AU2004257351B2 - Methods and compositions for controlling ectoparasites - Google Patents

Description

WO 2005/007188 PCT/AU2004/000955 METHODS AND COMPOSITIONS FOR CONTROLLING ECTOPARASITES 5 BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to methods and compositions for controlling ectoparasites. In 10 particular, the invention relates to methods and compositions for inhibiting hatching of an ectoparasite egg. The invention also provides methods and compositions for preventing or treating ectoparasite infestation. The invention also relates to methods for identifying compounds that can inhibit ectoparasite egg hatching. 15 DESCRIPTION OF THE PRIOR ART Ectoparasites including some insects cause significant pest problems in a wide variety of animals and plants. In particular, ectoparasites typically can annoy, bite, and cause infections to humans and domesticated animals. Of particular concern is the presence and 20 effect of such parasites on humans, household pets or companion animals, such as dogs and cats, and other domesticated animals, such as horses. Various compositions and application techniques are known for controlling or eliminating biting or blood-sucking pests (ectoparasites), such as fleas, ticks, flies, lice and mites. 25 Over the years a host of aerosols and space sprays, liquids, soaps, shampoos, wettable powders, granules, baits, and dusts, have been proposed for the control of such ectoparasites. Conventional control measures for ectoparasites have relied on the use of chemical 30 insecticides, for example chlorinated hydrocarbons (DDT, endosulfan etc), and synthetic pyrethroids (cypermethrin, deltamethrin). Problems associated with the use of chemical WO 2005/007188 PCT/AU2004/000955 -2 pesticides include the development of resistance by target ectoparasites, the persistence of the chemicals in the environment and in plant and animal tissues, and the harmful effects on host and non-target organisms. 5 Other types of ectoparasiticides include insecticides, such as insect growth regulators (IGRs) that are known to interfere with chitin synthesis and insecticidal bacterial toxins (eg. Bacillus thuringiensis (Bt) toxins). More useful groups of insecticides are those having high insecticidal activity and low environmental persistence, such as organophosphates and synthetic pyrethroids. However, a significant problem associated 10 with these insecticides is the development of resistance by target insects. For example, insecticidal agents used to treat lice are described in EP 0191236 and U.S. Pat. No. 5,288,483. A significant disadvantage of using these agents is that lice can become resistant. The need for further treatment increases the exposure to these harsh 15 agents and increases the cost. Additionally, clinicians and parents are reluctant to treat children with agents that can also prove toxic to human beings. Moreover, many of these compounds have unpleasant odors or other undesirable properties, causing noncompliance by the patient, leading to re-infestation of the individual, and spreading of the infestation to others. In addition, the harshness of these agents makes them unsuitable for use as 20 prophylactics. In the case of head lice infestation, home remedies such as application of corn oil, olive oil, eucalyptus oil, neem oil, coconut oil, mayonnaise, or petroleum jelly for a period of time sufficient to kill the lice (e.g. overnight) are not practical or completely effective. A 25 further disadvantage of methods to treat head lice is the requirement of removing the nits from the hair in a separate treatment step. The removal of nits has typically been done by hand using special fine-tooth combs. Use of combing alone to treat head lice has the disadvantage that the lice can hold onto the hair shafts using their claws or escape by crawling away from the area being combed. This labor intensive method requires daily 30 combing, is painful, and is unpleasant since the lice are active, visible and crawling.

WO 2005/007188 PCT/AU2004/000955 -3 There is a significant need for improved control of lice throughout the world. In particular, there are well-documented failures of products aimed at treating lice. The development of resistance of lice to many of the currently used chemicals including permethrin, pyrethrin and malathion is considered a major factor in treatment failures. In addition, inappropriate 5 formulations containing suboptimal actives are also believed to be in part responsible for resistance development. More recently there has been significant growth in the market for herbal products for treating head lice however there is very little published evidence from properly conducted trials to enable an effective assessment of these products to be made. Furthermore while a number of products claim to possess ovicidal activity the evidence for 10 this in the field is far from convincing hence it is common for products to recommend that following an initial treatment a second treatment should be given between 7-14 days later to kill newly emerged nymphs. Recently attention has focused on insect proteases that may provide a possible means of 15 ectoparasite control. Proteases perform a variety of functions in the organism including the regulation and breakdown of proteins and peptides, and thus assist with digestion. They are also involved in tissue reorganization during embryo development, molting and pupation. Proteases are a widely variable group of enzymes and include digestive proteases that vary considerably both in number and in catalytic properties within and 20 between species. For example, trypsin-like seine proteases have been recognized to be involved in the key growth regulatory area of molting (Samuels R.I. and Paterson C.J., Comparative Biochemistry and Physiology, 1995, 11 OB: 661-669). Protease inhibitors have been suggested to be a useful alternative to the chemical control 25 methods, particularly where the ectoparasites have become resistant to these chemical pesticides. In particular, serine and cysteine protease inhibitors have been shown to reduce the larval growth and/or survival of various insects (Dymock et. al., New Zealand Journal of Zoology, 1992, 19: 123-131). Growth inhibition has been achieved with inhibitors of principal digestive enzymes of the gut and have been targeted at ectoparasite larvae or 30 mature parasites. However, little is known about other types of activity and function of various classes of protease inhibitors. A common problem of existing ectoparasiticides is WO 2005/007188 PCT/AU2004/000955 -4 that they do not effect the ectoparasite eggs and therefore application of the parasiticides to hosts often require repeated treatment or prolonged exposure to the parasiticide for it to be effective. This is not only inconvenient but also increases risks to the environment and to the host. 5 Accordingly, there remains a need for providing alternative methods and compositions that are effective in inhibiting ectoparasite egg hatching to provide efficient control of ectoparasites.

WO 2005/007188 PCT/AU2004/000955 -5 SUMMARY OF THE INVENTION In an aspect of the invention there is provided a method for inhibiting hatching of an ectoparasite egg comprising exposing the ectoparasite egg to at least one metal chelating 5 agent, wherein the metal chelating agent is a compound comprising at least two heteroatoms able to simultaneously coordinate with a metal ion, at least one of the two heteroatoms being selected from nitrogen, sulfur, oxygen and phosphorus, wherein the compound comprises at least one carbocyclic ring substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom, or the compound comprises at 10 least one heterocyclic ring containing at least one heteroatom, wherein said heterocyclic ring is optionally substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom. In another aspect of the invention there is provided a method for inhibiting hatching of an 15 ectoparasite egg, the method comprising exposing the ectoparasite egg to an effective amount of a metalloprotease inhibitor. The present applicants have identified metal chelating agents and metalloprotease inhibitors as effective agents for inhibiting ectoparasite egg hatching. The use of metal 20 chelating agents or metalloprotease inhibitors for inhibiting ectoparasite egg hatching has the advantage of preventing breeding cycles of ectoparasites thereby controlling ectoparasite infestation. In another aspect there is provided a method of treating or preventing ectoparasite 25 infestation in a host comprising applying an effective amount of at least one chelating agent, wherein the metal chelating agent is a compound comprising at least two heteroatoms able to simultaneously coordinate with a metal ion, at least one of the two heteroatoms being selected from nitrogen, sulfur, oxygen and phosphorus, wherein the compound comprises at least one carbocyclic ring substituted with at least one heteroatom 30 and/or with a substituent containing at least one heteroatom, or the compound comprises at least one heterocyclic ring containing at least one heteroatom, wherein said heterocyclic WO 2005/007188 PCT/AU2004/000955 -6 ring is optionally substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom. In yet another aspect of the invention there is provided a method of treating or preventing 5 ectoparasite infestation in a host, the method comprising applying an effective amount of at least one metalloprotease inhibitor to a subject. In yet another aspect of the invention there is provided a method of inhibiting the hatching of a louse egg, comprising exposing the louse egg to an effective amount of at least one 10 compound of formula (Ia): R2 RI R' R2 R3 X

R

3 ' (Ia) N N R4 R4' wherein X is selected from a covalent bond, -C(R 5

)

2 -, -Z- or -C(R5)2-Z-C(R)2 15 R1 and R" are independently selected from hydrogen, C 1

-

6 alkyl, C 2

.

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C1.

6 alkoxy, thiol, CI- 6 alkylthio, CO 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH1 2 ,

NHCI

6 alkyl or N(C 1

.

6 alkyl) 2 ; R2, R2, R3, R', R4 and R4' are independently selected from hydrogen, C1.

6 alkyl,

C

2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, CI 6 alkylthiol, CO 2 H, C0 2

C

1

.

6 alkyl, 20 SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NHIC 1 6 alkyl or N(C 1

.

6 alkyl) 2 , or -CH 2

CHNH(CO

2 H); or

R

2 and R 3 or R3 and R 4 and/or R 2 ' and R 3 ' or R3' and R 4 ' taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring; each Rs is independently selected from hydrogen, C 1

.

6 alkyl, C 2

.

6 alkenyl, C 2

.

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , 25 NHC 1

.

6 alkyl or N(C 1

.

6 alkyl) 2 ; and Z is selected from a covalent bond, -NH-, -0-, -S-, -C(O)- and -C(S)-; WO 2005/007188 PCT/AU2004/000955 -7 or a pharmaceutically, veterinary or agriculturally acceptable salt thereof. In a further aspect of the invention there is provided a composition for inhibiting hatching of an ectoparasite egg comprising an effective amount of at least one metal chelating agent, 5 wherein the metal chelating agent is a compound comprising at least two heteroatoms able to simultaneously coordinate with a metal ion, at least one of the two heteroatoms being selected from nitrogen, sulfur, oxygen and phosphorus, wherein the compound comprises at least one carbocyclic ring substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom, or the compound comprises at least one 10 heterocyclic ring containing at least one heteroatom, wherein said heterocyclic ring is optionally substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom and a suitable diluent, excipient or carrier. In yet a further aspect of the invention there is provided a composition for inhibiting 15 hatching of an ectoparasite egg, the composition comprising an effective amount of at least one metalloprotease inhibitor and a suitable diluent, excipient or carrier. In another aspect of the invention there is provided a method of identifying a compound which inhibits hatching of an ectoparasite egg, the method comprising assessing the ability 20 of the compound to inhibit a metalloprotease present in the ectoparasite egg. In yet another aspect of the invention there is provided a metal chelating agent and/or metalloprotease inhibitor identified by the method described above.

WO 2005/007188 PCT/AU2004/000955 -8 BRIEF DESCRIPTION OF THE FIGURES Figure 1: shows a gelatine substrate SDS-PAGE analysis of protease activity of washings obtained from various samples of hair and lice eggs following staining of the gel with 5 Coomassie blue and destaining. Lane 1 shows protease activity detected in the washings obtained from unhatched lice eggs within 12 hours of hatching. Protease activity was in the higher molecular weight region of the SDS gel. Lane 2 shows protease activity detected in the washings from hair indicating the presence of a number of highly active and stable proteases likely to be of maternal origin. Lane 3 shows a hair sample that was 10 washed with a 1% solution of sodium hypochlorite for 1 minute followed by a number of water washes in an attempt to remove these contaminating proteases. This treatment was able to remove the maternal proteases resulting in no protease species being detected in the hair only sample. Lane 4 shows protease activity detected in the washings from eggs within 12 hours of egg hatching treated with sodium hypochlorite (as described above). 15 This treatment removed the protease activity that was observed in the unwashed sample (compare to lane 1). Lane 5 shows the presence of one or two high molecular weight protease species in egg washings from lice eggs that had been pretreated with sodium hypochlorite and allowed to hatch. These proteases were specifically associated with the lice eggs at the time of egg hatching. 20 Figure 2: shows a Coomassie stain of inhibitor treated gelatine SDS-PAGE gels of the egg shell washings from lice eggs following hypochlorite treatment. Three bands were evident at approximately 25-30 kDa (bracketed). Lane 1, ESW positive control no inhibitor treatment, lane 2, ESW after treatment with 10mM 1,10-phenanthroline, lane 3, 25 ESW after treatment with 5 mM PMSF and lane 4 ESW after treatment with 10pM E-64. Incubation was performed at 37*C for 3 hours. Note the significant reduction in protease activity following treatment with 1,10-phenanthroline (lane 2 bracketed region). No reduction in protease activity of the ESW was observed when the aspartic inhibitor pepstatin was used (data not shown). 30 WO 2005/007188 PCT/AU2004/000955 -9 Figure 3: shows the effect of 1,10-phenanthroline on egg hatching in lice. Eggs were treated 5 days post laying and then hatching observed over time. Figure 4: shows the effect of Bestatin on egg hatching in lice. Eggs were treated 5 days 5 post laying and then hatching observed over time.

WO 2005/007188 PCT/AU2004/000955 - 10 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As used herein, the term "metal chelating agent" refers to a compound comprising at least two heteroatoms able to simultaneously coordinate with a metal ion, at least one of the two 5 heteroatoms being selected from nitrogen, sulfur, oxygen or phosphorus, wherein the compound comprises at least one carbocyclic ring substituted with at least one heteroatom and/or a substituent containing at least one heteroatom, or the compound comprises at least one heterocyclic ring containing at least one heteroatom, and wherein said heterocyclic ring is optionally substituted with at least one heteroatom and/or a substituent containing at 10 least one heteroatom. Preferably the metal chelating agent contains an aryl or heteroaryl ring. More preferably, the metal chelating agent comprises at least one nitrogen heteroatom. Preferably the metal chelating agent is non-intercalating. As used herein, the term "metalloprotease inhibitor" refers to a molecule, compound, 15 protein or agent that inhibits the activity of a metalloprotease associated with ectoparasite egg hatching. The inhibition may be inhibition of the expression of the metalloprotease or inhibition of the enzymatic activity of the metalloprotease. Preferred metalloprotease inhibitors are metal chelating agents. 20 Preferred metal chelating agents and metalloprotease inhibitors are selected from biaryl compounds, peptides and amino acid derivatives, tetracyclic antibiotics and thioureas. Preferred biaryl compounds include bipyridyl compounds and 1,10-phenanthroline compounds. 25 In one embodiment the metal chelating agent or metalloprotease inhibitor is a compound of formula (I): WO 2005/007188 PCT/AU2004/000955 -11 R2 R1 R1' R2'

R

3 X R3' ( I) N N R4 R4' wherein X is selected from a covalent bond, -C(R 5

)

2 -, -Z- or -C(R)2-Z-C(R5)2-; RI and R" are independently selected from hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

.

6 alkynyl, 5 hydroxy, C 1

.

6 alkoxy, thiol, CI- 6 alkylthio, CO 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 ,

NHC

1

.

6 alkyl or N(C 1

.

6 alkyl) 2 , or RI and R" taken together are -C(R 5

)

2 -, -C(R 5

)

2

-C(R

5

)

2 -,

-CR

5

=CR

5 -, C(O), C(S) or NH; 2 2 4 R, R', R 3 , R 3 , R 4 and R4' are independently selected from hydrogen, CI 6 alkyl,

C

2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, C0 2

C

1

.

6 alkyl, 10 SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NHC 1

.

6 alkyl or N(C 1

.

6 alkyl) 2 , or -CH 2

CHNH(CO

2 H); or

R

2 and R 3 or R3 and R 4 and/or R 2 and R 3 ' or R 3 ' and R 4 ' taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring; each R5 is independently selected from hydrogen, C 1

.

6 alkyl, C 2

.

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, C0 2

C

1

.

6 alkyl, SO 3 H, SO 3 C1.

6 alkyl, NH 2 , 15 NHC 1

.

6 alkyl or N(C 1

.

6 alkyl) 2 ; and Z is selected from a covalent bond, -NH-, -0-, -S-, -C(O)- and -C(S)-; or a pharmaceutically, veterinary or agriculturally acceptable salt thereof. Preferred compounds of formula (I) have at least one of the following features: 20 R1 and R" are independently selected from C 1

-

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy,

C

1

.

6 alkoxy, thiol, C 1

.

6 alkylthio, CO 2 H, C0 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NHC1. 6 alkyl or N(C1.6alkyl)2,, more preferably hydrogen or C 1

-C

3 alkyl, even more preferably hydrogen or methyl;

R

2 and R2' are independently hydrogen or C 1

.

3 alkyl, more preferably hydrogen; WO 2005/007188 PCT/AU2004/000955 -12

R

3 , R", R 4 and R 4 ' are independently selected from hydrogen, Ci- 6 alkyl, C 2

-

6 alkenyl,

C

2

-

6 alkynyl, Ci- 6 alkoxy, C 1

.

6 alkylthiol or CO 2

C

1

.

6 alkyl, preferably hydrogen or C 1

-

3 alkyl, more preferably hydrogen or methyl; each R 5 is independently selected from hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, 5 C 1

.

6 alkoxy, C1- 6 alkylthiol or CO 2

C

1

.

6 alkyl, preferably hydrogen or C 1

.

3 alkyl, more preferably hydrogen or methyl; X is a covalent bond, -CH 2

-Z-CH

2 - or Z, preferably a covalent bond; and Z is -NH-, -0- or -S-, preferably -NH-. 10 Preferred compounds of formula (I) include 2,2'-dipyridyl, 6,6'-dimethyl-2,2'-dipyridyl, and 5,5'-dimethyl-2,2'-dipyridyl, or a phannaceutically, veterinary or agriculturally acceptable salt thereof. 15 In another embodiment, the metal chelating agent or metalloprotease inhibitor is a compound of formula (II): 20 wherein X' is selected from a covalent bond, -C(R") 2 -, Z' or C(R13)2-Z'-C(R")2-; U is selected from N or C(R1 3 ); W is selected from -NH-, -S- or -0-; Z' is selected from a covalent bond, -NH-, -0-, -S-, -C(O)-, or -C(S)-; 25 R 1 0 is selected from hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

..

6 alkylthiol, C0 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NH(C 1

.

6 alkyl),

N(C

1

.

6 alkyl) 2 , or -(CH2)nR14; WO 2005/007188 PCT/AU2004/000955 - 13 R1 is selected from (CH 2 )maryl or (CH 2 )mheteroaryl wherein each aryl or heteroaryl is optionally substituted with one or more C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1 . 6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NH(C 1 . 6 alkyl), N(C 1

.

6 alkyl) 2 , or halo; 5 each R 12 is independently selected from hydrogen, C 1

.

6 alkyl, C 2

.

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C1.

6 alkylthiol, CO 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 ,

NH(C

1

.

6 alkyl), N(C 1

-

6 alkyl) 2 , or -(CH2)nR14; or

R

1 0 and R1 together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring; 10 each R 13 is independently selected from hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, CO 2

C

1 6 -alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 ,

NH(C

1

.

6 alkyl), N(C 1

.

6 alkyl) 2 , or -(CH2)nRI4;

R

1 4 is selected from NH 2 , OH, SH or CO 2 H; m is 0 or an integer from 1 to 4; and 15 n is an integer from I to 4; or a pharmaceutically, veterinary or agriculturally acceptable salt thereof. Preferred compounds of formula (II) have at least one of the following features: X is a covalent bond or -CH 2

-Z-CH

2 -; 20 U is N; W is NH or S; Z' is NH;

R

10 is hydrogen, C 1

..

6 alkyl, C 2

.

6 alkenyl, C 2

-

6 alkynyl, or (CH 2 )nR 14 , preferably hydrogen,

C

1

.

3 alkyl or (CH 2 )nR14; 25 R" is phenyl, phenyl substituted with C 1

.

3 alkyl or halo, thiophene, pyridine, pyridinylmethyl, imidazole or imidazole substituted with one or two C 1

.

3 alkyl;

R

2 is hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

.

6 alkynyl, or (CH 2 )nR 4 , preferably hydrogen,

C

1

-

3 alkyl or (CH 2 )R 14; or R10 and R1 together with the carbon atoms to which they are attached form a fused phenyl 30 ring; R1 3 is hydrogen or C1.

3 alkyl, preferably hydrogen or methyl; WO 2005/007188 PCT/AU2004/000955 - 14 144 R14 is NIH 2 or CO 2 H; m is 0 or 1; and n is 1 or 2. 5 In another embodiment the metal chelating agent or metalloprotease inhibitor is selected from a compound of formula (III): R220 H Ar N-- R24 P (III) R21 O R23 10 wherein Ar is phenyl, naphthyl or indolyl optionally substituted with one or more

C

1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, C0 2

C

1

.

6 alkyl, S0 3 H, S0 3

C

1

.

6 alkyl, NH 2 , NH(C 1

.

6 alkyl), N(C 1

.

6 alkyl) 2 ; R2 is selected from NH 2 , NHR2 or -CH 2 SR2; R is selected from hydrogen, hydroxy or C 1

.

6 alkoxy; 15 R is selected from hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl or C 2

-

6 alkynyl; R is selected from OH, OR2, NH 2 , NHC 1

.

6 alkyl or N(C 1

-

6 alkyl) 2 ; R5 is selected from hydrogen, C(O)C 1

-

6 alkyl wherein the alkyl is optionally substituted with -SH or -OH; R is selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl or benzyl; and 20 p is 0 or 1, or a pharmaceutically, veterinary or agriculturally acceptable salt thereof. Preferred compounds of formula (III) have at least one of the following features: Ar is phenyl or naphthyl; 25 R2 is NH 2 , -NHC(O)C 1

.

6 alkyl optionally substituted with SH, -CH 2

SC(O)C

1

-

6 alkyl or

CH

2 SH; R 2 is hydrogen or hydroxy; WO 2005/007188 PCT/AU2004/000955 - 15 R 23 is hydrogen or C 1

.

3 alkyl, preferably hydrogen or methyl;

R

24 is OH, NH 2 or Obenzyl; and p is 0 or 1. 5 Preferred compounds of formula III include Bestatin and Thiorophan or a pharmaceutically, veterinary or agriculturally acceptable salt thereof. In yet another embodiment, the metal chelating agent or metalloprotease inhibitor is a compound of formula (IV): 10 R32 H Ar R33 - . )rl(IV)

R

31 0 wherein Ar is phenyl, naphthyl or indolyl optionally substituted with one or more C1.

6 alkyl, C 2

-

6 alkenyl, C 2

.

6 alkynyl, hydroxy, CI- 6 alkoxy, thiol, C1.

6 alkylthiol, CO 2 H, 15 C0 2

C

1

.

6 alkyl, SO 3 H, S0 3

C

1

.

6 alkyl, NH 2 , NH(C 1

.

6 alkyl), N(C 1

.

6 alkyl) 2 ;

R

3 1 is selected from CO 2 H, C0 2

C

1

.

6 alkyl, C0 2

C

2

-

6 alkenyl, CO 2

C

2

.

6 alkynyl, CONH 2 ,

CONH(C

1

.

6 alkyl) or CON(C 1

-

6 alkyl) 2 ; R is selected from hydrogen, C1.6alkyl, C 2

-

6 alkenyl, C2-6alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C 1

.

6 alkylthiol, CO 2 H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NH(C1-6alkyl), 20 N(C 1

.

6 alkyl) 2 , CH 2

CH

2

CO

2 H, CH 2

CH

2

CONH

2 , CH 2

CH

2 OH, CH 2

CH

2 SH; and

R

33 is selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol,

C

1

.

6 alkylthiol, CO 2 H, C0 2

C

1

.

6 alkyl, SO 3 H, SO 3 C1.

6 alkyl, NH 2 , NH(C 1

.

6 alkyl),

N(C

1

.

6 alkyl) 2 , CH 2

CO

2 H, CH 2

CO

2

C

1

.

6 alkyl, CH 2

CONH

2 , CH 2 OH, or CH 2 SH, or a phannaceutically, veterinary or agriculturally acceptable salt thereof. 25 Preferred compounds of formula (IV) have at least one of the following features: Ar is phenyl or indolyl,

R

3 1 is CO 2 H or CONH 2

,

WO 2005/007188 PCT/AU2004/000955 - 16 R1 2 is C 1

-

6 alkyl, CH 2

CH

2

CO

2 H, CH 2

CH

2

CONH

2 , CH 2

CH

2 OH, or CH 2

CH

2 SH,

R

33 is CH 2

CO

2 H, CH 2

CONH

2 , CH 2 OH, or CH 2 SH. In yet another embodiment, the metal chelating agent or metalloprotease inhibitor is a 5 compound of formula (V): 0 S NN R (V) wherein R 41 and R 42 are independently selected from hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, 10 C 2

.

6 alkynyl or R 41 and R 42 taken together with the nitrogen to which they are attached form a 5 or 6 membered heterocyclic ring which is optionally substituted with one or more

C

1

.

6 alkyl, C 2

-

6 alkenyl or C 2

.

6 alkynyl groups; and

R

43 is selected from hydrogen, C 1

.

6 alkyl, C 2

.

6 alkenyl, C 2

.

6 alkynyl, hydroxy, C 1

.

6 alkoxy, thiol, C1.

6 alkylthiol, C02H, CO 2

C

1

.

6 alkyl, SO 3 H, SO 3

C

1

.

6 alkyl, NH 2 , NHC 1

.

6 alkyl or 15 N(C - 6 alkyl) 2 ; or a pharmaceutically, veterinary or agriculturally acceptable salt thereof. Preferred compounds of formula (V) have at least one of the following features:

R

4 1 and R 42 are independently selected from CI- 6 alkyl or taken together with the nitrogen 20 to which the are attached form a piperidine, piperazine, N-methylpiperazine or morpholine group;

R

43 is hydrogen, C 1

.

6 alkyl, C 2

-

6 alkenyl or C 2

-

6 alkynyl. In yet a further embodiment the metal chelating agent or metalloprotease inhibitor is a 25 tetracyclic antibiotic selected from the group consisting of tetracycline, doxycycline or minocycline or a pharmaceutically, veterinary or agriculturally acceptable salt thereof.

WO 2005/007188 PCT/AU2004/000955 -17 In yet a further embodiment, the metal chelating agent or metalloprotease inhibitor is selected from 1-[(2S)-3-mercapto-2-methyl-1-oxopropyl]-L-proline (Captopril) or N-(alpha-rhamnopyranosyloxy-hydroxyphosphinyl)-L-leucyl-L-tryptophan (phosphoramidon), or a pharmaceutically, veterinary or agriculturally acceptable salt 5 thereof. As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group and may have a specified number of carbon atoms. For example,

C

1

-C

6 as in "C 1

-C

6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbons in a linear or 10 branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 5-methylpentyl, 2-ethylbutyl and 3-ethylbutyl. 15 As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group having one or more double bonds between carbon atoms and may have a specified number of carbon atoms. For example, C 2

-C

6 as in "C 2 -COalkenyl" includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, 20 pentenyl and hexenyl. As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group having one or more triple bonds between carbon atoms, and may have a specified number of carbon atoms. For example, C 2

-C

6 as in "C 2 -COalkynyl" includes groups having 25 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl. As used herein the term "halo" or "halogen" refers to fluorine (fluoro), chlorine (chloro), 30 bromine (bromo) and iodine (iodo).

WO 2005/007188 PCT/AU2004/000955 - 18 The term "alkyloxy" as used herein represents an alkyl group as defined above attached through an oxygen bridge. Examples of suitable alkyloxy groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, n-pentyloxy and n-hexyloxy. 5 The term "alkylthio" as used herein represents an alkyl group as defined above attached through a sulfur bridge. Examples of suitable alkylthio groups include, but are not limited to, methylthio, ethylthio, propylthio, i-propylthio, butylthio, i-butylthio, t-butylthio, pentylthio, hexylthio. 10 The term "carbocyclic ring" as used herein refers to a 3 to 10 membered ring or fused ring system, in which all of the atoms that form the ring are carbon atoms. The C 3

.

1 0 carbocyclic ring may be saturated, unsaturated or aromatic. Examples of suitable carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 15 cyclopentenyl, cyclohexyl, cyclohexenyl, phenyl, naphthyl and tetrahydronaphthyl. The term "heterocyclic ring" as used herein refers to a 3 to 10 membered ring or fused ring system in which at least one of the atoms that form the ring is a heteroatom. Preferably the heteroatom is selected from nitrogen, oxygen, sulfur and phosphorus. The C 3

.

10 20 heterocyclic ring may be saturated, unsaturated or aromatic. Examples of suitable heterocyclic rings include, but are not limited to, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazoyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, 25 naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4 dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, 30 thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, WO 2005/007188 PCT/AU2004/000955 - 19 dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, 5 methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom. As used herein, the term "aryl" is intended to mean any stable, monocyclic or bicyclic carbon ring of up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples 10 of such aryl groups include, but are not limited to, phenyl, naphthyl and tetrahydronaphthyl. The term "heteroaryl" as used herein, represents a stable monocyclic or bicyclic ring of up to 6 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains 15 from 1 to 4 heteroatoms selected from the group consisting of 0, N and S. Heteroaryl groups within the scope of this definition include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. 20 The compounds of the invention may be in the form of pharmaceutically, veterinary or agriculturally acceptable salts. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of 25 pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. 30 WO 2005/007188 PCT/AU2004/000955 - 20 Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. 5 Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. It will also be recognised that many compounds of the invention possess asymmetric 10 centres and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, 15 or by chiral resolution. A number of metal chelating agents and metalloprotease inhibitors useful in the present invention can be obtained commercially from speciality chemical companies. Those not commercially available can be synthesised from commercially available starting materials 20 using reactions known to those skilled in the art. For example, substituted 2,2-bipyridyls and 1,10-phenanthrolines may be obtained from suitable halogenated 2,2-bipyridyls or 1,10-phenanthrolines. For example, 2,2'-bipyridin 6,6'-dicarboxylic acid may be obtained from 6,6'-dibromo-2,2'-dipyridyl by halogen-metal 25 exchange with butyl lithium, treatment with dry ice and acidification [Buhleier et. al., Chein. Ber., 1978, 111: 200-204]. Monosubstitution of a bipyridyl, for example with

CH

2

CHNH

2

(CO

2 H) at the 6 position, can be obtained by treatment of 6-methyl-2,2' bipyridyl with N-bromosuccinimide followed by alkylation with N-protected-glycine ester. The protecting groups can then be removed by acid hydrolysis, (Imperiali B. and Fisher 30 S.L., J. Org, Chem., 1992, 57: 757-759).

WO 2005/007188 PCT/AU2004/000955 -21 2,2'-Dipyridyls can undergo nucleophilic substitution at the C6 and C4 positions to introduce substituents. This reaction is more favorable when a halogenated dipyridyl is used as. the starting material. For example an amine may be introduced at C6 and/or C6' by using 6-mono or di-halogenated 2,2'-dipyridyl and reacting this starting material with 5 ammonia. Bipyridyl-sulfonic acids can be prepared from 2,2'-bipyridyl by heating with either oleum (a solution of sulfur trioxide in concentrated sulfuric acid) or mercury (II) sulfate/concentrated sulfuric acid at 300 0 C. 10 Unsymmetrically substituted bipyridyls can be obtained from symmetrical bipyridyls, for example, 6'-methyl-2,2'-bipyridyl-6-carboxylic acid can be prepared from 6,6'-dimethyl 2,2'-bipyridyl by oxidation with selenium dioxide followed by treatment with silver nitrate (Al-Saya et. al., European J. Org. Chem., 2004, 173-182). 15 Compounds of formulae (III) and (IV) can be prepared from commercially available amino acids, for example phenylalanine and tryptophan, using known coupling reactions with amino acid carboxylic acids or amine groups (Jones J., Amino Acid and Peptide Synthesis, Oxford Chemistry Press, 1992). Suitable protection and deprotection steps may be 20 required as known in the art and shown in Jones, 1992, Supra or Green T.W. and Wutz P., Protecting Groups in Organic Synthesis, John Wiley & Son, 3 rd Ed., 1999. Thioureas of formula (V) may be prepared by reaction of a suitable benzamide with butyl lithium followed by thiophosgene. The resulting product can then be reacted with a 25 suitable amine or amino acid as shown in Scheme 1.

WO 2005/007188 PCT/AU2004/000955 - 22 0 S 1. BuLi, THF N Cl 2. Thiophosgene R - ' -H R

H

2 N k R R 0 S N ) N R' HI Scheme 1 In the present specification, the term "ectoparasite" is taken to include any parasitic animal species that externally infests a host and that reproduces by egg laying. Preferred 5 ectoparasites of the invention include a species from an order selected from the group consisting of Lepidoptera, Hemiptera, Orthoptera, Psocoptera, Hymenoptera, Isoptera, Coleoptera, Dictyoptera, Thysanoptera, Homoptera, Diptera, Anaplura, Malophaga, Siphonaptera and Arachnida. 10 Suitable ectoparasites that may be controlled using the methods of the present invention include: (a) from the order of the lepidopterans (Lepidoptera), for example, Adoxophyes orana, Agrotis ypsilon, Agrotis segetum, Alabama argillacea, Anticarsia gemmatalis, 15 Argyresthia coijugella, Autographa gamma, Cacoecia murinana, Capua reticulana, Choristoneura fumiferana, Chilo partellus, Choristoneura occidentalis, Cirphis unipuncta, Cnaphalocrocis medinalis, Crocidolomia binotalis, Cydia pomonella, Dendrolinus pini, Diaphania nitidalis, Diatraea grandiosella, Earias insulana, Elasmopalpus lignosellus, Eupoecilia ambiguella, Feltia subterranea, Grapholitha 20 funebrana, Grapholitha molesta, Heliothis armigera, Heliothis virescens, Heliothis WO 2005/007188 PCT/AU2004/000955 -23 zea, Hellula undalis, Hibernia defoliaria, Hyphantria cunea, Hypononeuta nalinellus, Keiferia lycopersicella, Lambdina fiscellaria, Laphygmna exigua, Leucoptera scitella, Lithocolletis blancardella, Lobesia botrana, Loxostege sticticalis, Lyinantria dispar, Lymantria nonacha, Lyonetia clerkella, Manduca sexta, 5 Malacosoma neustria, Manestra brassicae, Mocis repanda, Operophthera brumata, Orgyia pseudotsugata, Ostrinia nubilalis, Pandeinis heparana, Panolis flaminea, Pectinophora gossypiella, Phthorimaea operculella, Phyllocnistis citrella, Pieris brassicae, Plathypena scabra, Platynota stultana, Plutella xylostella, Prays citri, Prays oleae, Prodenia sunia, Prodenia ornithogalli, Pseudoplusia includens, 10 Rhyacionia frustrana, Scrobipalpula absoluta, Sesamia inferens, Sparganothis pilleriana, Spodoptera frugiperda, Spodoptera littoralis, Spodoptera litura, Syllepta derogata, Synanthedon myopaeform is, Thauinatopoea pityocampa, Tortrix viridana, Trichoplusia ni, Tryporyza incertulas, Zeiraphera canadensis; 15 (b) from the order of the hemipterans (Hemiptera), for example, Aphis, Bemisia, Phorodon, Aeneolamia, Enipoasca, Parkinsiella, Pyrilla, Aonidiella, Coccus, Pseudococcus, Helopeltis, Lygus, Dysdercus, Oxycarenus, Nezara, Aleyrodes, Triatoma, Psylla, Myzus, Megoura, Phylloxera, Adelges, Nilaparvata, Nephotettix or Cimwx spp.; 20 (c) from the order of the orthopterans (Orthoptera), for example, Gryllotalpa gryllotalpa, Locusta migratoria, Melanoplus bivittatus, Melanoplus femur-rubrum, Melanoplus mnexicanus, Melanoplus sanguinipes, Melanoplus spretus, Nomadacris septemfasciata, Schistocerca americana, Schistocerca peregrina, Stauronotus maroccanus, 25 Schistocerca gregaria; (d) from the order of the psocopterans (Psocoptera), for example, Peripsocus spp.; (e) from the order of the hymenopterans (Hymenoptera), for example, Athalia rosae, Atta 30 cephalotes, Atta sexdens, Atta texana, Hoplocanipa mninuta, Hoplocampa testudinea, WO 2005/007188 PCT/AU2004/000955 - 24 Iridonyrmes hum ilis, Iridomyrnex purpureus, Monomoriumn pharaonis, Solenopsis geminata, Solenopsis invicta, Solenopsis richteri, Technomyrmex albipes; (f) from the order of the termites (Isoptera), for example, Calotermes flavicollis, 5 Coptotermes spp, Leucotermes flavipes, Macrotermes subhyalinus, Nasutiterines spp such as Nasutitermes walkeri, Odontotermes formosanus, Reticulitermes lucifugus, Ternes natalensis; (g) from the order of the beetles (Coleoptera), for example, Anthonoinus grandis, 10 Anthonoinus pomorum, Apion vorax, Atomaria linearis, Blastophagus piniperda, Cassida nebulosa, Cerotoma trifurcata, Ceuthorhynchus assimilis, Ceuthorhynchus napi, Chaetocnema tibialis, Conoderus vespertinus, Crioceris asparagi, Dendroctonus refipennis, Diabrotica longicornis, Diabrotica 12-punctata, Diabrotica virgifera, Epilachna varivestis, Epitrix hirtipennis, Eutinobothrus brasiliensis, Hylobius abietis, 15 Hypera brunneipennis, Hypera postica, Ips typographus, Lema bilineata, Lema melanopus, Leptinotarsa decenlineata, Linonius californicus, Lissorhoptrus oryzophilus, Melanotus cominmunis, Meligethes aeneus, Melolontha hippocastani, Melolontha inelolontha, Oulenia oryzae, Ortiorrhynchus sulcatus, Otiorrhynchus ovatus, Phaedon cochleariae, Phyllopertha horticola, Phyllophaga sp., Phyllotreta 20 chrysocephala, Phyllotreta nemorum, Phyllotreta striolata, Popillia japonica, Psylliodes napi, Scolytus intricatus, Sitona lineatus; (h) from the order Dictyoptera, for example, from the families Polyphagidae, Bladberidae, Blattidae, Epilampridae, Chaetecsidae, Metallycidae, Mantoididae, Amorphoscelidae, 25 Eremiaphilidae, Hymenopodidae, Mantidae and Empusidae; (i) from the order of the thrips (Thysanoptera), for example, Frankliniella fusca, Frankliniella occidentalis, Frankliniella tritici, Haplothrips tritici, Heliothrips haenorrhoidalis, Scirtothrips citri, Thrips oiyzae, Thrips palni, Thrips tabaci; 30 WO 2005/007188 PCT/AU2004/000955 -25 (j) from the order of the homopterans (Homoptera), for example, Acyrthosiphon onobrychis, Acyrthosiphon pisum, Adelges laricis, Aonidiella aurantii, Aphidula nasturtii, Aphis fabae, Aphis gossypii, Aphis pomi, Aulacorthum solani, Bemisia tabaci, Brachycaudus cardui, Brevicoryne brassicae, Dalbulus naidis, Dreyfusia 5 nordmannianae, Dreyfusia piceae, Dysaphis radicola, Empoasca fabae, Eriosoma lanigerum, Laodelphax striatella, Macrosiphuin avenae, Macrosiphum euphorbiae, Macrosiphon rosae, Megoura viciae, Metopolophium dirhodum, Myzus persicae, Myzus cerasi, Nephotettix cincticeps, Nilaparvata lugens, Perkinsiella saccharicida, Phorodon huniuli, Psylla mali, Psylla piri, Psylla pyricola, Rhopalosiphum maidis, 10 Schizaphis graminum, Sitobion avenae, Sogatella furcifera, Toxoptera citricida, Trialeurodes abutilonea, Trialeurodes vaporarioruin, Viteus vitifolii; (k) from the order of the dipterans (Diptera), for example, Anastrepha ludens, Ceratitis capitata, Contarinia sorghicola, Dacus cucurbitae, Dacus oleae, Dasineura brassicae, 15 Delia coarctata, Delia radicum, Hydrellia griseola, Hylemyia platura, Lirionyza sativae, Liriomyza trifolii, Lucilia Sp., Mayetiola destructor, Musca sp., Orseolia oryzae, Oscin ella frit, Pegomya hyoscyami, Phorbia antiqua, Phorbia brassicae, Phorbia coarctata, Rhagoletis cerasi, Rhagoletis pomonella; 20 (1) from the order Anaplura, for example, Pthirus pubis, Pediculus humanus capitus, Pediculus hunianus humanus; (m) from the order Mallophaga, for example, from the genera Bovicola, Dainalania, Trichodectus and Menopon; 25 (n) from the order of the siphonapterans (Siphonaptera), for example, Ctenocephalides or Pulex spp. (o) from the order Arachnida, for example, Ixodes holocyclus, Boophilus microplus, 30 Rhipicephalus sanguineus, Sarcoptes scabiei and Dermatophagoides spp.

WO 2005/007188 PCT/AU2004/000955 - 26 It is preferred that the ectoparasite egg which is prevented from hatching by the present invention is selected from the group consisting of louse, flea, tick, fly and other biting or blood-sucking ectoparasite eggs. In a preferred embodiment, the ectoparasite egg is a louse egg, more preferably head louse egg. Lice are a parasite that feed on animal skin and 5 blood and they deposit their digestive juices and faecal material into the skin. These materials, as well as the puncture wound itself, cause skin irritation and lesions from the resulting scratching, and can cause a serious infection with ganglionic inflammation. Lice are also vectors of certain diseases, such as exanthematic or epidemic typhus and recurrent fever. The adult female louse has a life span of about one month and can lay up to ten eggs 10 a day. Lice that infect humans may include the species of crab louse (Pthirus pubis) and the separate species Pediculus humanus which is composed of two subspecies, Pediculus hunanus capitis or head lice and Pediculus humanus humanus or clothing lice (Busvine, Antenna, 1993, 17: 196-201). The above subspecies of lice are closely related and are known to successfully interbreed (Busvine, Cutaneous Infestations and Insect Bites, 1985, 15 163-174). The head louse Pediculus humanus var. capitis, is a host-specific ectoparasite that lives exclusively on human heads and feeds via sucking blood from the scalp. Following a blood meal, mature adult female lice will lay up to 10 eggs close to the scalp over a 24 hr 20 period. The eggs are attached firmly to the hair shaft via a glue. Seven to ten days post laying depending on temperature and humidity, the eggs will hatch and the newly emerged nymphs begin to feed. The nymphs progress through three moults (1st instar, 2 "d instar, 3 rd instar) with each moult taking between 3-5 days to complete. Following the final moult the adult male or female emerges with mating taking place as early as two days later. 25 Within hours of feeding, eggs will be produced and the cycle continues. The entire life cycle from egg to egg takes approximately 20-30 days to complete depending on conditions of warmth and humidity. Following egg hatching the egg shell remains attached to the hair shaft and will gradually move away from the scalp as the hair lengthens. Hatched eggs (nits) are relatively easily detected due to their refractive nature 30 appearing white under artificial light, in contrast unhatched eggs are a light pale brown in WO 2005/007188 PCT/AU2004/000955 -27 color enabling them to blend in to most hair colors and therefore making them more difficult to detect. In one embodiment of the present invention, the methods and compositions of the 5 invention are to cure a subject of lice by inhibiting hatching of louse eggs. The present applicants have identified metal chelating agents and metalloprotease inhibitors as an effective agent for inhibiting ectoparasite louse egg hatching. The use of metal chelating agents or metalloprotease inhibitors for inhibiting ectoparasite louse egg hatching has the advantage of preventing breeding cycles of ectoparasites thereby controlling ectoparasite 10 infestation. The term "metalloprotease" as used herein is taken to refer to a protease involved in ectoparasite egg hatching, wherein the protease has an active metal ion that acts as a catalyst. Preferably, the metalloprotease contains a zinc ion that participates in catalysis by 15 polarizing a water molecule to attack a substrate-peptide bond. More preferably, the metalloprotease is sensitive to metal chelating agents that are capable of blocking their activity. The metalloprotease preferably is involved in inducing egg hatching by acting on the operculum of an egg to facilitate egg hatching. Suitable metalloprotease involved in ectoparasite egg hatching can include endoproteases (enzymes that cleave within the 20 peptide chain) and exoproteases (enzymes that cleave amino acid(s) from the termini of peptides). Exoproteases can further be categorised as carboxyproteases (which cleave amino acid(s) from the C terminus) or aininopeptidase (which cleave amino acids from the N terminus). Metallo-carboxyproteases require a bivalent cation (usually Zn2+) for activity, while aminopeptidases are generally classified according to their dependence on metal ions 25 (Zn 2 + or Mg 2 +). They exist in both free and membrane-bound forms and favour activity at high (8-10) pH. One method of detecting metalloproteases associated with egg hatching can involve collecting either the fluid surrounding the developing embryo at the time of egg hatching or by washing the empty egg shells shortly after egg hatching and analyzing the sample for the presence of proteases using gelatine substrate SDS-PAGE analysis. 30 Having shown the presence of proteolytic activity from the sample it is then possible to incubate the sample in the presence of a metalloprotease inhibitor, for example, 1,10- WO 2005/007188 PCT/AU2004/000955 - 28 phenantholine, and then reanalyze the treated sample to determine if the activity of the proteases extracted from the egg have been inhibited. Having shown inhibition of the activity of the metalloprotease(s) obtained from the hatched egg, it is then possible to expose unhatched eggs to the same inhibitor and assess whether inhibition of egg hatching 5 occurs. Metalloproteases involved in egg hatching may also be identified by identification of a gene encoding a metalloprotease, silencing that gene and showing that the egg is unable to hatch by methods known to those skilled in the art. The phrase "inhibiting hatching of an ectoparasite egg" as used herein is taken to mean that 10 hatching of an ectoparasite egg is prevented. In the present invention an ectoparasite egg is exposed to a metal chelating agent or a metalloprotease inhibitor that is capable of preventing egg hatching when compared to an untreated ectoparasite egg. Egg hatching is characterised by the hatchflap or operculum of an egg opening and shortly thereafter the emergence of a nymph. In the case of lice, the head appears first followed by the thorax to 15 which the legs are attached. Finally, the abdomen emerges and the nymph moves free from the egg. Egg hatching is taken to exclude damage or accidental breakage of an eggshell. Preferably, the metal chelating agent or metalloprotease inhibitor is a compound capable of inhibiting egg hatching when it is applied to the egg at any time between laying and 20 hatching. The ectoparasite egg is preferably present on, but not limited to, a host organism, such as on the skin, hair, coat or fleece of an animal or head hair of a human. In alternative embodiments of the invention the ectoparasite egg may be present on host animals 25 including humans, host plants including cereal crops, fruit trees, cotton, oil seed crops, ornamental plants, flowers, vine crops, root crops, pasture plants and vegetables, or other breeding sites, such as, but not limited to, houses and buildings, enclosures for domestic and farming animals, carpets, blankets, curtains and furniture. 30 According to the present invention, the ectoparasite egg may be exposed to a metal chelating agent or a metalloprotease inhibitor by any suitable means. A person skilled in WO 2005/007188 PCT/AU2004/000955 -29 the art will appreciate that these means may vary widely, depending upon whether the inhibitor is to be applied to a host, such as a plant or animal or various other breeding sites, and depending on the nature and type of ectoparasite targeted. Suitable means for exposing ectoparasite eggs present on animals to metal chelating agents or metalloprotease 5 inhibitors, include, but are not limited to, direct topical application, such as by dipping or spraying, implants, delayed release fonnulations or devices. Where the invention is applied to humans, formulations suitable for topical application include but are not limited to sprays, aerosols, shampoos, mousses, creams and lotions, and formulations suitable for internal application include but are not limited to tablets, capsules or liquid formulations. 10 In some situations parenteral administration by injection may be the most suitable means of treatment for humans or animals. Where the metal chelating agent or metalloprotease inhibitor is to be applied to plants, suitable means include but are not limited to sprays, dusts, pellets, or aerosols. The method of the invention also encompasses the concurrent or successive use of two or more metal chelating agents or metalloprotease inhibitors or the 15 use of one or more metal chelating agents and/or metalloproteases in conjunction concurrently or successively with other known agents that control ectoparasites. In yet another aspect of the invention, the methods and compositions may include other ectoparasiticides that control hatching, nymphs or adult ectoparasites. For example, 20 suitable ectoparasiticides which may be used in conjunction, either simultaneously or separately, with the metal chelating agents or metalloprotease inhibitors of the present invention include organophosphates such as malathion, synthetic pyrethroids (cypermethrin, deltamethrin) insect growth regulators, including juvenile hormone analogues, chitin synthesis inhibitors and triazine derivatives, insecticidal bacterial toxins, 25 chlorinated hydrocarbons (DDT, endosulfan) or insecticidal agents described in EP 0191236, US 5,288,483 and US 6,727,228. Other useful insecticides include dimethicone copolyols, such as those described in US 6,663,876 and US 6,607,716, which have low toxicity. The advantage of such a combination is that only one application may be required to control the ectoparasite over all of its life cycle. 30 WO 2005/007188 PCT/AU2004/000955 - 30 The metal chelating agent or the metalloprotease inhibitor may be applied to the hair or skin of a host, preferably in a region that is infested with an ectorparasite. The ectoparasite infestation may preferably be due to ectoparasites selected from the group consisting of lice, fleas, ticks, flies and other biting or blood-sucking ectoparasites, and combinations 5 thereof. Most preferably, the ectoparasite infestation is due to lice. The metal chelating agent or the metalloprotease inhibitor may be applied topically in the form of ointments, aqueous compositions including solutions and suspensions, creams, lotions, aerosol sprays or dusting powders. 10 The term "effective amount" means a concentration of at least one metal chelating agent or at least one metalloprotease inhibitor sufficient to provide treatment or prevention of ectoparasite infestation in a host. The effective amount of a metal chelating agent or metalloprotease inhibitor used in the methods of the present invention may vary depending on the host and the type and level of ectoparasite infestation. The metal chelating agent or 15 metalloprotease inhibitor is preferably applied to the scalp of a person suffering from head lice infestation and are left on the treated person for a period of time to prevent hatching of the louse eggs. Preferably the period of time is between 5 and 15 minutes, The metal chelating agent or metalloprotease inhibitor is preferably used at a concentration of between about 0.0001mM to IM, preferably 0.01mM and 100mM, more preferably in the 20 range of 0.1mM and 30mM. The effective amount depends on the metal chelating agent or metalloprotease used. However, some dipyridyl compounds may suitably applied in the range of 5mM to 15mM, especially at a level of about 10mM. Since a significant number of mammalian proteases require zinc for their activity and may be effected by metal chelating agents and/or metalloprotease inhibitors, it would be necessary to ensure that the 25 metal chelating agent or metalloprotease inhibitor was used in a safe and effective amount and is preferably specifically targeted to ectoparasite eggs. The host treated by the methods of the invention may be selected from, but is not limited to, the group consisting of humans, sheep, cattle, horses, pigs, poultry, dogs and cats. The 30 methods of treatment or prevention of the present invention may be applicable to plants and or other breeding sites of ectoparasites. Plants treated by the methods of the invention WO 2005/007188 PCT/AU2004/000955 -31 are preferably selected from the group consisting of cotton, oil seed crops, ornamental plants, flowers, fruit trees, cereal crops, vine crops, root crops, pasture plants and vegetables. 5 The compositions of the present invention may be formulated as solutions and emulsions. Suitable excipients, such as emulsifiers, surfactants, stabilizers, dyes, penetration enhancers and anti-oxidants may also be present in the compositions. Suitable carriers that may be added in the compositions can include, water, salt solutions, alcohols, polyethylene glycols, gelatine, lactose, magnesium sterate and silicic acid. The compositions may 10 include sterile and non-sterile aqueous solutions. The compositions are preferably in a soluble form and the metal chelating agent or metalloprotease inhibitor are preferably, diluted in a soluble sterile buffered saline or water solution. The compositions can also be fonnulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension and may also 15 contain stabilizers. The solutions may also contain buffers, diluents and other suitable additives. The compositions can include other adjunct components that are compatible with the activity of the metal chelating agent or metalloprotease inhibitor. The compositions of the present invention may be formulated and used as foams, emulsions, microemulsions, shampoos, mousses, creams and jellies. The formulations of the above 20 compositions described would be known to those skilled in the field of ectoparasiticides. In a preferred embodiment, the composition comprises a metal chelating agent at a concentration of about 0.0001mM to 1M, preferably between 0.1mM to 100mM, more preferably in the range of 0.1mM to 30mM. Compositions containing some metal 25 chelating agents or metalloprotease inhibitors, for example, the dipyridyl compounds, may preferably contain between 5 and 15mM of compound, especially at a level of about 10mM. The invention also provides a method of identifying a compound which inhibits hatching 30 of an ectoparasite egg, the method comprising assessing the ability of the compound to inhibit a metalloprotease present in the ectoparasite egg.

WO 2005/007188 PCT/AU2004/000955 - 32 The effect of the compound on the activity of the metalloprotease may be assessed in a number of ways, however, in general the assessment preferably involves comparison of metalloprotease enzyme activity in the presence and absence of the test compound. One method of detecting metalloproteases associated with egg hatching can involve collecting 5 either the fluid surrounding the developing embryo at the time of egg hatching or by washing the empty egg shells shortly after egg hatching and analyzing the sample for the presence of proteases using gelatine substrate SDS-PAGE analysis. Having shown the presence of proteolytic activity from the sample it is then possible to incubate the sample in the presence of a metalloprotease inhibitor, for example, 1,10-phenantholine, and then 10 reanalyze the treated sample to determine if the activity of the proteases extracted from the egg have been inhibited. Having shown inhibition of the activity of the metalloprotease obtained from the hatched egg, it is then possible to expose unhatched eggs to the same inhibitor and assess whether inhibition of egg hatching occurs. Metalloproteases involved in egg hatching may also be identified by identification of a gene encoding a 15 metalloprotease, silencing that gene and showing that the egg is unable to hatch by methods known to those skilled in the art. In a preferred embodiment the method further comprises testing the compound in a biological ectoparasite egg hatching assay. 20 A suitable biological ectoparasite egg hatching assay preferably comprises exposing a control sample of ectoparasite eggs to a control buffer solution whilst at the same time exposing a test sample of ectoparasite eggs to a solution comprising a test compound. 25 A compound that is effective in inhibiting ectoparasite egg hatching is identified when egg hatching is observed in the control sample and a lower level of hatching is observed in the test sample. In the preferred biological egg hatching assay of the present invention, the ectoparasite eggs are selected from the group consisting of louse, flea, tick, fly and other biting or blood-sucking ectoparasite eggs. In a preferred embodiment the sample of 30 ectoparasite eggs are lice eggs. Preferably, the egg samples (control and test samples) used WO 2005/007188 PCT/AU2004/000955 - 33 are no more than post 6-7 days after being laid. Most preferably, the egg samples used are no more than 1 day after being laid. The control buffer solution may include, but is not limited to, sterile phosphate buffered 5 saline or water. The compound tested is preferably a metal chelating agent and/or a metalloprotease inhibitor. In the biological egg hatching assay egg hatching is observed when the hatchflap or operculum of the egg opens and shortly thereafter the nymph begins to emerge. In the case of lice, the head appears first followed by the thorax to which the legs are attached. Finally, the abdomen comes out and the nymph moves free from the egg. 10 In the case of head lice, the eggshell then remains cemented to the hair shaft. In another aspect of the invention there is provided a use of at least one metal chelating agent in the manufacture of a composition for inhibiting hatching of an ectoparasite egg or for treating or preventing ectoparasite infestation, wherein the metal chelating agent is a 15 compound comprising at least two heteroatoms able to simultaneously coordinate with a metal ion, at least one of the two heteroatoms being selected from nitrogen, sulfur, oxygen and phosphorus, wherein the compound comprises at least one carbocyclic ring substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom, or the compound comprises at least one heterocyclic ring containing at least one 20 heteroatom, wherein said heterocyclic ring is optionally substituted with at least one heteroatom and/or with a substituent containing at least one heteroatom. In yet another aspect of the invention there is provided use of at least one metalloprotease inhibitor in the manufacture of a composition for inhibiting hatching of an ectoparasite egg 25 or for treating or preventing ectoparasite infestation. Also encompassed by the present invention are agents comprising at least one metal chelating agent and/or at least one metalloprotease inhibitor as described herein, for inhibiting hatching of an ectoparasite egg or for treating or preventing ectoparasite 30 infestation.

C:\NRPorbl\DCC\AZB\261542MI.DOC-7/12/2m1" - 34 Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. 5 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of 10 endeavour to which this specification relates. The invention will hereinafter be described by way of the following non-limiting Figures and Examples.

WO 2005/007188 PCT/AU2004/000955 -35 EXAMPLES Example 1 -assessment of the mechanism of lice egg hatching: 5 The mechanism of lice egg hatching was assessed under a dissecting microscope. Female clothing lice were fed for half an hour on a rabbit before being transferred to a petri dish containing human hair. The petri dish was then placed in an incubator at 32'C; 42% relative humidity. Within 5 hours of feeding the female lice begin to lay their eggs. Each female lays up to 10 eggs at a sitting. The eggs develop over the next 7-9 days. Within the 10 last 12 hrs prior to hatching the following changes were observed. The eyes of the developing embryo could be clearly detected inside the egg with the developing embryo orientated so that it has its head is adjacent to the hatch flap or operculum. The embryo can be observed moving within the egg. Hatching takes place when the operculum opens and shortly thereafter the embryo begins to emerge. The head appears first followed by the 15 thorax to which the legs are attached. Finally, the abdomen comes out and the nymph moves free from the egg that remains cemented to the hair. There are no obvious structures associated with the head of the newly emerged nymph visible under light microscopy, that would facilitate hatching (ie no egg tooth is present). This observation suggests that while physical movement of the nymph within the egg probably contributes to egg hatching, 20 other specific biochemical events are involved. Example 2 -detection of protease activity in lice egg extracts: Within 12 hours of hatching 50 body lice eggs (Pediculus humanus hunanus) were 25 removed from the hair and placed in a 1 ml eppendorf tube. 20 LL of distilled water was added to the unhatched eggs and the preparation incubated for 30 minutes at 32'C. The 20 L was recovered and stored at -70'C. A number of other samples were also collected as described. Hair samples from which unhatched lice eggs had been removed were also collected and incubated as described above. In addition, a sample of unhatched eggs and a 30 sample of hair from which lice eggs had been removed were collected 7 days post laying (within 24 hrs of egg hatching). Both samples were washed in 10 mls of a 1% solution of WO 2005/007188 PCT/AU2004/000955 - 36 sodium hypochlorite for 1 minute followed by a 5 x 1 minute washes in 25mls in distilled water to remove the hypochlorite. These samples were then incubated in 20pLL of distilled water as described above. In addition, a group of 25 lice eggs that were within 24 hours of hatching, were pretreated with 1% sodium hypochlorite, washed as described above and 5 left to hatch. Within 1-2 hours after hatching the empty egg shells were incubated in 20ptL of distilled water as described above, the washings collected from the hatched egg shells and stored at -70'C. All of the extract samples were then freeze-dried overnight. The freeze-dried samples were then resuspended in 15pL of non-reducing SDS sample buffer, centrifuged at 10,000g for 2 minutes and the entire 15[LL loaded on to 10% gelatine 10 substrate SDS-PAGE gels. Gels were run at 4'C for 10 minutes at 10mA followed by a further 25 minutes at 15mA per gel. They were then incubated for 2 x 20 minutes in a 2.5% Triton-X 100 solution followed by a three hour incubation in 0.1M Tris/HCl containing 1mM CaCl 2 pH 8.0. Activity was detected as clear areas on the gel the result of protease activity degrading the gelatine within the gel. 15 The results from these studies indicated that proteolytic activity was present in a number of different preparations as analysed using substrate SDS-PAGE. Protease activity was detected in the washings obtained from unhatched lice eggs within 12 hours of hatching (Figure 1, lane 1). This activity was in the higher molecular weight region of the gel. When 20 hair samples that had had lice eggs removed were analysed on gelatine substrate SDS PAGE a significant amount of protease activity was detected (Figure 1, lane 2). The most likely explanation for this activity was that it was of maternal origin being produced at the time of laying. Treatment of hair samples with sodium hypochlorite completely removed the contaminating proteases (Figure 1, lane 3). In addition treatment of unhatched eggs was 25 also able to remove this protease activity (Figure 1, lane 4). It was therefore decided to treat all eggs prior to hatching with 1% Na Hypochlorite as described above in order to remove these maternal proteases. Analysis of freshly hatched egg shells (Egg Shell Washings) indicated the presence of two high molecular weight species (Figure 1, lane 5). Note only 25 lice eggs were used in this collection, possibly contributing to the lower level 30 of protease activity. These results demonstrate the presence of protease activity directly associated with freshly hatched lice eggs.

WO 2005/007188 PCT/AU2004/000955 - 37 In conclusion the hatching process in lice was studied by light microscopy. Egg hatching appears to be associated with physical activity of the developing nymph within the egg. However, the lack of any specialised structures for piercing or loosening the hatch flap or operculum indicates that the hatching process may also involve a biochemical component. 5 While highly active proteases were detected around the time of egg hatching in lice the primary source of these proteases appears to be of maternal origin. Removal of this activity prior to egg hatching was achieved using sodium hypochlorite with the lice progressing through to successfully hatch. Subsequent analysis of the ESW from freshly hatched lice indicated the presence of a limited number of protease species that were further 10 investigated as targets for inhibiting egg hatching in lice. Example 3: characterisation of proteases in egg shell washings: In order to evaluate the potential of lice hatching proteases in the egg shell washings as 15 targets for inhibiting egg hatching it was first necessary to characterize the nature of the hatching proteases. Inhibitors of the 4 major classes of proteases were used to classify the proteases in the ESW. 10% SDS-PAGE gelatine substrate gels were loaded with freeze dried egg shell washings 20 from 100 lice eggs that had been resuspended in 50 1ii of non-reducing sample buffer with samples run at 10 1d per lane. Gels were run at 4'C for 10 minutes at 10 mA per gel followed by a further 25 minutes at 15 mA per gel. Gels were then cut into strips and each strip incubated for 2 x 20 minutes in a 2.5% Triton-X 100 solution containing a specific inhibitor. The inhibitors used were the serine protease inhibitor PMSF (5mM), the 25 metalloprotease inhibitor 1,10-phenanthroline (10mM), the aspartic protease Pepstatin (5ptM) and the cysteine inhibitor E-64 (1OpIM). The gel strips were then incubated in 0.1M Tris/HC1 containing lmM CaCl 2 pH 8 containing the different protease inhibitors for 3 hrs at 37'C, before being stained in Coomassie blue and destained as previously described. In contrast to Figure 1, lane 5 that shows a predominance of proteolytic activity around 25-30 30 kDa (refer to Figure 2 brackets). Subsequent analysis of numerous preparations of ESW indicated that this triplet of proteolytic activity around 25-30 kDa was highly reproducible.

WO 2005/007188 PCT/AU2004/000955 - 38 The results from the inhibitor studies indicate a significant reduction in protease activity following treatment with 1,10-phenanthroline (lane 2 bracketed region). No reduction in protease activity of the ESW was observed when the serine protease inhibitor PMSF or the cysteine protease inhibitor E64 was used. In addition, the aspartic inhibitor pepstatin did 5 not show any reduction in protease activity (data not shown). Example 4: development of an in vitro bioassay for measuring lice egg hatching: To evaluate the potential effects of protease inhibitors on lice egg hatching it was 10 necessary to develop a reliable in vitro bioassay. Male and female clothing lice were fed on a rabbit as previously described. Female and male adult lice in a ratio of 3:1 were then transferred to a clean petri dish containing nylon cloth approximately 3 x 3 cm 2 and left for 12 hrs at 32'C. During this period the female lice laid their eggs and attached them to the woven cloth. All lice would then be removed and the eggs permitted to incubate for the 15 following 5 days. On Day 6 the cloth containing the eggs would be placed for 1 minute in a 1% sodium hypochlorite solution and then washed extensively. The eggs would then progress through to their final stages of development and hatch. In untreated control eggs a reliable average percentage hatch of between 85-95 percent was obtained using the in vitro egg hatch assay. It was subsequently found that for the egg hatching assay it was not 20 necessary to pre-treat the lice eggs with sodium hypochlorite. Example 5: identification of compounds that can inhibit the activity of lice hatching proteases: 25 (a) Testing ofprotease inhibitors using Lice egg-hatching bioassay. Having refined a bioassay for measuring egg hatching in lice, the next phase of the research was to use this bioassay as a means of testing the effects of different protease inhibitors on egg hatching. 30 WO 2005/007188 PCT/AU2004/000955 -39 Lice eggs were laid onto cloth as described above. Five days post laying the cloth containing lice eggs was removed and immersed in a 1% sodium hypochlorite solution before being washed extensively in distilled water and blotted dry on tissue paper. Lice eggs were counted under a dissecting microscope and the cloth cut into batches of between 5 10-30 eggs with 3-5 replicates used per treatment. The cloth containing lice eggs was then immersed in a protease inhibitor solution for a period of between 2-10 minutes, placed on tissue paper for 1 minute to dry before being transferred to a clean petri dish and incubated until hatching. The eggs were observed at regular time intervals for evidence of eggs hatching over the next 1-2 days by which time the control eggs had hatched. Protease 10 inhibitor solutions were typically prepared as stock solutions and added fresh at the appropriate concentration. Specifically stock solutions were prepared as follows: 1,10 phenanthroline (200mM in methanol) and Bestatin (5mg/ml in methanol). In addition, the equivalent levels of the solvent were added to the non-inhibitor containing controls eggs to test for any buffer alone effects. Percentage hatch inhibition was calculated as the 15 percentage reduction in egg hatch compared to the untreated control. The untreated control was assigned a percentage hatch of 100%. The addition 1,10-phenanthroline, a metal chelating agent and a metalloprotease inhibitor significantly inhibited egg hatching in lice at 10mM while at 1mM the level of inhibition 20 was approximately 30% compared to that of the controls (refer to Figure 3). Bestatin, a metal chelating agent and an inhibitor of metalloproteases and more specifically aminopeptidase M and N, was also able to significantly inhibit lice egg hatching at 5mM (Figure 4). 25 These results provide data on the effect of specific metal chelating agents and metalloprotease inhibitors on egg hatching in lice. It was however noted that when either 1,10-phenanthroline or Bestatin were added within 24 hours of hatching, variable inhibition of egg hatching was observed (data not shown). This variability in hatch inhibition could be due to a number of factors that relate to the specific developmental 30 stage of the louse. Furthermore these studies indicated that it is very difficult to predict the exact time of egg hatch and therefore the choice of a single time point in which to treat the WO 2005/007188 PCT/AU2004/000955 -40 eggs may be problematic when assessing the effects of a specific inhibitor on egg hatch. The in vitro assay system was therefore modified to account for this variability in lice development. 5 (b)Time course experiment using in the in vitro hatching assay. A series of time-course experiments was conducted as a means of assessing inhibitors of lice egg hatching. Eggs were laid onto cloth as previously described and then at 24 hr intervals an inhibitor was added to a new group of eggs for eggs up to 120 hrs post laying. 10 The eggs were then incubated at 28'C for a further 8 days to permit egg hatching. This method of assaying inhibitors more closely mirrors the field situation where lice eggs will be at various stages of development. The results of these studies are shown in Table 1. Significant inhibition by 1,10 15 phenanthroline was demonstrated at varying concentrations over the course of lice hatching. A degree of concentration dependence was also observed with the inhibitory effects of 1,10-phenanthroline. The results indicate that time-course experiments provide a more reliable means of assessing the effects of specific inhibitors on lice egg hatching. The addition of Bestatin resulted in significant inhibition, but only when applied approximately 20 24 hrs prior to egg hatch. Table 1. Percent inhibition of egg hatching following treatment with different concentrations of 1,10-phenanthroline and 5mM Bestatin at 24 hr intervals post egg laying.

WO 2005/007188 PCT/AU2004/000955 - 41 Time post egg laying (hr) Inhibitor 24 hr 48 hr 72 hr 96 hr 120 hr 144 hr 10 mM 1,10- 100 100 100 100 100 100 phenanthroline 5 mM 1,10- 100 100 100 100 93 96 phenanthroline 2.5 mM 1,10- 100 100 85 85 89 60 phenanthroline 5 mM Bestatin - - - - - 53 Results from the above studies indicate that lice hatching enzymes are proteases of the metallo class as judged by the ability of metal chelating agent and metalloprotease 5 inhibitor 1,1 0-phenanthroline to inhibit their activity. Furthermore this compound was able to significantly inhibit egg hatching in lice at all time points examined with some evidence of a dose dependent effect particularly when eggs were treated with the lower concentrations around the time of hatching. 1,10-phenanthroline exerts its effects through its ability to chelate metal ions, preferably zinc and thereby inhibiting zinc dependent 10 proteases. The data for Bestatin also indicated that the compound could partially inhibit lice egg hatching when administered to eggs in the late developmental stage. Bestatin is a cyclic compound comprising an aryl ring substituted with a substituent containing an amine, a 15 hydroxy group, an amide and a carboxylic acid group. Bestatin is an antibiotic of microbial origin, which is used for treating various forms cancer including nonlymphocytic leukemia and also different forms of solid tumors including, lung, stomach, bladder, head, neck and oesophagus where it is used under the name of ubenimex. It can be administered with low toxicity to cultured cells, intact animals and humans. While Bestatin is normally used as an 20 inhibitor of purified proteases at micromolar concentrations (10pM and 1301M) the data obtained thus far show it being effective at 5mM. This result may be due to a number of WO 2005/007188 PCT/AU2004/000955 - 42 factors including the ability of Bestatin to penetrate the egg and its specificity for the lice hatching proteases. Example 6: screening of protease inhibitors to inhibit lice egg hatching: 5 Lice eggs were laid onto cloth as described in Example 4. A series of time-course experiments were set-up as described in Example 5(part (b)). A considerable number of commercially available protease inhibitors/metal chelators were tested in the lice hatching assay to determine the effect of individual protease inhibitors on lice egg hatching (Table 10 2). Percentage hatch inhibition was calculated as the percentage reduction in egg hatching compared to the untreated control. The untreated control was assigned a percentage hatch of 100%.

WO 2005/007188 PCT/AU2004/000955 -43 Table 2. Percentage inhibition of lice egg hatching following treatment with various protease inhibitors. The percentage inhibition refers to the maximum egg hatch obtained over the time-course of the experiment. Number Inhibitor %Inhibition* 1 1-10 100 phenanthroline (10mM) 2 2,2-dipyridyl 100 (10mM) 3 6,6'-Dimethyl- 100 2,2'-dipyridyl (10mM) 4 5,5'-Dimethyl- 100 2,2'-dipyridyl (10mM) 5 Captopril 27 (23mM) 6 D-L Thiorophan 20 (1mg/ml) 7 Phosphoramidon 41 (5mM) 8 Actinonin 0 (5mM) 9 Bestatin 58 (5mM) 10 NitroBestatin 0 (1mg/ml) 11 Amastatin 26 (5mM) WO 2005/007188 PCT/AU2004/000955 - 44 12 Leuhistin 0 (5mM) 13 Ebelactone 0 (1, 2 and 5mM) 14 L-leucinthiol 0 (5mM) 15 Fumagillin 0 (5mM) 16 Carboxypeptidase 26 inhibitor (5mM) 17 N-CBZ-PRO- 0 LEU-GLY Hydroximate (10mM) 18 Tetracycline 89 (5mg/ml) 19 Doxycycline 69 (5mg/ml) 20 Minocycline 55 (5mg/ml) 21 GM 1489 0 22 GM 6001 0 23 Inhibitor IV 0 24 Chlorhexidine 0 dihydrochloride *The percentage inhibition of egg hatching of the different protease inhibitors has been calculated relative to the appropriate controls and represents the maximum egg hatch inhibition observed.

WO 2005/007188 PCT/AU2004/000955 - 45 A number of protease inhibitors were shown to markedly inhibit lice egg hatching. The most effective inhibitors tested included metal chelating agents and metalloptotease inhibitors such as 1-10 phenanthroline, 2,2-dipyridyl and 6,6'-Dimethyl-2,2'-dipyridyl, 5,5'-dimethyl-2,2'-dipyridyl (100% inhibition at 10mM each). Bestatin, a metalloprotease 5 inhibitor was also able to significantly inhibit egg hatching (58% at 5mM). Naturally derived matrix metalloprotease (MMP) inhibitors and metal chelating agents that are tetracyclic compounds in which one ring is an aryl ring and wherein the tetracyclic structure is substituted with a number of hydroxy groups, carbonyl groups, an amine and 10 an amide, included: Tetracycline (89% inhibition at 5mg/ml), Doxycycline (65% inhibition at 5mg/ml) and Minocycline (55% at 5mg/ml). These metal chelators showed inhibitory activity towards egg hatching, however the results for these compounds were more variable in magnitude and appeared to be time dependent. The overall usefulness of the hydroxamate inhibitors may be limiting due to the drive to reduce the use of antibiotics in 15 the general environment. These results indicate that MMP inhibitors may also exert an inhibitory effect on lice egg hatching. Other protease inhibitors that were tested included EDTA at 100mM, EGTA at 10mM and Triethanolamine at 5%. The results indicated that these inhibitors did not appear to have an effect on egg hatching at the concentrations used (results not shown). 20 Example 7: effect of washing eggs post treatment with 1-10 phenanthroline: An experiment was undertaken to determine whether washing of the eggs would effect the inhibitory activity of 1,10-phenanthroline (Table 3). A control group (5% methanol) was 25 also set up. Percentage hatch inhibition was calculated as the percentage reduction in egg hatch compared to the untreated control. The untreated control was assigned a percentage hatch of 100%. The results from this experiment indicate that 1,10-phenanthroline is still highly efficacious at inhibiting lice egg hatching following washing of eggs in water. In later stage eggs that are approaching egg hatch (day 5) the effects appear to reflect a 30 concentration dependence similar to that observed when lower concentrations of the WO 2005/007188 PCT/AU2004/000955 -46 inhibitor were used. It was also noted that a proportion of eggs treated with 1,10 phenanthroline had embryos that appeared to develop normally yet failed to hatch. Table 3. Percent inhibition of egg hatching following treatment with 10mM 1-10 5 phenanthroline at 24 hr intervals post egg laying in lice. Lice eggs were treated with inhibitor for 10 minutes and left unwashed or treated and washed for 1 minute and then left to hatch. Time post laying (hr) 24 hr 48 hr 72 hr 96hr 120 hr Treated/not 100 100 100 100 100 washed Treated/was 100 100 100 97 62 washed 10 Example 8: inhibition of hatching of head lice eggs with 1-10 phenanthroline: Tests were carried out to determine if metal chelating agent and metalloprotease inhibitor 1,10-phenanthroline could inhibit head lice egg (Pediculus humanus capitus) hatching as opposed to body lice. Head lice eggs were obtained by placing groups of both 1-2 adult 15 male and 6-8 adult female head lice in separate wells in a 24 well petri dish containing cotton cloth. The petri dish was transferred to a humid incubator at 324C, 70% RH for 12 hours to permit the female lice to lay their eggs. After 12 hours, all adult lice were removed from the petri dish wells and a series of time-course experiments conducted. A group of eggs (24 hr old) was treated for 10 minutes with 2 00 t 1 of a 10mM solution of 20 1,10-phenanthroline. A control (ie no inhibitor treatment) group of eggs was also included. The eggs were removed from the inhibitor, blotted dry on tissue paper, placed at 32'C, 70% RH and left to hatch. A second group of eggs, (48 hr old) were treated as previously described and also left to hatch. This process was repeated at 24 hr intervals on head lice eggs up to 120hr post laying. This method of assaying inhibitors more closely mirrors the WO 2005/007188 PCT/AU2004/000955 -47 field situation where lice eggs will be at various stages of development on the head and permits the inhibitory effects to be observed on these different stages of the parasite. The results from the above studies indicate that 1,10-phenanthroline can significantly 5 inhibit egg hatching in head lice (Table 4). Table 4. Percent inhibition of egg hatching following treatment with 10mM 1,1 0-phenanthroline at 24 hr intervals post egg laying in lice relative to the control. Days post laying 1 2 3 4 5 Treated 100 87 88 100 100 10 These results strongly suggest that body lice are an effective model for assaying the effects of protease inhibitors in egg hatching of head lice. Example 9: inhibition of lice egg hatching with metal chelators: 15 Experiments were conducted using two metal chelating agents that can act as metalloprotease inhibitors to determine their effects on lice egg hatching. These compounds were tested in the standard lice assay to determine their ovicidal effects (refer to example 5 and 6 on methods used to test inhibitors). The following metal chelating 20 agents were evaluated: 2,2'-dipyridyl and 6,6'-Dimethyl-2,2'-dipyridyl. The results of this study are shown in Tables 5 and 6. Table 5. Results of egg hatching following treatment with 2,2'-dipyridyl at 24 hr intervals post egg laying. The results are indicated for: N (number of eggs per replicate), H (number 25 of eggs successfully hatched) and Ph (number of eggs partly hatched).

WO 2005/007188 PCT/AU2004/000955 -48 Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H Ph N H Ph N H Ph N H Ph 1 7 0 0 6 0 0 7 0 0 13 0 0 10 0 0 2 8 0 0 14 0 0 7 0 0 9 1 0 10 0 0 3 11 0 0 - - - 14 0 0 10 0 0 13 0 0 Table 6. Results of egg hatching following treatment with 6,6'-Dimethyl-2,2'-dipyridyl at 24 hr intervals post egg laying. The results are indicated for: N (number of eggs per 5 replicate), H ( number of eggs successfully hatched) and Ph (number of eggs partly hatched). Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H Ph N H Ph N H Ph N H Ph 1 10 0 13 0 0 15 0 0 25 0 0 23 00 2 10 0 0 11 0 0 16 0 0 22 0 0 9 0 0 3 11 0 0 6 0 0 10 0 0 18 0 0 - - The results from these studies indicate that both 6,6'-Dimethyl-2,2'-dipyridyl and 10 2,2'-dipyridyl displayed very strong ovicidal activity whereby lice egg hatching was completely inhibited at all time points examined. Both 6,6'-Dimethyl-2,2'-dipyridyl and 2,2'-dipyridyl are metal chelating agents and metalloprotease inhibitors that are non-intercalating. 15 Example 10: comparative assessment of commercial lice products with 1,10 phenanthroline The ovicidal properties of three major commercial head lice products were evaluated in the standard lice egg-hatching assay. The 3 commercial head lice products were as follows: 20 1. KP-24@ Nelson Laboratories, active ingredients 1% maldison (malathion); WO 2005/007188 PCT/AU2004/000955 - 49 2. RID@ Bayer, active ingredients, 1% pyrethrins; and 3. NIX@ Pfizer, active ingredients, 1% permethrin. 5 These three products were tested according to manufacturer's recommendations. Groups of eggs (24 hr old) were treated with the different products according to manufacturer's recommendations for the appropriate period of time (5-10 minutes) followed by a rinse for 1-2 minutes in 32'C water. A positive controls (lOmM 1,10-phenanthroline) and two negative controls (no treatment and 20% Methanol) were also incorporated. Post exposure 10 to the different products, the eggs were rinsed with warm water at 32'C before being blotted dry on tissue paper and placed at 32'C, 70% RH and left to hatch. A second group of eggs, (48hr old) were treated as previously described and also left to hatch. This process was repeated at 24 hr intervals on head lice eggs up to 120 hr post laying. This method of assaying inhibitors more closely mirrors the field situation where lice eggs will be at 15 various stages of development on the head and permits the inhibitory effects to be observed on these different stages of the parasite. The results of these studies are shown in Table 7. Table 7. Results of egg hatching following treatment with 3 commercial head lice 20 products, 10mM 1,10-phenanthroline and controls at 24 hr intervals post egg laying. The results are indicated for: N (number of eggs per replicate), H (number of eggs successfully hatched) and Ph (number of eggs partly hatched). NIX-Pfizer Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H Ph N I Ph N H Ph N H Ph 1 16 12 2 9 7 0 18 3 3 12 8 3 19 12 3 2 10 4 3 6 2 3 10 3 3 15 7 5 18 8 7 3 10 7 2 9 4 3 17 5 7 - - - 36 215 WO 2005/007188 PCT/AU2004/000955 -50 RID-Bayer Replicates 24hr 48 hr 72hr 96hr 120hr N H Ph N H Ph N H Ph N H Ph N H Ph 1 8 03 12 3 4 7 0 0 8 0 0 14 0 1 2 8 2 5 7 0 1 5 1 2 8 00 - - 3 5 0 2 10 0 2 6 1 3 11 0 0 - - KP24KP24 Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H Ph N H Ph N H Ph N H Ph 1 7 7 0 10 10 0 10 1 3 10 0 0 10 0 0 2 6 6 0 10 9 0 0 0 0 7 0 0 8 0 0 3 9 8 0 - - - - - - - - - 12 0 1 1,10-phenanthroline (10mM) Replicates 24hr 48hr 72hr 96hr 120hr N HPhN HPh N H Ph NHPh NHPh 1 13 0 0 5 0 0 7 0 0 10 0 0 9 0 0 2 9 0 0 15 0 0 7 0 0 10 0 0 6 40 3 - - - 8 0 0 9 00 - - - 7 1 0 Control (20% Methanol) Replicates 24hr 48hr 72hr 96hr 120hr NHPh N H PN HP N H PN H Ph 1 - - - 14 14 0 10 10 0 10 10 0 13 13 0 2 - - - 5 4 0 8 8 0 10 9 0 7 7 0 3 - - - - - 0 9 7 0 4 4 0 10 10 0 WO 2005/007188 PCT/AU2004/000955 -51 Control (Untreated) Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H P N H P N H P N H Ph 1 10 9 0 11 11 0 25 24 0 10 8 0 20 20 0 2 20 18 0 8 7 0 10 10 0 11 10 0 20 18 0 3 - - - 8 8 0 - - - 10 10 0 - - Results from the testing of 3 commercial pediculicides indicate that they displayed inconsistent levels of ovicidal activity across the different stages of lice egg hatching. 5 Whereas, the compound 1,10-phenanthroline was highly effective at inhibiting lice egg hatching. Example 11: Assessment of additional commercial lice products 10 The ovicidal properties of two major commercial head lice products were evaluated in the standard lice egg-hatching assay. The 2 commercial head lice products were as follows: 1. Pronto Plus@ Shampoo Del Laboratories, active ingredients 0.33% Pyrethrins; and 15 2. Pronto Plus@ Mousse Shampoo Del Laboratories, active ingredients, 0.33% Pyrethrins. These two products were tested according to manufacturer's recommendations. Groups of eggs (24 hr old) were treated with the different products according to manufacturer's 20 recommendations for the appropriate period of time (5-10 minutes) followed by a rinse for 1-2 minutes in 32'C water. Two negative controls (no treatment and 20% ethanol) were also incorporated. Post exposure to the different products, the eggs were blotted dry on tissue paper and placed at 32'C, 70% RH and left to hatch. A second group of eggs, (48hr old) were treated as previously described and also left to hatch. This process was repeated 25 at 24 hr intervals on head lice eggs up to 120 hr post laying. This method of assaying WO 2005/007188 PCT/AU2004/000955 -52 inhibitors more closely mirrors the field situation where lice eggs will be at various stages of development on the head and permits the inhibitory effects to be observed on these different stages of the parasite. The results of these studies are shown in Table 8. 5 Table 8. Results of egg hatching following treatment with 2 commercial head lice products and controls at 24 hr intervals post egg laying. The results are indicated for: N (number of eggs per replicate), H (number of eggs successfully hatched) and Ph (number of eggs partly hatched). Pronto Plus Shampoo Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H P N H P N H P N H Ph 1 14 10 2 11 9 0 30 27 0 35 30 0 40 38 2 2 20 15 3 21 18 0 19 16 0 42 36 0 38 29 5 3 - - - - - -- - - - - - - - Pronto Plus Mousse Shampoo Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H P N H Ph N Ph N H Ph 1 10 8 0 18 15 0 47 31 9 63 8 34 51 7 40 2 15 13 0 10 6 0 30 14 8 29 5 10 50 8 30 3 11 9 0 - - - 34 13 17 21 1 15 31 1 17 Control (ethanol) Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H P N H P N H P N H P 1 12 10 0 18 16 0 40 36 1 21 20 0 49 47 0 2 11 9 0 21 18 0 41 37 0 28 26 0 39 36 0 3 11 11 0 13 11 0 75 70 0 29 27 0 36 34 0 10 WO 2005/007188 PCT/AU2004/000955 - 53 Control (untreated) Replicates 24hr 48hr 72hr 96hr 120hr N H Ph N H P N H P N H P N H P 1 10 9 0 27 26 0 61 60 0 50 49 1 48 46 0 2 - - - - - - - - - - - - - - 3 - - - - - - - - - - - - - - Results from the testing of 2 commercial pediculicides indicate that they displayed very poor and inconsistent ovicidal activity across the different stages of lice egg hatching. 5 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not 10 restrictive. All publications discussed above are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which was 15 included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in any country before the priority date of each claim of this application.

WO 2005/007188 PCT/AU2004/000955 - 54 References: Al-Sayah, M.H., McDonald, R., Branda, N.R., Euro. J. Org. Chem., 2004, 173-182. 5 Buhleier, E., Wehner, W., V6gthe, F., Chem. Ber., 1978, 111, 200-204. Busvine, J.R.,. Entomology and evolution. Antenna. 1993, 17: 196-201. Busvine, J.R, Biology of the parasites. Cutaneous Infestations and Insect Bites (M. Orkin 10 and H.I. Maibach, eds).1985, pp.163-17 4 . New York: Marcel Dekker. Dymock, J.J., Laing W.A., Shaw B.D., Gatehouse A.M.R, Cristellar. J.T. New Zealand Joumal of Zoology, 1992, 19: 123-131. 15 Green, T.W. and Wutz, P., Protecting groups of organic synthesis, John Wiley & Son, 3 rd Edition, 1999. Imperiali, B. and Fisher, S.L., J. Org. Chem., 1992, 57, 757-759. 20 Jones, J., Amino acid synthesis andpeptide synthesis, Oxford Chemistry Press, 1992. Samuels R. I. and Paterson J. C. Comparative Biochemistry and Physiology. 1995 110B: 661-669.

Claims (21)

1. A method for inhibiting hatching of an ectoparasite egg comprising the step of exposing the ectoparasite egg to at least one metal chelating agent, wherein the 5 metal chelating agent is a compound represented by the structure of formula I, or a pharmaceutically, veterinary or agriculturally acceptable salt thereof: R

2 R 1 R R R 3 RT (I) N N R 4 R4' 10 wherein X is selected from a covalent bond, -C(R 5 ) 2 -, -Z- or -C(R5)2-Z-C(R5)2-; R' and R' are independently selected from hydrogen, Ci- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, hydroxy, C 1 - 6 alkoxy, thiol, C 1 . 6 alkylthio, CO 2 H, C0 2 CI. 6 alkyl, SO 3 H, SO 3 C 1 . 6 alkyl, NH 2 , NHC 1 - 6 alkyl or N(CI 6 alkyl)2; R2, R , R, R , R 4 and R are independently selected from hydrogen, Ci. 6 alkyl, 15 C 2 . 6 alkenyl, C 2 - 6 alkynyl, hydroxy, C 1 . 6 alkoxy, thiol, C 1 . 6 alkylthiol, CO 2 H, C0 2 CI. 6 alkyl, SO 3 H, SO 3 CI- 6 alkyl, NH 2 , NHC 1 . 6 alkyl or N(CI- 6 alkyl) 2 , or -CH 2 CHNH(CO 2 H); or R 2 and R 3 or R 3 and R 4 and/or R 2 ' and R 3 ' or R 3 ' and R4' taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or 20 heterocyclic ring; each R 5 is independently selected from hydrogen, C 1 - 6 alkyl, C 2 - 6 alkenyl, C2. 6 alkynyl, hydroxy, C 1 . 6 alkoxy, thiol, CI- 6 alkylthiol, CO 2 H, CO 2 CI-6alkyl, SO 3 H, S0 3 CI. 6 alkyl, NH 2 , NHCI 6 alkyl or N(CI. 6 alkyl) 2 ; and Z is selected from a covalent bond, -NH-, -0-, -S-, -C(0)- and -C(S)-; 25 thereby inhibiting the hatching of the ectoparasite egg. C:NRPonbrDCC\AZB\2952949 I.DOC-I2/05/2010 - 56 2. The method according to claim 1, wherein R', R', R 2 and R 2 are independently hydrogen or CI. 3 alkyl.

3. The method according to claim 1 or claim 2, wherein R 3 , R 3 ', R 4 and R 4 are 5 independently selected from hydrogen, CI. 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, CI. 6 alkoxy, Ci. 6 alkylthiol or C0 2 CI. 6 alkyl.

4. The method according to claim 1 or claim 2, wherein each R 5 is independently selected from hydrogen, Ci. 6 alkyl, C 2 - 6 alkenyl, C 2 . 6 alkynyl, CI. 6 alkoxy, C 1 . 10 6 alkylthiol or CO 2 C 1 . 6 alkyl.

5. The method according to claim I or claim 2, wherein X is a covalent bond, -CH 2 -Z CH 2 - or Z. 15

6. The method according to claim 1 or claim 2, wherein Z is -NH-, -0- or -S-.

7. The method according to any one of claims 1 to 3 or 5, wherein the compound is selected from: 2,2'-dipyridyl, 20 6,6'-dimethyl-2,2'-dipyridyl, and 5,5'-dimethyl-2,2'-dipyridyl, or a pharmaceutically, veterinary or agriculturally acceptable salt thereof.

8. The method according to any one of claims I to 7, wherein the ectoparasite egg is 25 laid by an ectoparasite of a species from the order of Lepidoptera, Hemiptera, Orthoptera, Psocoptera, Hymenoptera, Isoptera, Coleoptera, Dictyoptera, Thysanoptera, Homoptera, Diptera, Anaplura, Malophaga, Siphonaptera and Arachnida. 30

9. The method according to claim 8, wherein the ectoparasite is from the order Anaplura (lice). C:\NRPobIlDCC\AZB\2952949 I.DOC-12J15f/2010) -57

10. A method of inhibiting the hatching of a louse egg on a host, comprising topically exposing the affected area of the host to an effective amount of at least one compound represented by the structure formula (I): 5 R2 R1 R1' R2' R 3 XR 3 (I) N N R4 R4' wherein X is selected from a covalent bond, -C(R 5 ) 2 -, -Z- or -C(R')2-Z-C(R')2 R' and R" are independently selected from hydrogen, Ci- 6 alkyl, C 2 - 6 alkenyl, 10 C 2 - 6 alkynyl, hydroxy, Ci- 6 alkoxy, thiol, Ci- 6 alkylthio, CO 2 H, C0 2 Ci- 6 alkyl, SO 3 H, S0 3 CI. 6 alkyl, NH 2 , NHCI- 6 alkyl or N(CI- 6 alkyl) 2 ; R 2, R , R', R", R' and R 4 ' are independently selected from hydrogen, CI. 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, hydroxy, CI. 6 alkoxy, thiol, CI. 6 alkylthiol, CO 2 H, C0 2 CI. 6 alkyl, SO 3 H, SO 3 CI. 6 alkyl, NH 2 , NHCI. 6 alkyl or N(CI- 6 alkyl) 2 , or 15 -CH 2 CHNH(CO 2 H); or R2 and R 3 or R 3 and R 4 and/or R 2 and R 3 or R 3 ' and R4' taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring; each R 5 is independently selected from hydrogen, CI- 6 alkyl, C 2 - 6 alkenyl, 20 C 2 - 6 alkynyl, hydroxy, C1. 6 alkoxy, thiol, Ci- 6 alkylthiol, CO 2 H, CO 2 CI- 6 alkyl, SO 3 H, S0 3 CI- 6 alkyl, NH 2 , NHCI- 6 alkyl or N(CI- 6 alkyl) 2 ; and Z is selected from a covalent bond, -NH-, -0-, -S-, -C(O)- and -C(S)-; thereby inhibiting the hatching of the louse egg. C:\NRPonbl\DCC\AZBU952949 I DOC.1212)5/20 10 - 58

11. The method of claim 10, wherein the compound is selected from 2,2'-dipyridyl, 6,6'-dimethyl-2,2'-dipyridyl and 5,5'-dimethyl-2,2'-dipyridyl or pharmaceutically, veterinary or agriculturally acceptable salt. 5

12. The method of claim 10, wherein said method inhibits hatching of human head lice.

13. The method of any one of claims I to 12, wherein said compound is formulated for use in a direct topical application in a form selected from the group consisting of a dip, spray, aerosol, shampoo, mousse, emulsion, solution cream, dust, and lotion. 10

14. A method according to any one of claims I to 11, wherein said ectoparasite egg is human head lice eggs and the compound is formulated for use in a topical application to human scalp and is in a form selected from the group consisting of a dip, spray, aerosol, shampoo, mousse, emulsion, solution cream, dust, and lotion. 15

15. A topical composition when used for topically inhibiting hatching of an ectoparasite egg comprising an effective amount of at least one compound of formula (I): R2 R I Rl' R2' R3 X R 3 ' N N R4 R4' (I) 20 wherein X is selected from a covalent bond, -C(R 5 ) 2 -, -Z- or -C(R5)2-Z-C(R5)2-; R' and R" are independently selected from hydrogen, Ci- 6 alkyl, C 2 - 6 alkenyl, C 2 . 6 alkynyl, hydroxy, CI- 6 alkoxy, thiol, C 1 . 6 alkylthio, CO 2 H, C0 2 Ci- 6 alkyl, SO 3 H, 25 SO 3 CI- 6 alkyl, NH 2 , NH Ci- 6 alkyl or N(C 1 . 6 alkyl) 2 ; C:NRPonbIDCC\AZB\2952949 J DOC-12/11512010 - 59 R2, R", R', R", R 4 and R4' are independently selected from hydrogen, C 1 . 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, hydroxy, CI- 6 alkoxy, thiol, C 1 . 6 alkylthiol, CO 2 H, CO 2 CI. 6 alkyl, SO 3 H, SO 3 Ci. 6 alkyl, NH 2 , NHCI. 6 alkyl or N(Ci. 6 alkyl) 2 , or -CH 2 CHNH(CO 2 H); or 5 R 2 and R 3 or R 3 and R 4 and/or R 2 and R 3 or R 3 and R 4 ' taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring; each R 5 is independently selected from hydrogen, CI. 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, hydroxy, CI- 6 alkoxy, thiol, CI- 6 alkylthiol, CO 2 H, C0 2 CI- 6 alkyl, SO 3 H, 10 SO 3 CI- 6 alkyl, NH 2 , NHCi- 6 alkyl or N(Ci- 6 alkyl) 2 ; and Z is selected from a covalent bond, -NH-, -0-, -S-, -C(O)- and -C(S)- and a suitable diluent, excipient or carrier.

16. The composition of according to claim 15, further comprising a second 15 ectoparasiticide, wherein the second ectoparasiticide is selected from the group consisting of organophosphates, synthetic pyrethroids, insect growth regulators, chitin synthesis inhibitors, triazine derivatives, insecticidal bacterial toxins, chlorinated hydrocarbons and dimethicone copolyols. 20

17. Use of at least one compound of formula (I): R 2 R' R I R 2 R X3R N N R4 R4' (I) wherein X is selected from a covalent bond, -C(R 5 ) 2 -, -Z- or -C(R 5 ) 2 -Z-C(R 5 )2-; C \RPortbIlDCC\AZBU952949- .DOC-12/115/2010 - 60 R' and R" are independently selected from hydrogen, Ci. 6 alkyl, C 2 - 6 alkenyl, C 2 6 alkynyl, hydroxy, CI- 6 alkoxy, thiol, CI. 6 alkylthio, CO 2 H, CO 2 Ci. 6 alkyl, SO 3 H, S0 3 CI. 6 alkyl, NH 2 , NHCI. 6 alkyl or N(CI. 6 alkyl) 2 ; R 2, R , R 3, R 3 , R 4 and R 4 are independently selected from hydrogen, Ci. 6 alkyl, 5 C 2 . 6 alkenyl, C 2 - 6 alkynyl, hydroxy, Ci. 6 alkoxy, thiol, Ci. 6 alkylthiol, CO2H, CO 2 Ci. 6 alkyl, SO 3 H, S0 3 Ci. 6 alkyl, NH 2 , NHCI. 6 alkyl or N(Ci. 6 alkyl) 2 , or -CH 2 CHNH(CO 2 H); or R2 and R 3 or R 3 and R 4 and/or R and R or R and R 4 taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or 10 heterocyclic ring; each R 5 is independently selected from hydrogen, Ci- 6 alkyl, C 2 . 6 alkenyl, C 2 6 alkynyl, hydroxy, Ci- 6 alkoxy, thiol, CI- 6 alkylthiol, CO 2 H, CO 2 CI. 6 alkyl, SO 3 H, S0 3 CI. 6 alkyl, NH 2 , NHC 1 . 6 alkyl or N(C 1 - 6 alkyl) 2 ; and Z is selected from a covalent bond, -NH-, -0-, -S-, -C(O)- and -C(S)- and a suitable 15 diluent, excipient or carrier in the manufacture of a topical composition for inhibiting hatching of an ectoparasite egg.

18. The use of claim 17 wherein the at least one compound of formula (I) is applied simultaneously, separately or sequentially with a second ectoparasiticide wherein 20 the second extoparasiticide is organophosphates, synthetic pyrethroids, insect growth regulators, chitin synthesis inhibitors, triazine derivatives, insecticidal bacterial toxins, cholorinated hydrocarbons or dimethicone copolyols..

19. The use of claim 15 or claim 17 wherein said compound is formulated for use in a 25 direct topical application in a form selected from the group consisting of a dip, spray, aerosol, shampoo, mousse, emulsion, solution cream, dust, and lotion.

20. A method according to any one of claims I to 3, 5 and 7 to 14, a composition according to claim 15 or 16 or a use according to any one of claims 17 to 19, 30 wherein the compound is 5,5'-dimethyl-2,2'-dipyridyl or a pharmaceutically, veterinary or agriculturally acceptable salt. C ±NRPonbl\DCC\AZB\2952949_ DOC-I 2/05/2010 -61

21. A method according to either one of claims 1 or 10, or a composition according to claim 15 or a use according to claim 17, substantially as hereinbefore described and/or exemplified.