become extinct at six weeks of treatment, despite the decreases in ICmg of > 50 percent.

Table 9a TEST TIME RC RCX + 2% OXY + 2% T (weeks) n = l n = 3 IC g 71.3 34.9 (+ SEM) (+1.6) ?% -5% • 68% ?% + 955% -99% Table 9a: Individual average consumption (ICmg) and percentage of change (?%) In the average number of population in colonies of German cockroaches of the susceptible VPI strain to which food was offered without (RC ), and with 1 percent xanthine (RCX) and 2 percent oxipurinol (OXY and 2 percent trimethoprim (T) (w / w) over time (weeks).

Table 9b TEST TIME RC RCX + 2% OXY + 2% T (weeks) n = l n = 3 3 ICmg 72 34.1 (+ SEM) (+0.6) 3?% -2.4% -76% i »20 6?% + 1416% -98% Table 9b: Individual average consumption (ICmg), and percent change (?%) In the average population number in colonies of German cockroaches of the Hawthorne resistant strain at 25 which were offered food without (RC), or with 1 per xanthine percent (RCX) and with 2 percent oxipurinol (OXY) and 2 percent trimethoprim (T) (weight / weight), over time (weeks).

Example 12 - Evaluation of Purines with Oxipurinol or Trimethoprim Cockroach colonies of the susceptible VPI strain were prepared as described above. The diets offered were rat food alone (CR), rat food (weight / weight) with 1 percent xanthine and 3 percent trimethoprim (RCX + T), rat food with 1 percent hypoxanthine and 3 percent trimethoprim (HX + T), rat food with 1 percent guanine and 3 percent trimethoprim (G + T), and rat food with 1 percent hypoxanthine and 1 percent of oxipurinol (HX + OXY). Individual consumption (ICmg), and the change in population numbers were calculated as above with the results shown in Table 10 presented below. The results with hypoxanthine and guanine replacing the xanthine component of the diet mixtures are closely compared to those obtained with xanthine. This was the case both with trimethoprim and oxipurinol, with the population growing controlling until the colonies were extinct. Some inhibition of feeding occurred in all trimethoprim mixtures.

Table 10 TEST TIME RC RCX + T HX + T G + T HX + OXY (weeks) n = 2 n = 2 n = 2 n = 2 n = l 3 ICmg 54 26 25 29 42 (+ SEM) (+0) (+8.0) (+7.0) (+8.0) - - 3?% -5.5 -68 -74 -71 -17 6?% +152 -99 -91 -94 -83 9?% +1426 -100 -100 -100 -100 Table 10: Average individual consumption (ICmg), and percentage of change (?%) In the average numbers of the population in ... colonies of German cockroaches of the susceptible VPI strain to which food was offered without (RC), or with 1 percent of a purine (in weight / weight) and either 3 percent of trimethoprim (T), or 1 percent oxipurinol (OXY), over time (weeks). The purines were xanthine (X), hypoxanthine (HX), or guanine (G).

Claims (12)

CLAIMS:

1. A composition comprising a purine and a second component selected from the group consisting of a xanthine oxidase inhibitor, a dihydrofolate reductase inhibitor and mixtures thereof.

2. A composition according to claim 1, wherein the purine is selected from the group consisting of guanine, xanthine, hypoxanthine and mixtures thereof.

3. A composition according to claim 1, wherein the xanthine oxidase inhibitor is a 6-unsubstituted pyrazolo [3,4-d] pyrimidine compound.

4. A composition according to claim 1, wherein the dihydrofolate reductase inhibitor is selected from the group consisting of trimethoprim, methotrexate and mixtures thereof.

A composition according to claim 3, wherein the pyrazolo [3,4-d] pyrimidine is selected from the group consisting of allopurinol, oxipurinol, and mixtures thereof.

6. A method to control a harmful insect that saves or collects, stores or excretes its nitrogenous waste through the metabolic access path of y- -. purine, which comprises contacting the harmful insect, a quantity of growth control of a composition comprising a purine and a second component which is selected from the group consisting of a 5 xanthine oxidase inhibitor, a dihydrofolate reductase inhibitor and mixtures thereof.

7. A method according to claim 6, wherein the insect is a cockroach.

8. A method according to claim 6, wherein the purine is selected from the group consisting of guanine, xanthine, hypoxanthine, and mixtures thereof.

9. A method according to claim 6, wherein the oxidase inhibitor of Xanthine is a 6- [substituted] pyrazolo [3,4-d] pyrimidine compound.

10. A method according to claim 9, wherein the pyrazolo (3,4-d) pyrimidine compound is selected from the group consisting of Allopurinol, oxipurinol and mixtures thereof.

11. A method according to claim 6, wherein the dihydrofolate reductase inhibitor is selected from the group consisting of trimethoprim, methotrexate and mixtures thereof.

12. The method according to claim 6, wherein the composition is administered by incorporation into a bait or an attractant product for harmful insects that is ingested by the harmful insects.