US2884357A - Methods for destroying animal parasites using indandione derivatives - Google Patents

Description

United States Patent METHODS FOR DESTROYING ANIMAL PARA- SITES USING INDANDIONE DERIVATIVES Lloyd W. Hazleton, Falls Church, Va., and Warren H. Dolben, Summit, N .J.

No Drawing. Application June 21, 1955 Serial No. 517,095

17 Claims. (Cl. 167-53) This invention relates to a new method for destroying blood-sucking parasites which infest mammals. Such parasites may be external or internal.

Conventional parasiticides are generally specifically formulated to be effective against either external parasites, or against internal parasites. Parasiticides for external use may be in the form or dusts, sprays, dips, aerosols, etc. Sheep dip is a typical external parasiticide. Parasiticides for internal administration generally depend for their action on a toxic principle which frequently affects the host, particularly at elevated dosage levels. Both types of parasiticide sufier the disadvantage that neither affects the other type of parasite. Dips are difiicult to apply, and are seldom as effective as is desirable. The same is true of dusts, aerosols, and sprays. Internally administered parasiticides may be used more readily, but frequently are ineffective and inefiicient at levels which are safe for use with the host.

' An object of the present invention is to provide a parasiticide which is toxic both to many external parasites and to many internal parasites.

Another object of this invention is to provide a method for controlling blood-sucking parasites of Mammalia through the administration of a member or members selected from a class of parasiticidal compounds which are also characterized by their anticoagulant properties, together with the administration of an antidote which effectively counteracts the anticoagulant properties of the parasiticides, while either promoting the parasiticidal action by independent parasiticidal properties or in synergistic fashion, or having no detrimental effect upon the parasiticidal action.

These and other objects of the invention, which will become more obvious from a detailed consideration of this specification, are achieved by the administration to a parasite-infested mammal of one or more of a class of compounds characterized as being extremely toxic toward blood-suc ing parasites, while also possessing anticoagulant properties affecting the. host mammal. The anticoagulant properties are antidoted by the approximately simultaneous administration to the host mammal of an anti-anticoagulant, such as, for example, an activator for prothrombin, a compound known to possess vitamin K activity.

The preferred parasiticides are 2-pivalyl-1,3-indandione and its sodium and calcium salts. These compounds are most effective against all types of parasites. The parasiticidal activity is a property which is distinct from the anticoagulant activity. Other suitable parasiticides may be 'found in the class of chemicals designated by the following structural formula:

where H is a hydrocarbon radical; and, as well, in the enolate salts of this class of compounds:

where Me may be an alkali metal or an alkaline earth metal. Other parasiticides which may be employed include the following compounds:

2-acetyl-1,3 -indandione 2-propionyl-1,3 -indandione Z-butyryl-l ,3-indandione 2-isobutryl-1,3-indandione 2-n-valeryl-1,3-indandione 2-is0valeryl-1,3indandione 2-trimethylacetyl-1,3 -indandione 2-caproyl- 1,3 -indandione 2-hexahydrobenzoyl-l ,3 -indandione 2-(2-phenylethyl) -1,3-indandione 2-benzoyl-1,3-indandione In addition, the following alkali metal salts of substituted indandiones have been tested and found to be eifec' tive parasiticides:

In addition to the alkaline earth metal salts of the indandiones mentioned above, the following alkaline earth metal salts have been found to be effective as parasiticides according to the teachings of this invention:

The calcium salt of 2-isovaleryl-1,3-indandione The strontium salt of 2-isobutyl-1,3-indandione The barium salt of 2-isobutyl-1,3-indandione The strontium salt of 2-ethyl-1,3-indandione The barium salt of 2-ethyl-1,3-indandione The calcium salt of 2-ethyl-1,3-indandione The strontium salt of 2-cyclopropyl-l,3-indandione The barium salt of 2-cyclopropyl-1,3-indandione The calcium salt of 2-cyclopropyl-l,3-indandione A preferred antidote for approximately simultaneous administration with the parasiticide is vitamin K since it is eifective in counteracting the anticoagulant efiect of the parasiticides, is inexpensive, and is readily available. Many other substances may be employed, however, which are efiective in offsetting the anticoagulant tendencies of these parasiticides, such as:

2 methyl-1,4 naphthohydroquinone-3 sodiumsulfonate Hykinonej) 2-methyl-3-hydroxy-1,4-naphthoquinone (Phthiocol) 2-methyl-l,4-dihydroxy-3-naphtholaldehyde 2-methyl-3-farnesyl1,4-naphthoquinone 5,8-dihydrovitamin K Z-hydroxy-3-dimethylalkyl-1,4-naphthoquinone (Lapachol) Z-methyI-I-naphthylamine 2-methyl-5,8-dihydro-1,4-dihydroxynaphthalene Vitamin K hydroquinone diacetate 1-hydroxy-2-methyl-4-methoxy-naphthalene 2-methyl-1,4-dibenzoxynaphthalene Z-methyl-1,4-diacetoxynaphthalene Sodium 2-methyl-1,4-naphthohydroquinone diphosphate Sodium 2-methyl-1,4-naphthohydroquinone disulfate Vitamin K -2,3-oxide Z-methyl-l,4-naphthoquinone-2,3-oxide {{Vitamin K hydroquinone diacetate Othe'r compounds possessing anti-anticoagulant activity .ticular animal, andwith the typeof parasite whichgmust ..be eliminated .1 Asimple method for computing proper dosages is based upon the body weight: of the mammal.

Through experimentation, it hasbeen found that a single dosage level of 1 mg. per kg. of body weight has a mild parasiticidal effect, over the entire class of compounds referred to above. The effect is too mildto be of substantial commercial importance at this level. 'At 'a single dosage level of 5 mg./kg., considerably better results are obtained. In the case of 2-pivalyl-1,3-indandione, and

its alkali metal and alkaline earth metal salts, this dosage level is productive of highly effective results. For example, three days after a feeding of a dose of 5 mg./kg. of body weight of 2-pivalyl-l,3-indandione to rabbits, the kill of lice on the rabbits was between 90% and 100%. After a single dosage of 300 mg./kg. of body weight, a 100% kill was obtained. For the entire class of compounds, comparably good results are obtained at a 300 nag/kg. single dosage rate.

Where cumulative dosing is employed, as by incorporating a small amount of a parasiticide in the daily food ration, extremely small daily doses are effective because of the cumulative toxicity which is a characteristic of this class of parasiticides. For example, a daily dosage of 0.25 mg./kg. of 2-pivalyl-1,3-indandione in the food of rabbits produced 100% elimination of louse ectoparasites. In each case, it is necessary to administer an anti-anticoagulant along with the parasiticide in order to eliminate danger to the host. With cumulative dosing, the administration of the anti-anticoagulant may prove to be somewhat burdensome, since parenteral (intravenous) administration is preferred.

The invention may become more clear from a few specific examples of the use of the parasiticides in combination with an anti-anticoagulant. However, in setting forth the following examples, the purpose is to explain the invention, and it should be understood that the broad scope of the invention is that set forth above, rather than the more limited scope defined by the following, necessarily restricted examples.

EXAMPLE 1 Group 1.--Control.

Received a single oral dose of 5.0 cc. of corn oil. 1

Group 2.Recelved a single oral dose of 5.0 mg. per

kg. of (Z-pivalyl-1,3-indandione) (Pival).

Group 3.Received a single oral dose of 5.0 mg. per kg.

of 2-pivalyl-1,3-indandione (Pival) and a single intravenous injection of 1.0 mg. per kg. of vitamin K (Mephyton, Merck).

The 2-pivalyl-1,3-i.ndandione (Pival) was administered in a single dose as a 0.2% weight by volume solution in corn oil, and the vitamin K as a 1.0% suspension in distilled water. The corn oil and the parasiticide solution were administered orally by stomach tube, while the vitamin K was introduced into the marginal ear vein.

Twenty-four hours after the administration of the indicated materials, a group of approximately 40 adult body lice, Pediculus humanus corporis, was applied to the closely-clipped abdominal skin of each of the six rabbits.

The lice were allowed to feed for approximately'thirty minutes, after which each group of lice was placed in a beaker and stored at a temperature of approximately 75 to 85 F. and at a relative humidity of approximately 50% to A final mortality count was made twentyfour hours after the lice hadbeen allowed to feed.

This procedure was duplicated at 2, 3, 5, 6, 7, 8, and 10 days following the oral administration of the indicated material to the rabbits, each feeding being with a fresh group of lice. While not being used for experi mental feedings, the louse colony was fed at least once every twenty-four hours on a stock rabbit host.

The rabbits were carefully observed for gross signs of systemic toxicity for a period of sixteen days following oral administration of the indicated material. At the end of the sixteen-day period, the rabbits were sacrificed and gross autopsies were performed. Prothrombin times were determined initially and at l, 2, 3, 5, 7 and 16 days following the oral administration. Coagulation times were measured initially, and at 1, 2, 7, and 16 days.

Table No. 1 presents louse mortality data for each batch of lice fed on the control and experimental animals. The mortality among the lice fed on the control animals appears to. fall within normal expected limits. Mortality among lice fed on the rabbits which received Pival alone or Pival plus vitamin K was significantly higher than the control values for seven days following the oral administration.

Throughout the experiment, the control animals ex hibited normal behavior and appearance, and at the conclusion of the experiment, both control animals showed an increase over their initial body weight.

7 Of the two rabbits which receives the parasiticide alone, rabbit No. 3 exhibited normal behavior and appearance throughout the experimental period. Rabbit No. 4 appeared somewhat emaciated for several days following the oral administration of the parasiticide, and showed a slight loss in body weight. However, after the ninth day, this animal improved in appearance and showed a slight gain in weight. At the time of sacrifice, both animals which had received the parasiticide alone showed a slight increase over their initial body Weight.

Both of the animals which received the parasiticide in combination with vitamin K exhibited normal behavior and appearance throughout the entire experimental period. At the time of sacrifice, both rabbits showed an increase over their initial body weight.

Tables 2 and 3 present the prothrombin and coagulation times, respectively, taken at intervals throughout the study. The coagulation times for all control and experimental animals were within substantially normal limits throughout the experimental period. Both of the rabbits which received the parasiticide alone showed an increase in prothrombin time, which seemed to reach a maximum on the fifth day following oral administration of the parasiticide. As is indicated by the data, rabbits 3 and 4, which received the parasiticide alone showed abnormally high prothrombin times. Higher doses of tii p'aiasitieid'efalone' have been demonstrated :to cause death' by this mechanism. At .the timed sacrifice, the p'rothrombin times of 'both' animalswhich hadreceived theparas iticide alone'were within' normal limits.

Table' No. 1

- PERCENT OF LICE MORTALITY 5.0 mgJkg. 2- Control, 5.0 cc. 5.0 mgJkg. 2- pivalyl-1.3- corn oil pivalyl-1,3- indandione with indandione 1.0 mgJkg. Vita- Days After min K1 I.V.

Application Rabbit Rabbit Rabbit No. 1 No. 2 No. 3 No. 4 No. 5 IfIo. 6

5 a 77 as so 93 20 0 80 70 ,100 100 30 10 90 100 100. 100 0 0 90 100 90 100 30 60 90 90 90 90 0 10 80 100 80 40 0 0 50 100 60 10 10 20 90 80 50 Table No. 2 I PRO'IHROMBIN TIME, IN SECONDS 5.0 rngJkg. 2- Control, 5.0 cc. 5.0 mgJkg. 2- ivalyl-1,3- corn oil. pivalyl-1,3- in andione with indendione 1.0 mgJkg. Vita- Deys After min Kl I.V. Application nabbit Rabbit Rabbit No. 1 No. 2 No. 3 No. 4 N05 No 6 40 .9 Prothrombin times were determined by a modification of the Quick method, as re orted in the American Journal ct Medicinal Science, vol. 19 page 501, 1935.

.2. I I concurred-r1113," 1N Mmu'rnsf I Coagulation times were measured by the capillary tube method.

On the basis of the above data, it is'clear that a single oral dose of 5.0 mg. per kg. of 2-pivalyl-1,3-indandione, in combination with a single intravenous dose of 1.0 mg. per kg. of vitamin K administered to rabbits, produced a highly'significant percentage of mortality among laboratory reared lice which were fed upon the rabbits. The period of efiectiveness lasted at least seven days following the dosage. The rabbits which received the parasiticide alone exhibited an elevation in prothrombin time. The prothrombin times of those rabbits'which received theparasiticide in combination with'vitaniin K; were within normal limits.

EXAMPLE 2 The preceding" example demonstrated the effectiveness of Z-pivaIyI-LB-indandione against external parasites. The present example will demonstrate the eifectiven'ess'of the same compound against internal...parasites.

From a. large group of laboratory dogs selected, at random. five dogs were obtained which showed relatively heavy infestations of the hookworm, Ancylostoma caninum. Other helminth ova observed inthe feces of these dogs included the tapeworm Taenia taeniaformisi and the roundworm Toxacara cam's (ascarid) and Tri-- churis yulpis (whipworm). The results of an initial fecal examination of these five dogs are presented in Table No.4. a

Each of the five dogs was administered, by capsule, on an empty stomach, a dose of 25 mg. of 2-pivalyl- 1,3-indandione per kg. of body weight and 25 mg. of vitamin K (Mephyton, Merck) per kg. of body weight. The animals were then ofiered the regular laboratory Table No. 4

[Summary of ova observed initially in fecal specimens from a group of 5 dogs, and parasitic worms recovered at autopsy 21 days following one oral dose of 25 mg. of 2-pivelyl-1,3-indandione per kg. of body weight and 25 mg. of vitamin K1 (Mephyton,

Merck) per kg. of body weight.

0cc.-occasional.]

Dog N 0. 8-548 Dog No. 8-555 Dog No. 8-558 Dog No. 8-502 Dog No. 8-563 F F F M M OVA OBSERVED INITIALLY occ. hookworm. occ. hookworm occ. hookworm, occ. hookworm. iew hookworm,

occ. escerid i'ew occ. whipworm, few whipworm.

tapeworm T. many tapeworm taentajormir). (T. taeniujormia) (specimen contained a. portion of a mature tapeworm, T. taminformic).

WORMS RECOVERED AT AUTOPSY 5 ascarids. 8 escarids. 51 whipworms. i whipworm. 6 aecerids. 2 T. taeniaformia. 1 T. taeniaformia. 6 T. tamiaformia. 5 T. tamiaformil. 31 whipworme. 2 D. canium. 1D. canium. v 4 D. canium.

assess? diet. Throughout thejttidL-th'e animals were housed individually Twenty-one -.days following dosage with the 2-.piva.ly1-f1,3-.indandione, .the animals were sacrificed andgross autopsies were performed. At the time of autopsy, a thorough search for macroscopic helminths was made. The Worms recovered for each dog are summarized in Table No. 4.

As shown by the data in Table No. 4, the 2-pivaly1- 1,3-indan'dione efiected a one hundred percent removal of the 'hookworms which were initially present in each dog. The other ihelminths present do not fall within the category of blood-sucking parasites, and there appeared to belittle, if any eifect on the tapeworms, ascarids, and whipworms.

. .Prothrombin times were determined initially and at 3, 6, l0, and 14 days following the administration of the 2=pivalyl l;3-indandione. The results of these determinations are summarized in Table No. 5. Each dog exhibited a moderate rise in prothrombin time within three days following the oral administration of 2-pivalyl-- 1.3-indandione. Within six days, the elevation had become marked in three of the five dogs. It was evident from the prothrombin time that there was danger of internal bleeding, and in the case of any external injuries which might cause bleeding, there would be a definite danger of the animal bleeding to death. Consequently, on-the seventh day, each dog was administered intravenouslya dose of 10 :mg. of vitamin K in order to reduce the hypoprothrombinemia. At 10 and 14 days, the prothrombin times of all animals were within norma limits. 1

Table No.

[Rrothromhin-tinres. in seconds, of dogs that received asingle oral dose of 25 mg. of 2'-pivalyl=1,3indandione per kg. of body weight and 25 mg. of .vitamin. K1 '(Mephyton, Merck) per kg. of'body weight. A supplemental dose of vitamin K1 was administered intravenously, as indirated. Sae.= sacrificed.] v

'Days'Followmg Dog No. .Dog'No. Dog No. Dog No. Dog No.

Dosage 8 .5.48 18:555. 8-558 8-562 8-563 7. 4 7. 3 7. 9 25. 3 23. 9 24. 4 24. 7 58.0 12. 6 Kl adminttred intra-enously 7. 2 8.1 8. 2 8. 1 7. 8 9.3 Sac. ,Sac. Sac.

'PROTHROMBIN TIMES (IN SECONDS) or mononmnoos roLLowmo THE DAILY GESTION onnxrr (1% OF'BODY wnron'r) CONTAINING 0.025 PIiAL During the- :study the animals; exhibited generally normal behavior and appearance, and the body weight remained fairly constant. .At gross autopsy, one animal exhibited hemorrhagic areas of :the duodenal mucosa; A second animal exhibited pronounced hemorrhage and edema of the mucosa of the small and large intestines, with a small amount of blood being present in the cecum. The organs of the remaining three animals appeared within normal limits.

This experiment indicated that a single oral dose of 25 mg. of 2-pivalyl-1,3-iudandione per kg. of body weight removed one hundred percent of the hookworms present in each of the test animals. The experiment also indicated the efiicacy of intravenously administered vitamin K in 'antidoting the undesirable anticoagulant efi'ects produced in the animals by 2-pivalyl-1,3-indandione.

EXAMPLE 3 Further experiments were conducted to determine the subacute toxicity of 2-pivalyl-1,3-indandione, and to determine the efficacy of the antidote.

Literature research and experience with dogs indicated that the prothrombin time using undiluted plasma isnormally 7 to 12 seconds, and that the coagulation time is normally from 1 to 5 minutes. For the purpose of this experiment, it was arbitrarily decided that a prothrombin time of about 20 seconds and a coagulation time about 8 to 10 minutes would be indicative of bleeding tendencies andwould indicate a dangerous level. In this example, antidote was administered when the prothrombin time reached seconds andwhen the coagulation time was 8 minutes or longer.

Ten dogs were used to determine the subacute toxicity of .2-pivalyl-1,3-indandione. .Two dogs were used as a control group and were maintained on a normal diet of horse'meat calculated to be equivalent to approximately 1% of the. body Weight of the animal. The remaining 8 dogs were administered a daily bait of horse meat containing 0.025% .Pival by weight, .so that each dog consumed by oral ingestion 2.5 mg. per kg. of body weight of 2-pivalyl-.l,3-indandione per day.

Prothrombin and coagulation times were determined Table No. 6

{Group 1 received 2-pivalyl-l,3-indandione until death. 2-pivalyl-1,3-indandione was withdrawn after 6 days in groups 2, 3, and 4. After 2-pivaly1-1,3-indandione withdrawal, vitamin K (Synkayvite Roche) was administered intravenously to group 3 and vitamin K1 (Mephyton) to group 4.]

0-150 0-151 0-146 (3-149, 0-153 6-154 (J- 0-152 0-147 0448 F F .M F M M M F M F Days Controls Group 1 Group 2 Group 3 Group 4 8.6 8.4 9.0 8.6 8.6 9.4 8.7 8.4 21. 3- 24. 9 32. 0 28. 4 17. 4 23. 5 15.8 22. 6 14. 5 79. 8 42. 9 35. 0 51. 6 18. 2 40. 4 88. 2 18. 0 134. 4 128. 3 128. 3 l 65.5 I 720-840 I\ 60. 6 I 1, 200

I Iutravenouslniection of. antidote ,(1 mgJkgJ. 'Intravenous injection otantidoto (10 rug-.lkgJ.

In addition. to the bait of horse meat, each dog, after consuming initially and were followed closely after the animals had started on the bait.

The '10 dogs were divided into a control group and tour experimental groups. Experimental group 1 was fed 2-pivalyl-1,3-indandione bait each day until death of the animals. Groups 2, 3, and 4 were fed poisoned bait until definite toxic signs of markedly prolonged prothrombin and coagulation times developed, at which time the bait was withdrawn from each of groups 2, 3, and 4. In group 2, after the poisoned bait was discontinued, "the dogs received no further treatment except normal laboratory diet and care. In group 3, after the last consumption of the poisoned bait by the dogs in group 3, each dog received an intravenous injection of vitamin K amounting to v1 mg. per kg. 'of body weight of the dog. The vitamin .K administered to the two "dogs .in group 3 was Synkayvite Roche, which is the tetrasodium .salt of 2-.methyl-l,4-naphthohydroquinone disphosphoric acid. The two animals in group 4 also received an intravenous injection :of 1 .mg. per kg. of antidote immediately after the last poisoned bait had been consumed. (Mephyton). 'Tableswfi and 71present :the prothrorribin and coagulation Stimes of the dogs. These times increased progressively up to "the sixth day of the study in all but one dog, No. -146, where the peak wasnot :reached until :about the-eleventh'day.

The antidote used was vitamin K signs of the efiiect of 2-pivalyl-1,3-indandione in these animals were generally weakness, anorexia, bleeding at the sites of venipuncture, salivat ion, vomiting, bloody fluid in the mouth, bloody feces, progressive weakening, labored respiration, tremors, extensor spasms, and coma. 1. l

One of the dogs (C-147) in group 4 was given 1 mg. of vitamin K per kg. intravenously, and had normal prothrornbin and coagulation time values within 24 hours, while the second dog in group 4 (C448) show'eda slower return of these values to normal, during the week following the vitamin K injection.

The intravenous administration of 1 mg. per kg. of Synkayvite Roche to the dogs in group '3 was particularly effective in decreased prothrombin and coagulation time values within 24 hours. The decrease in prothrombin time was very slight in dog 33-145 in group 3-, and therefore, this animal was ejected with another '1 mg. per 'kg. dose of Synkayvite Roche. Signs of systemic toxicity and hemorrhages did not subside appreciably during the first 24 hours following the administration of Synkayvite Roche, and within 48 hours the coagulation times had increased to 1.8 and 25.5 minutes in dogs C-145 and C-152, respectively (Table 7). Each dog was therefore given 10 mg. per lg. of Synkayvite Roche intravenously. The coagulation and prothrornbin times fluctuated somewhat in these animals for the next. five days, but then dropped to within normal limits.

CDAGULAZIIQN TIMES .(IN MINU (DESlOFli/ION GRELDOGS .FOL'LOWIN G DAILYINGESTION 0F "BAIT (1% OF BODY 'WEIGHT) CONTAINING 0.025% PIVAL [fir 'oupi received 2mivaly'l-L3-indandione .uritihdea'th. Withdrawal of 2-pivalyl-.1,3-indandione after fidays in gronpsl2, ligand! was -lol1owed by vitamin K administration to group 3 and vitamin K to group 4.]

0-150 0-151 0-1146 0-149 0-153 0-154: 0-145 0-152 0-147 0-148 .F .F M F 1 M M M F M F Days I Controls Groupl Group 2 Group 3 Group 4 2-pivalyl-1,3-indandione discontinued 1. 5 o 2.75 3.0 4.25 4.0 4.75 1.0 1 as 3.0 4.75 3.25 3-5 1.5 4.5 2. 25 as 4. 0 2.5 2.5 4.0 2.25

s s s s lntravenous injection .01 antidote 1.mg./k Intravenous injection of antidote (10 mgJkg.) S *Sacrificed.

Determination .011 .0 day by Lee-White method. Other determinations by capillary tube-method.

No material change in either the prothrombin time or the coagulation time was observed in the case of the tworcontr'ol dogs.

The dogs in group .1 were :maintained 'on the poisoned bait until they died. No. -C-149 received a total of 15 mg. of :2-pivalyl-1,'3-indandione per Pkg. of .her body' weight and died on the seventh day. No. CE-146, the male dog in group 1., received a total of 32.5 mg. per kg. and died :on the thirteenth day.

The remaining six experimental dogs in groups 2, 3, and 4, .had each ingested a total of 1 7.5 mg. per kg. of 2 -piva'1yl-J.,B-indandione when the baitwa's removed. At that time, each of the animals had a'prothrombin time and a :coagulation time well above normal.

The two dogs in group 2 received no treatment except the .removal of the Pival bait after the sixth day. Neither .dog recovered. Prothrombin :and coagulation times decreased somewhat following the removal of the poisoned bait, but bothdogs died within a 'week. Gross received 2-pivalyl-1,3-indandione until they died, with no antidote being administered revealed a high incidence of subcapsular cortical infarcts in the kidneys, irritation in the gastrointestinal tract, and hemorrhages in the lungs. There was also exhibited some vascularization or hemorrhages involving the brain and its coverings.

In group 2, where the Z-pivalyl-l,3-indandione 'baiti was removed after 6 days, but no antidote 'was administered to the animals, the post-m-ortem examination revealed the sameconditions which were found in the 'two dogs in group 1.

In groups 3 and 4 where an antidote was administered on the sixth day to "each animal, *nodamage'was observed in the animals.

. .dione.

11' EXAMPLE 4 daily dosages, having a cumulative efiect, was demonstrated.

In Table No. 8', there are summarized the results obtained through administration to two groups of rats, orally, of a single dose amounting to 20 mg. per kg. of 2?pivalyl-l,3-indandione, and of 2-isovaleryl-1,3-indan- At 6 hours, 12 hours, and at 24 hour intervals after'ingestion of the parasiticide, controlled feeding of fleas on each rat was allowed. Mortality counts were taken on each group of fleas after a single blood feeding period of 30 to 45 minutes. Mortality rates of between about 10 percent were observed.

Table No. 8

EFFECT ON FLEAS ALLOWED ONE 131.0013 MEAL ON RATS DOSED WITH A SINGLE ORAL ADMINISTRATION OF THE rxnasrrrornn In Table No. 9, there are summarized the results obtained through administration to three rats, orally, of daily doses of 20 mg. per kg. each of 2-isovaleryl-l,3- indandione. At 6 hours, 12 hours, and at 24 hour intervals after initial ingestion of the parasiticide, controlled feeding of fleas on each rat was allowed. Mortality counts were taken on each group of fleas after a single blood feeding period of 30 to 45 minutes. Mortality rates of between 10 to 40 percent were observed.

Table No. 9

EFFECT ON FLEAS ALLOWED ONE BLOOD MEAL N RATS RECEIVING A DAILY DOSE OF A PARASITICIDE Percent mortality of fleas fed at indicated times after initial in- Dosage, Host gestion of parasitlcide mg.lkg. Chemical No.

12 24 48 72 hrs. hrs. hrs. hrs. hrs.

20 sodium salt of 2- 8 30 30 2O 20 20 lsovaleryl-l, 3- 9 20 20 20 10 'indandione. 10 40 20 Died In Table No. 10, data are summarized illustrating the results obtained from the oral administration to three rats of 20 mg./kg. doses of the sodium salt of 2-pivalyll,3-indandione daily. Unfortunately, two of the three rats immediately died, apparently from the toxic effects of the chemical. One group of fleas was allowed to feed repeatedly on the surviving rat, and the mortality count, basedon the number of fleas at'each new 24 hour interval, was observed. The repeated feeding simulated natural infestation.

12 Table No. 10

EFFECT ON FLEAS ALLOWED SEVERAL BLOOD MEALS ON RATS RECEIVING DAILY DOSES OF A PARASITICIDE Percent mortality of fleas fed at indicated intervals after Dosages, Chemical Host initial administration mgJkg. N o.

24 hrs. 48 hrs. 72 hrs. 96 hrs.

20 sodium salt of 2- 8 20 10 10 10 pivalyl-l, 3- in- 9 Died dandlone. 10 Died 1 The fleas employed were 14 days old.

The fleas employed in the above tests were oriental rat fleas, Xenophylla cheopis. These demonstrations indicated that the sodium salts tested possess definite toxic properties as chemotherapeutic agents. The more effective chemical of the two appears to be the sodium salt of 2-pivalyl-1,3-indandione. Young fleas appeared to be more susceptible to the toxic agents than older fleas.

EXAMPLE 5 Secondary toxicity studies were made by feeding mice which had been poisoned by ingestion of 2-pivalyl-l,3- indandione to cats. A diet containing 0.025% 2-pivalyl- 1,3-indandione in ground .rat meal was prepared and offered to forty albino mice housed collectively. The diet and water were available at all times. When signs of bleeding 'were apparent in some of themiceand one death had occurred, all of the surviving mice were sacrificed, weighed, frozen with liquid air, andwere homogenized in a Waring Blendor with 1000 ml. of distilled water. The homogenate was then frozen in an ice cube tray.

Three cats were selected and control coagulation times of blood taken from a slit in the ear pinna were determined by the capillary tube method. One cat was designated as a control and was given one normal live mouse each day. Each of the other two cats received aliquots of the whole mouse homogenate, equivalent to one 25 gr. mouse for thirteen days and portions equal to two mice for two days. The mouse homogenate was mixed with a small amount of commercial cat food. Additional cat food was offered freely to the cats daily. Coagulation times of each cat were determined at three-day intervals for the first twelve days and of the fourteenth day of the study. The cats were observed daily for gross signs of systemic toxicity and bleeding tendencies. After fifteen days the cats were sacrificed, blood was collected for prothrombin and coagulation times, and complete gross autopsies were performed. In the mice fed the 0.025% 2-pivalyl-l,3-indandione diet, intoxication was apparent within twenty-four hours when they exhibited signs of weakness and gross bleeding from the anus. After fortyeight hours one mouse had died and it became apparent that the animals had ingested nearly the maximum tolerable amount of the compound.

In the cats receiving portions of the poisoned mouse homogenate, no untoward effects were found. Both cats remained healthy with no significant changes in coagulation times over the fifteen-day period. Terminal coagulation and prothrombin times of the experimental and control animals were comparable.

At autopsy, no significant gross pathology was found which could be directly attributed to secondary intoxication.

The several specific examples set forth above demonstrate the effectiveness of the specified parasiticides against external and internal parasites. The present invention appears to offer great promise as a method of controlling parasitic infestation of man and animals.

While oral administration of the parasiticidal chemical parenterally by intravenous injection. Simultaneous parenteral administration ofboth the parasiticidal enemspecifically mentioned int'he "above description. For example, iit addition to tho'se animals sp'ecifioa lly m'ehtion'ed, it is .possiblei'to employ the present inventions-er the control of internal and external parasites :in all fort-its of domesticated 'animals, sudh as homes, "battle, goats, sheep, and other similar animals, as well as for animals such as the fox, chinchilla, and mink, which are raised for their valuable furs.

We claim:

1. A method of controlling lice and hookworm parasite infestations in a veterinary animal infested with at least, one of said parasites comprising rendering said animal systemically toxic through the administration to the animal of a parasiticidal compound selected from the group consisting of compounds having the formula where R is a radical selected from the group consisting of lower alkyl, cycloalkyl, aralkyl and aryl, and the alkali metal and alkaline earth metal salts of these compounds; and approximately simultaneously antidoting the anticoagulant effects of the parasiticidal compound by administering said animal a counteracting amount of an anti-anticoagulant factor.

2. The method of claim 1 where R is a lower alkyl radical.

3. The method of claim 1 where R is a butyl radical.

4. The method of claim 1 where R is a tertiary butyl radical. I

5. A method of controlling lice and hookworm parasite infestations in a veterinary animal comprising rendering the infested animal systemically toxic to said parasites by causing the animal to ingest at least 5 mg. per kg. of its body weight of a compound selected from the group consisting of compounds having the formula E i CH--ER l where R is a radical selected from the group consisting of lower alkyl, cycloalkyl, aralkyl and aryl, and the alkali metal and alkaline earth metal salts of these compounds; and approximately simultaneously counteracting the anticoagulant elfects of the compound by the parenteral administration to said' animal of an antihemorrhagic factor.

6. The method of claim Sin which R is a lower alkyl radical.

7. The method of claim in which R is a tertiary butyl radical.

8. A method for controlling lice and hookworm parasites infesting a domesticated animal comprising rendering the infested animal systemically toxic to said parasites by oral ingestion of an amount on the order of lected from the group consisting of compoundshaving the formula LE \CH--ER where R is a radical selected from the group consisting of lower alkyl, cycloalkyl, aralkyl and aryl, and the alkali metal and alkaline earth metal salts of these compounds; "and approximately simultaneously anti'dotin'g Tfhe anticoa ulant e'iiect of the -'compound by the parenteral administration of a substance having vitamin K activity.

9. The method of claim 8 in which R is a lower alkyl radical.

10. The method of claim 8 in which R is a tertiary butyl radical.

11. A method for controlling lice and hookworm parasitic infestation in a domesticated animal comprising rendering the infested animal systemically toxic to said parasites through the administration to the animal of a single massive dose on the order of about 300 mgs. per kilogram of body weight of the animal of a parasiticidal compound selected from the group consisting of compounds having the formula 0 II C where R is a radical selected from the group consisting of lower alkyl, cycloalkyl, aralkyl and aryl, and the al kali metal and alkaline earth metal salts of these compounds; and antidoting the anticoagulant effects of the parasiticidal compound by administering to the animal a counteracting amount of an anti-anticoagulant factor.

12. The method of claim 11 in which R is a lower alkyl radical.

13. The method of claim 11 in which R is a tertiary butyl radical.

14. A method for controlling lice and hookworm parasitic infestation in a domesticated animal comprising rendering the infested animal systemically toxic to said parasites through the administration to the animal of a plurality of small doses of a parasiticide having a cumulative effect selected from the group consisting of compounds having the formula where R is a radical selected from the group consisting of lower alkyl, cycloalkyl, aralkyl and aryl, and the alkali metal and alkaline earth metal salts of these compounds; and simultaneously antidoting the anticoagulant effects of the parasiticidal compound by administering to the animal a counteracting amount of an anti-coagw lant factor.

15. The method of claim 14 in which R is a lower alkyl radical.

16. The method of claim 14 in which R is a tertiary butyl radical.

17. A method for eliminating lice and hookworm parasitic infestation in a veterinary animal infested with at 0 cH-Ji-R O CH-iil-R about 5 mg. per kg. of body weight of a compound .seleast one of said parasites which comprises rendering the 2384357 '15 i -16 v infested animal systemically toxic to said parasites kali metal and alkalingearth nicta l salts of these comthrough the administration to the animal of a compound pounds and an anti-anticoagulant antidote for the antiselected from the group consisting of. compounds having coagulant efiect of the'compound administered. the formula o 5 g I References Cited in the file of this patent I5 FOREIGN PATENTS 0 112,639 Australia Feb. 27, 1941 OTHER REFERENCES C 10 Bram: Chem. Ahst., vol. 44 1950), p. 50540.

Kabat: The J. of Pharmacol. and Expt. Then, vol. 80, No. 2, February 1944. pp. 160-170 (pp. 160-164 pert.).

Crabtree: Pest Control, July 1953, pp. 22 and 24.

where R is a radical selected from the group consisting of lower alkyl, cycloalkyl, aralkyl and-aryl, and the 81-:

Claims (1)

1. A METHOD OF CONTROLLING LICE AND HOOKWORM PARASITE INFESTATIONS IN A VETERINARY ANIMAL INFESTED WITH AT LEAST ONE OF SAID PARASITES COMPRISING RENDERING SAID ANIMAL SYSTEMICALLY TOXIC THROUGH THE ADMINISTRATION TO THE ANIMAL OF A PARASITICIDAL COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS HAVING THE FORMULA.