AU782104B2 - Compositions and methods for inhibiting nematocyst discharge - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT(S): NIDARIA TECHNOLOGY, LTD.

ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: "Compositions and methods for inhibiting nematocyst discharge" The following statement is a full description of this invention, including the best method of performing it known to us: 1 COMPOSITIONS AND METHODS FOR INHIBITING NEMATOCYST

DISCHARGE

This application is a divisional application of Australian Patent 745348.

TECHNICAL FIELD This invention is in the field of compositions which repel and prevent the stinging of marine organisms, such as jellyfish. In particular, compositions and methods for inhibiting the discharge of the stinging structure (nematocyst or polar capsule) are provided.

BACKGROUND

Swimmers and divers throughout the world are familiar with the stinging organisms of the oceans such as jellyfish, sea anemone and coral. Jellyfish stings, although seldom fatal, are a major public health problem. Lotan et al. (1992) Marine Biology 112:237-242; Lotan et al. (1994) Marine Ecology Progress Series 109:59-65. In the summer months, it is estimated that over 500,000 swimmers and divers in the Chesapeake Bay area and 200,00 persons in Florida are stung by jellyfish, mainly by "sea nettles," the common name of Chrysaora quinquecirrha.

Burnett et al. (1992) MMJ41(6):509-513. Similarly, between the months of March and August, one in four Florida bathers are stung and more than 10,000 persons require emergency medical treatment for pruritic eruptions caused by contact with jellyfish larvae known as "sea lice." Tomchik et al. (1993) JAMA 269(13):1669-1672.

The members of the phylum Cnidaria jellyfish, sea anemone and coral) and the phylum Myxozoa are all equipped with stinging subcellular organelles, known as nematocysts, cnidocysts, or polar capsules. The nematocysts are located in specialized cells (nematocytes) and consist of a capsule containing a condensed tubule with potent toxins and threads. When nematocysts discharge, the tubule penetrates into its target organism and releases its toxins. The threads arrayed on the tubule enhance the anchoring and attachment of the nematocyst tubule to its target. Thus, nematocysts are involved in target recognition, toxin delivery, infection and attachment.

All members of the phyla Cnidaria and Myxozoa contain nematocysts of varying sizes, shapes and types. Mariscal, pp. 129-178, "Nematocysts" in COELENTERATE BIOLOGY: REVIEWS AND NEW PERSPECTIVES, eds. Muscatine and Lenhoff (Academic Press, New York, 1974). These different types of cnidocysts function in diverse biological roles including capture of prey, toxin delivery, recognition, attachment, adherence and infection. (see, Tardent (1995); Bioessays 17(4):351-362; Lotan et al. (1995) Nature 375:456; Lotan et al. (1996) Expt 'I Zool. 275:444-451; Lotan et al., pp. 132-144, "Toxicology and ecology and the Mediterranean jellyfish Rhopilema nomadica" in BIOCHEMICAL ASPECTS OF MARINE PHARMACOLOGY, eds. Lazarovici et al. (Alaken, Inc., Fort Collins, Colorado, 1996); Spaulding (1972) Biol. Bull. 143:440-453; Holstein and Tardent (1984) Science 223:830-833 and Mariscal, supra. Thus, although best known for their stinging capabilities, nematocytes also play a key role in recognition, attachment and infection. For example, parasites from the phylum Myxozoa use nematocysts (polar capsules) to recognize and infect their hosts. El- Matbouli et al. (1995) J. Fish Biol. 46:919-935 and Yokoyama et al. (1995) Diseases Aquatic Organ. 21:7-11.

The main body of the nematocyte cell consists of a dense capsule, the nematocyst, within which is a high folded eversible tubule. Discharge (eversion) of this tubule is driven by the build up of a high internal hydrostatic pressure of approximately 150 atmospheres within the capsule. The eversion of the internally folded tubule occurs within 3 microseconds at accelerations of up to 40,000 x g, one of the most rapid mechanical events in cell biology.

The nematocyte can be sub-divided into 3 morphological compartments with different functional entities: the capsule lumen and wall, the tubule and the sensory organelles. The capsule wall and lumen are the main components involved in developing the driving force for nematocyst discharge. The tension on the inner capsule wall during nematocyst discharge reaches up to 375 MPa.

Holstein (1994) Science 265:402-404. The strong capsule wa!! is highly permeable to water with a pore size of 600 Dalton. Within the resting nematocyst capsule, concentrations of up to 0.5 M of cations such as Ca Mg" or K' can be found. (see, Tardent, supra; Lubbock et al. (1981) PNAS 78(6):3624-3628; Godknecht et al. (1988) Marine Biology 100:83-92; Lubbock and Amos (1981) Nature 290(5806):500-501; Weber (1989) Int. J. Biochem. 184:465-476; Hidaka (1993) Biol. Bull. 184:97-104 and Gerke (1991) Hydrobiologia 216/217:661-669 for discussions of cations and nematocysts). The anionic counterparts are represented by poly-y-L-glutamatic acid (PGA) in varying degrees of polymerization. During nematocyst discharge, an extreme increase in internal capsule osmotic pressure occurs due to the influx of water. It has been suggested that the influx of water into the capsule is mediated by an internal release of the cations, such as Ca" in sea anemone or K' in hydra normally combined with PGA.

This osmotic pressure is translated into hydrostatic pressure causing the eruption and then evagination of the tubule form the nematocyst capsule (discharge). After the nematocyst discharge the internal cation concentration of the capsule is dissolved into the surrounded fluids.

The second compartment of the nematocyst is a highly condensed eversible tubule. This tubule serves the main role in nematocyte biological function; namely, the interaction or delivery of substances from the cnidarian or myxozoan into its target. The tubule, which is 200-850 pm when elongated, is twisted more than a hundred times around its axis and is packed into the 3-10 pm diameter of the nematocyst. Godknecht Tardent (1988) Marine Biol. 100:83- 92. Hollow barbs, arrayed on the inner surface of the tubule, become everted during discharge and play an important role in the penetration and anchoring of the tubule into its prey. Toxins, contained on the outer surface before discharge, are delivered through the barbs after the nematocyst is anchored. Lotan et al.

(1995), supra.

The sensory systems of the nematocyte are responsible for the control of nematocyst discharge. Discharge of nematocysts requires both chemical and 4 mechanical stimulation of the sensory organeles. In the sea anemone Aiptasia pallida, two classes of chemical receptors have been identified. One type of receptor is triggered by N-acetylated sugars, while the second chemoreceptor is triggered by certain amino acids. Thorington et al. (1988) Biol Bull. 174:163-171.

The mechanical sensory system is dependent on activation of the chemoreceptors.

These mechanoreceptors, however, can be adjusted by the organism, for instance, by tuning them to unique frequency signals emitted by a favored prey.

Certain fish diseases, including whirling disease, are caused by infection with an obligate parasite of the phylum myxozoan. (see for descriptions of myxozoan, Yokoyama et al. (1993) Dis. Aquat. Org. 17:223-228; Yokoyama et al. (1995) Dis. Aquat. Org. 21:7-11; Smothers et al. (1994) Science 265:1719- 172; and El-Matbouli et al. (1995) J Fish Biology 46:919-935). These parasites require two hosts, a fish and an aquatic annelid. The stages emerging from each host are infectious only for the other host, however, at each infectious stage, activated polar capsules (nematocyst) release a tubule that penetrates or adheres to the integument of the target. Host recognition is species-specific, indicating that nematocysts serve an important role in recognition of their host (El-Matbouli, supra).

In sum, nematocysts provide an effective method of delivering a substance deep into the target. Because nematocysts are able to penetrate their target so efficiently, it is difficult to remove them, or to treat after the toxin has penetrated.

Conventionally, nematocysts stings have been treated with antidotes such as steroids, aluminum sulfate/surfactant and antihistamines. Tomchik, supra. For example, Wasuwat (1970) Nature 225:758 describes how Thai fisherman use an extract made from the leaves of Ipomoea pea-caprae as an antidote to jellyfish poison. When the extract was analyzed, it was found to be mildly antihistaminic.

The extract exhibits the same effect against jellyfish poison as two commercially prepared antidotes containing antihistamines. In Australia, vinegar is recommended for treatment of lethal box jellyfish stings. Hartwick et al. (1980) Med J. Aust. 1:!5-20. Vinegar is not recommended for stings by othe species.

Fenner et al. (1993) Med. J. Aust. 158:498-501.

Since post-sting treatments for nematocyst stings are.often unsatisfactory, the search for ways to prevent nematocyst discharge has been ongoing. The most often prescribed method of preventing jellyfish stings is avoiding any contact with the nematocysts. Tomchik, supra. However, in the case of microscopic larvae, this often means foregoing all ocean activities during the months of high incidence, March through August in Florida). It would, therefore, be useful to have a means for inhibiting nematocyst discharge even when contact does occur.

Australian patent application 67563/94 (WO 94/17779) discloses topical hydrodispersion preparations that are reported to be effective in preventing nematocyst discharge as measured by scanning electron microscopy (SEM). The formulations contain inorganic micropigments incorporated into the lipid phase of the hydrodispersion; an optional UV filtering substance and are essentially free of emulsifiers.

Lubbock (1979) J. Exp. Biol. 83:283-292 describes how proteinaceous compounds tend to induce a stronger response leading to nematocyst discharge in sea anemones than either polysaccharides or lipids. The authors could determine no simple recognition basis and speculated that the process of nematocyst discharge was complex. Lubbock and Amos, supra, disclose that isolated nematocyst capsules do not discharge in 50 mM CaCl 2 The authors report that inhibition of nematocyst discharge occurs only if a solute that could not rapidly penetrate the capsule wall is used, for example, high molecular weight polyethylene glycol. Thus, calcium in the surrounding environment may stabilize nematocysts because it reduces the differential between the calcium concentration outside the capsule and inside the capsule. Normally, the calcium concentration inside the nematocyst capsule is approximately 600 mM. Normal calcium concentration in sea water is around 7 mM, about 100 fold less than inside the capsule. Thus, increasing the calcium concentration outside the capsule to 50 mM reduces the differential to around 10 fold and may be involved in inh)ibting nematocyst discharge.

Heeger et al. (1992) Marine Biology 113:669-678 tested the ability of three commercially available sunscreen lotions to inhibit jellyfish nematocyst discharge on samples of live human skin. Two of the three lotions were effective at reducing the number of nematocysts discharged. The authors concluded that glycerol and oil components of the lotions could be masking or suppressing the effects of natural stimuli of the skin, however, even the lotion which did not inhibit nematocyst discharge contained these substances. Hartwick et al. (1980) Med J. Australia 1:15-20 report that commercial sting remedies provide do not inhibit nematocyst discharge.

Thorington et al. Biol. Bull. 174:163-171 (1988) describe two classes of nematocyst chemoreceptor. One class of chemoreceptors is specific for Nacetylated sugars, and is unaffected by antihistamines. The second class of chemoreceptors is specific for amino acids and is inhibited by antihistamines. The authors hypothesized that the N-acetylated sugar receptors are the initial trigger for nematocyst discharge and that the antihistamine-affected receptors are probably activated only upon leakage of amino acids from the puncture wound created in the prey by the sugar-triggered nematocyst. It is therefore, unexpected that the present invention has shown that antihistamines alone are effective at inhibiting nematocyst discharge.

The present invention provides novel compositions that effectively inhibit nematocyst discharge. The compositions described herein are extremely effective at inhibiting nematocyst discharge when applied topically prior to exposure to nematocysts. In addition, it was the surprising discovery of the inventor that placing antihistamines and/or cations into the aqueous environment surrounding nematocyst-bearing organisms effectively inhibits nematocyst discharge. Thus, the present invention provides both protection for swimmers, divers and fishermen, as well as protection from nematocyte-facilitated infection in contexts such as fish farms where the surrounding environment can be treated.

DISCLOSURE OF THE INVENTION In accordance with one embodiment of the present irivention, a composition for inhibiting the discharge of nematocysts comprising an effective amount of an antihistamine is provided. In accordance with another embodiment of the present invention, compositions for inhibiting the discharge of nematocysts comprising an effective amount of at least one cation are provided. In accordance with yet another embodiment of the present invention, compositions for inhibiting the discharge of nematocysts comprising effective amounts of an antihistamine and at least one cation are also provided.

The nematocysts may be discharged from organisms selected from the group consisting of Hydrozoa, Anthozoa, Myxozoa and Schyphoza, preferably from the group consisting of Aurelia sp., Pelagia sp., Chrysaora sp., Anthoplaura sp, Rhopilema sp., Physalia sp., Cyanea sp., Linuche sp., Catostylus, Carybdea sp., Chironex sp., Stomolophus sp., Rhiozostoma and Corinactis sp., more preferably from the group consisting of Aurelia aurita, Corynactic californica, Anthopleura elegantissima, Pelagia noctiluca., Chrysaora quinquecirrha and Anthoplaura sp In one embodiment, the antihistamine is present in a concentration of from about 0.0005% to about preferably from about 0.001% to about The antihistamine can be any antihistamine, preferably diphenhydramine, cimetidine or tripelennamine, more preferably diphenhydramine.

In yet a further embodiment, the composition comprising an antihistamine further comprises at least one cation. The cation can be Ca", Na', Mn", Co", Mg", or Fe and is present in a concentration of from about 50 mM to about 1 M, preferably from about 50 mM to about 500 mM, more preferably from about mM to about 200 mM.

In another embodiment, the composition comprises at least one cation.

The cation can be Ca", Na Mn", Co-, Mg", or Fe" and is present in a conccntration of11111 about 50 JIvl to abOU i 1IV, preferably from about 50 mM to about 500 mM, more preferably from about 50 mM to about 200 mM.

In another embodiment, compositions within the present invention are incorporated into an ointment base. In a preferred embodiment, the ointment base does not comprise a glycoprotein, and is a lipid or a silicone polymer. In yet a further embodiment, the ointment base further comprises a sunscreen.

In yet a further embodiment of the present invention, methods are provided for inhibiting nematocyst discharge comprising applying inventive compositions within the present invention to the skin of a subject prior to contact with nematocysts. In another embodiment, method are provided for inhibiting nematocyst discharge in an environment surrounding the nematocyst.

In another embodiment, the use of antihistamines and/or cations is provided for the prevention or inhibition of nematocyst or polar capsule discharge.

As will become apparent, preferred features and characteristics of one aspect of the invention are applicable to any other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing three steps activation of nematocysts. Chemical secretions of stimulants from the skin bind to sensors.

Signals from the sensors activate the capsule. High internal pressure builds up in the capsule and leads to ejection of the tubule.

Figure 2 is a schematic diagram depicting how the formulation will prevent or inhibit discharge of nematocysts. In this depiction, a topical ointment containing the inventive composition is applied to the skin. Secretions of stimulants from the skin are thereby reduced or prevented. The sensors and capsule discharge mechanism are chemically inactivated by the inventive composition.

/%ATC\ThC UcD r' A DDVTATC'K' rT T'T Tr' TT rr- T-T/11 T IVmJLL, 1iU L I I 'I.J UU I 1IL UN V CIN II'JN Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation. -The disclosures of the publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

As used herein, certain terms will have defined meanings.

The term "nematocyst" or "cnidocyst" or "polar capsule" is used in the conventional sense to refer to the subcellular stinging structures found in the nematocytes or cnidocytes. Nematocysts are described in detail in Tardent, supra and Mariscal, supra. These stinging organelles are capable of penetrating, anchoring into and injecting a substance such as a toxin into a target organism.

The terms "nematocyte" or "cnidocyte" or "cnidoblast" are intended to refer to the specialized cell which contain the nematocysts. Non-limiting examples of classes of the phylum Cnidaria which possess cnidocytes include, for example, Hydrozoa, Anthozoa, Myxozoa and Scyphozoa. Examples of jellyfish include, but are not limited to, Aurelia sp., Pelagia sp., Chrysaora sp., Anthoplaura sp., Rhopilema sp., Physalia sp., Cyanea sp., Linuche sp., Catostylus sp., Carybdea sp., Chironex sp., Stomolophus sp., Rhiozostoma sp., Corinactis sp. and the like.

The term "antihistamine" refers to the general class of compounds which inhibit histamine binding. There are generally two types of histamine receptors, H, and Antagonists of both types of histamine receptors are effective at decreasing or preventing nematocyst discharge.

The term "cation" refers to any positively charged ion. The term "cation source" refers to any substance which is capable of supplying positively charged ions. Preferably, the cations are metal cations. Even more preferably, they are alkali cations, for instance, Li*, Na' or or alkali earth cations, for instance, Mg", Ca" and the like. Sources of these cations are known to those of skill in the art and are commercially available. Preferably, the source is a salt. Non-limiting examples include CaCI,, MgCI, and KCI and the like.

The term "ointment base" refers to a vehicle for substances intended for external application to the body. Generally, the base will comprise the main ingredient of a topically applied composition.

The term "glycoprotein" refers to a conjugated protein containing one or more covalently linked carbohydrate residues.

The term "effective amount" refers to an amount sufficient to effect beneficial or desired results. For purposes of thi.s invention, an effective amount of an antihistamine and/or a cation is an amount sufficient to inhibit, limit, repress, palliate or prevent the discharge of nematocysts.

The term "final concentration" refers to the concentration of the ingredient after the composition has been prepared, including, for instance, the concentration of the composition after it has been placed in an aqueous environment.

The term "surrounding environment" refers to the aqueous medium which surrounds stinging marine organisms. It includes, for example, fish ponds, aquariums, ocean enclosures and the like.

The present invention includes chemicals that inhibit nematocyst discharge. Without being bound to one theory, it is believed that the present invention acts to inhibit discharge by interfering with nematocyst discharge' at three levels: by reducing secretions of stimulants from the skin; by acting as an antagonist to the chemoreceptor trigger of discharge and by interfering with the build up of hydrostatic pressure required for discharge.

Inhibition of Nematocyst Discharge Compositions of the present invention are effective at inhibiting discharge of nematocysts. In one embodiment, the present invention includes compositions comprising an effective amount of antihistamine. In another embodiment, the present invention includes composition comprising an effective amount of antihistamine and an effective amount of at least one cation. Preferably, the antihistamine is diphenhydramine, tripelennamine or cimetidine. These antihistamines are commercially available from various suppliers. In a preferred embodiment, dinhenhvdramne is used. The antfhistamine is added to a fina concentration of at least about 0.0005% (or 15 ptM). Preferably, the final concentration is from about 0.0005% to about More preferably, the concentration is between about 0.001% and about Even more preferably, the concentration is between about 0.001% and Preferably, the concentration of cations is from about 50 mM to about 1 M. More preferably, the concentration is from about 50 mM to about 500 mM. Even more preferably, the concentration is from about 50 mM to about 200 mM. Concentrations greater than 1 M are also likely to be effective. It is believed that higher concentrations of the antihistamine or cations would be effective, but in view of cost considerations and any potential side-effects, the amounts specified above are preferred.

Hydrostatic pressure is the driving force for nematocyst discharge. To generate the needed pressure, bound cations Mg", NH 4 Na', and/or Co") are released from the internal nematocyst capsule. For example, the Ca" concentration in internal resting nematocysts is 300-600 mM, this Ca" is bound to the PGA matrix. Releasing the Ca" during nematocyst discharge increases the tonicity of the internal capsule and transforms osmotic pressure into hydrostatic pressure. Ca" concentration in the nematocyte cell, outside of the nematocyst capsule, and in sea water is 7 mM. When the Ca" concentration in the surrounding fluids of the capsule reaches 50 mM, the osmolarity of the nematocyst is reduced and discharge is less likely to occur. Because the cations involved in nematocyst discharge may differ between species, the present invention encompasses adding a mixture of cations. In this way, inhibition can be effected for a broader range of species, for example, in both hydra and sea anemones. In another aspect, an effective amount of only one cation is used.

Preferably, the cations used are Ca", Na", Mn", Co", Mg", and Fe". Even more preferably, they are Ca", K' and Mg". These cations can be obtained from various commercial vendors.

In yet another embodiment, compositions comprising an effective amount of at least one cation are provided. Preferably, the concentration of cations is from about 50 mM to about 1 M. More referably, the concentratio is from cn- rcenltfaioll is from about 50 mM to about 500 mM. Even more preferably, the concentration is from about 50 mM to about 200 mM. Concentrations greater than I M are also likely to be effective. In a preferred embodiment, at least one cation is included in the ointment base detailed in the following section.

In yet a further aspect, the invention includes adding the compositions described herein to the aqueous environment surrounding the nematocystcontaining organism. An effective amount of the composition is added to the aqueous environment surrounding the nematocysts. The concentrations described above are preferable.

Topical Formulations In another aspect, compositions of the present invention are formulated for topical application. Preferably, topical formulations containing compositions within the present invention create physical barriers to minimize skin stimulant secretion and, are themselves, not stimulants. Formulations should be water-proof so as to be useful for swimmers and divers. Ca" binding compound such as sodium citrate, EDTA, amino acids and glycoproteins generate nematocysts discharge and are, therefore, not to be used. Examples of suitable carriers include, but are not limited to, dextran, dextran sulfate, agarose, phosphatidyl ethanolamine, cholesterol, cholesterol palmitate, palmitic acid, oleic acid, lysolecitin, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, squalene, phenyl trimethicone, stearic acid, propylene glycol sterate, petrolatum, stearoxytrimethyl-silane, stearyl alcohol. Other carriers are known to those in the art. Silicone polymers are preferred as they provide good barriers, are water-proof and do not stimulate nematocyst discharge.

Optionally, the compositions described herein may contain a known sunscreen. Preferably, the sunscreens contain at least one UVA filter and at least one UVB filter. Oil-soluble UVB products include 3 -benzylidenecamphor derivatives, 4-aminobenzoic acid derivatives, esters of cinnamic acid, derivatives of benzophenone, esters of benzylidenemalonic acid. Water soluble IIVA filters include salts of 2-phenylbenzimidazole-5-sulphonic acid, sulphonic acid derivatives of benzophenones and sulphonic acid derivativesof 3benzylidenecamphor. Effective amounts of sunscreens will be known to those of skill in the art. Preferably, sunscreens will comprise between about 0. 1% and by weight of the total preparation.

The following examples are intended to illustrate but not to limit the subject invention.

EXAMPLES

Example 1: Decrease in Nematocyst Discharge Using Antihistamine in the Aqueous Environment To initially test inhibition ofnematocyst discharge, the following test was performed. It is typical behavior for jellyfish or sea anemone tentacles to adhere to and paralyze newly hatched nauplii of Aremia sp. The adhesion and subsequent paralysis is characteristic of nematocyst discharge by the tentacles.

Small 2 to 3 mm fragments of jellyfish or sea anemone tentacles were prepared and placed in a 12-well plate containing filtered sea water. The tentacle fragments were washed by changing the filtered sea water three times. One mL of filtered sea water was left in each well after the final wash. In 6 of the 12 wells, the antihistamine diphenhydramine hydrochloride was added to final concentration of 0.02%. The 6 wells without the antihistamine served as controls. Ten Artemia sp.

were added to each well in a total volume of between 50 and 100 uL of filtered sea water. After 2 minutes, the number of attached or paralyzed Artemia in each well was measured. Results are shown in Table 1.

*11- 1. 1 iauic I Surrounding water No. paralyzed Artemia Total No. Artemia sea water antihistamine 2 sea water antihistamine 1 8 sea water antihistamine 2 12 sea water antihistamine 3 14 sea water antihistamine 2 sea water antihistamine 1 sea water alone 8 sea water alone 9 13 sea water alone 9 11 sea water alone 7 sea water alone 9 11 sea water alone 8 9 Taken together, these results show that 11 out of 64 Artemia were paralyzed by nematocysts when antihistamine was added to their environment. In contrast, 50 of 64 Artemia were paralyzed by nematocysts when no antihistamine was present. Diphenhydramine significantly decreased the number of Aremia paralyzed by nematocysts.

Example 2: Inhibition of Nematocyst Discharge Into Human Skin Using Topically-applied Antihistamine To test the ability of antihistamines to inhibit nematocyst discharge, an assay using human skin was performed as described herein.

1. Preparation of Skin Samples Normal human skin (HS) was obtained from neonatal elective circumcision. The skin was immediately placed into sterile phosphate buffered saline (PBS) containing antibiotic/antimycotic. The tissue was frozen embedded in Tissue-TekTM. The tissue was then sectioned into 50 pm sections using a cryostat. The sections were attached to glass slides coated with polylysine and maintained at -70 0 C until time of use. All sections were examined to ensure that they were perpendicular to the plane of the epidermis, exposing cross-sections of epidermis, dermis and sub-cutaneous tissue.

2. Immunoassay to Detect Nematocyst Discharge A polyclonal antibody specific for nematocyst toxin was prepared, as described in Lotan et al. (1996), supra. Briefly, the HPLC fraction containing the toxin phospholipase A2 from the jellyfish Rhopilema nomadica was isolated and injected into rabbits as described in Vaitukaitis, pp. 46-52, "Production of Antisera with Small Doses of Immunogen: Multiple Intradermal Injections, in METHODS IN ENZYMOLOGY, VOL. 73, ed. Larson Vunkis (Academic Press, New York, 1981). Serum was collected from the rabbits after two injections, and the presence of toxin examined on immunoblots.

The slides prepared and frozen as described above were thawed at room temperature. The slides were submersed in sea water for approximately seconds and then attached to jellyfish or sea anemones tentacles for approximately seconds. The tentacles were detached, the slides washed two times with PBS, and blocked using 5% Fetal Bovine serum (FBS), and 0.1% Triton X100 in PBS (blocking solution). All washing and incubation steps were conducted on a tilting shaker at room temperature. The slides were incubated in 10 mL of blocking solution containing a 1 2,500 dilution of the anti-toxin antibody for one hour.

After incubation, the slides were washed five times for five minutes each wash with 100 mL of 0.1% TritonX100 in PBS (Wash solution). The slides were then incubated in 10 mL blocking solution containing a second antibody, Goat anti rabbit FITC (Fluorescent marker) for 30 minutes. The excess PBS was removed by shaking the slides. One drop of glycerol was added to each slide and the slides covered with a glass coverslip. The slides were observed under a conventional light microscope or under an ultraviolet (UV) microscope at least at 200x magnification.

3. Inhibition Nematocyst Udiscarge inio Human Skin Samples To monitor nematocyst discharge, three types of live jellyfish, Aurelia aurita, Pelagia noctiluca, and Chrysaora quinquecirrha and two species of sea anemone, Anthoplaura elegantissima and Corynactis californica were used in this experiment.

Four frozen human skin slides were thawed at room temperature. Two slides were submersed in glycerol (control) and the other two were submersed in glycerol containing 0.2% diphenhydramine. The slides were placed vertically on a glass dish to allow the glycerol to drip off. The slides were then exposed to one of the jellyfish or sea anemone species noted above and the number of nematocysts discharged was determined using immunohistochemical analysis. On the glycerol only slides, 135 and over 500 nematocysts were counted per slide.

On the glycerol plus antihistamine (diphenhydramine) 10 and 52 nematocysts were counted. These results demonstrate that the antihistamine diphenhydramine is effective at inhibiting nematocyst discharge into human skin samples.

To closely parallel the natural conditions under which jellyfish sting humans, the experiment described above was performed at the Monterey Bay Aquarium with the Chrysaora quinquicirrha and Pelagia noctiluca species of jellyfish. A total of 35 human slides were submersed in control or test substances and exposed to the jellyfish. Six untreated slides, seven slides submersed in glycerol, and seven submersed in glycerol plus 0.2% diphenhydramine were exposed to each jellyfish species for approximately 10 to 15 seconds. The human skin slides exposed to Chrysaora quinquecirrha showed that diphenhydramine decreases nematocyst discharge by 2.5-5 fold. In Pelagia noctiluca significant reduction in nematocyst discharged was observed. A ten-fold decrease in nematocyst discharge was observed with Aurelia (N=3 for the test and 3 for each control), however, a low number of nematocysts was discharged in the control (less than 100) thus leading to less striking results as compared with those obtained with the other species.

17 Example 3: Inhibition of Nmatocyst Discharge using Antihistaimine and Cations Frozen human skin slides are thawed at room temperature and submersed in glycerol containing 0.2% diphenhydramine and 100 mM CaCI 2 KCI or MgCl,.

Control slides are submersed in glycerol only. The slides are placed vertically in a glass dish to allow the glycerol to drip off. The slide is then exposed to jellyfish or sea anemone for about 15 seconds. The number of nematocysts that adhere to the skin sample is counted using the procedure described in Example 2.

Example 4: Inhibition of Myxozoan infection Host recognition and attachment of parasites to their host organisms is effected by nematocyst discharge. For example, the triactinomixon stage of infection recognizes and infects specific fish hosts by extrusion of a polar capsule.

Yokoyama, supra. The filaments of the polar capsule penetrate the fish epidermis and anchor the parasites to its host.

To prevent parasitic infections, a final concentration of 0.02% antihistamine diphenhydramine hydrochloride and 100 mM of a cation, e.g., CaClI, MgCIl, or KCI, is added to the fish pond or aquarium. The cation is selected based on the cation that is normally found in the lumen of the polar capsule. Unlike cnidarian nematocysts, triactinomyxon are exposed to the surrounding environment. Yokoyama, supra have previously shown that the ionic strength of the aqueous environment interferes with the internal structure of the triactinomyxon. Thus, the present invention involves preventing fish infection by two mechanisms: antihistamines to block recognition of the host and (2) cations to inactive the polar capsule.

Fish between 14 and 21 days old that are susceptible to triactinomyxon are exposed to an average of 10,000 waterborne triactinomyxon for approximately 1 hour in a total volume of 100 mL of water. In test aquariums, the water contains 0.02% diphenhydramine hydrochloride and 100 mM KC1, MgCI, or CaCI,.

Control aquariums have no antihistamine or cation. The level of triactinomyxon infection is determined by microscopy, or by immunoassay as described in Example 2. The same concentrations of antihistamine and cation can be used in larger scale fish ponds or aquariums.

Example 5: Inhibition of Nematocyst Discharge Into Human Skin Using Topically-applied Cations To test the ability of cations to inhibit nematocyst discharge, an assay using human skin is performed as described herein.

1. Preparation of Skin Samples Normal human skin (HS) is obtained from neonatal elective circumcision.

The skin is immediately placed into sterile phosphate buffered saline (PBS) containing antibiotic/antimycotic. The tissue is frozen embedded in Tissue-TekTM The tissue is then sectioned into 50 plm sections using a cryostat. The sections are attached to glass slides coated with polylysine and maintained at -70 0 C until time of use. All sections are examined to ensure that they are perpendicular to the plane of the epidermis, exposing cross-sections of epidermis, dermis and subcutaneous tissue.

2. Immunoassay to Detect Nematocyst Discharge A polyclonal antibody specific for nematocyst toxin is prepared, as described in Lotan et al. (1996), supra. Briefly, the HPLC fraction containing the toxin phospholipase A2 from the jellyfish Rhopilema nomadica is isolated and injected into rabbits as described in Vaitukaitis, pp. 46-52, "Production of Antisera with Small Doses oflmmunogen: Multiple Intradermal Injections, in METHODS IN ENZYMOLOGY, VOL. 73, ed. Larson Vunkis (Academic Press, New York, 1981). Serum is collected from the rabbits after two injections, and the presence of toxin examined on immunoblots.

The slides prepared and frozen as described above are thawed at room temperature. The slides are submersed in sea water for approximately 10 seconds and lthenl aLttached to jellyfLsh or sea anmo, es tentacles for a ipproximatel y seconds. The tentacles are detached, the slides washed two times with PBS, and blocked using 5% Fetal Bovine serum (FBS), and 0.1% Triton X100 in PBS (blocking solution). All washing and incubation steps are conducted on a tilting shaker at room temperature. The slides are incubated in 10 mL of blocking solution containing a 1 2,500 dilution of the anti-toxin antibody for one hour.

After incubation, the slides are washed five times for five minutes each wash with 100 mL of 0.1% TritonX100 in PBS (Wash solution). The slides are then incubated in 10 mL blocking solution containing a second antibody, Goat anti rabbit FITC (Fluorescent marker) for 30 minutes. The excess PBS is removed by shaking the slides. One drop of glycerol is added to each slide and the slides covered with a glass coverslip. The slides are observed under a conventional light microscope or under an ultraviolet (UV) microscope at least at 200x magnification.

3. Inhibition of Nematocyst discharge into Human Skin Samples A. Inhibition with Calcium To monitor nematocyst discharge on human skin samples, the experiment described herein was performed at the Monterey Bay Aquarium with the Chrysaora quinquecirrha species of jellyfish.

Twenty-three frozen human skin slides were thawed at room temperature.

Seven untreated slides, five slides submersed in silicone gel and slides submersed in silicone gel containing 200 mM CaCl 2 were exposed to the jellyfish species for approximately 10 to 15 seconds. The slides were analyzed to determine the number of nematocysts discharged using immunohistochemical analysis. The untreated slides had an average of 464 nematocyts discharged per sample. The slides treated with silicone gel prior to exposure to the jellyfish showed an average of 316 nematocysts per sample. The slides treated with silicone gel containing 200 mM CaCl showed a 7.8-fold decrease in the number of nematocysts, an average of 59 per slide, compared to the untreated slides and those treated with silicone gel only.

B. Inhibition with Calcium and Magnesium To closely parallel the natural conditions underwhich jellyfish sting humans, the experiment described herein are performed at the Monterey Bay Aquarium with the Pelagia noctiluca, and Chrysaora quinquecirrha species of jellyfish.

Thirty frozen human skin slides are thawed at room temperature. Ten untreated slides, ten slides submersed in glycerol and ten slides submersed in glycerol containing 200 mM CaCl2 plus 200 mM MgCI 2 are exposed to each jellyfish species for approximately 10 to 15 seconds. The slides are analyzed to determine the number of nematocysts discharged using immunohistochemical analysis. The untreated slides and the slides treated only with glycerol prior to exposure to the jellyfish show equivalent results with an average of 500 nematocysts per sample. The slides treated with glycerol containing 200 mM CaCI plus 200 mM MgCl show a 5-fold decrease in the number of nematocysts, an average of 100 per slide, compared to the untreated slides and those treated with glycerol only.

Example 6: Inhibition of Nematocyst Discharge Using Cations on Human Subjects Three square centimeters of the left inner arm of a human subject is covered with glycerol containing 200 mM CaCI 2 plus 200 mM MgCl,. The arm is submersed in sea water and after 5 seconds exposed to the fishing tentacles of Chrysaora quinquecirrha (sea nettle) for approximately 10 to 15 seconds. The same experiment is repeated using the right arm covered with glycerol alone as a control. Five human subjects are exposed to Chrysaora quinquecirrha and additional subjects are exposed to the fishing tentacles ofPelagia noctiluca. The degree of skin damage in each subject is evaluated 5 minutes and one hour after exposure. SevPn of te ten cijiert tstPd shnw no sn dama n th, eft IFrm while three exhibit minor damage to the left arm. Moderate to severe skin damage is observed on the right arm of these subjects.

Example 7: Inhibition of Myxozoan infection To prevent parasitic infections, a final concentration of 200 mM of a cation, CaCIl, MgCl 2 or KCI, preferably at least two cations such as CaCI 2 MgCI 2 is added to the fish pond or aquarium. The cation is selected based on the cation that is normally found in the lumen of the polar capsule. Unlike cnidarian nematocysts, triactinomyxon are exposed to the surrounding environment.

Yokoyama, supra have previously shown that the ionic strength of the aqueous environment interferes with the internal structure of the triactinomyxon. Thus, the present invention involves preventing fish infection by two mechanisms: (1) antihistamines to block recognition of the host and cations to inactive the polar capsule.

Fish between 14 and 21 days old that are susceptible to triactinomyxon are exposed to an average of 10,000 waterborne triactinomyxon for approximately 1 hour in a total volume of 100 mL of water. In test aquariums, the water contains 200 mM KCI, MgCI, or CaCl2, or preferably 200 mM of both CaC1, and MgCl,.

Control aquariums have no cation. The level of triactinomyxon infection is determined by microscopy, or by immunoassay as described in Example 2. The same concentrations of cation can be used in larger scale fish ponds or aquariums.

P Nopcrjgcdsi cliin of 2242(PA) doc-2nAAW -22- Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form or suggestion that that prior art forms part of the common general knowledge in Australia.

It would be appreciated by a person skilled in the art the numerous variations and/or modifications may be made to the invention as shown 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 restrictive.

Claims (12)

1. 5-05; 2:37PM;DAVIES COLLISON CAVE -23- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 5/ 11 a a. a a a a a a 1. A method of inhibiting the discharge of nematocysts or polar capsules, comprising applying a composition having at least one cation to the skin of a subject prior to contact with nematocysts or polar capsules, so as to prevent infection and toxic effects of nematocyst or polar capsule discharge. 2. The method of claim 1, wherein said at least one cation is a metal cation. 3. The method according to claim 2, wherein said metal cation is selected from the group consisting of Ca K Na*, Mn Co*, Mg"* and Fe". 4. The method according to claim 1, wherein said composition contains two cations. 15 5. The method according to claim 4, wherein said two cations are Ca and Mg-.
2. 6. The method according to claim 1, wherein the composition is suitable for topical application. 20 7. The method according to claim 1, wherein the composition further comprises a sunscreen.
3. 8. The method according to claim 1, wherein the nematocysts are discharged from a stinging marine organism selected from the phylum consisting of Cnidaria and Myxozoa.
4. 9. The method according to claim 1, wherein the stinging marine organism is selected from the group of Cnidaria consisting ofjelly fish, sea anemone and coral. COMS ID No: SBMI-01257069 Received by IP Australia: Time 14:50 Date 2005-05-20 5-05: 2:37PM:DAVIES COLLISON CAVE# 6/1 6/ 1 1 ?P OERUpeAMM.Ct 4- I -24- A method of inhibiting the discharge nemnatocyst or polar capsules comprising adding an effective amount of at least one cation to the environment surrounding the nematocyst or polar capsule, so as to prevent infection and toxic effects of nematocyst or polar capsule discharge, wherein said cation is selected from the group consisting of Ca Nae, M6", Mg and Fe'.
5. 11. The method according to claim 10, wherein the final concentration of the cation is from 50mM to IM,
6. 12. The method according to claim 10, comprising adding two cations.
7. 13. The method according to claim 12, wherein the two cations are Ca' and Mg'.
8. 14. A method of preventing a Myxozaa parasite infection in a fish, comprising the step 15 of inhibiting the discharge of the Myxozoa parasite nematocysts or polar capsules. The method of claim 14, wherein the discharge of nernatocysts or polar capsules is inhibited by adding at least one cation at a final concentration of from 50mM to IM, to the aqueous environmnent surrounding the fish, thus preventing infection.
9. 16. cation. The method according to claim 15, wherein said at least one cationi is a metal
10. 17. The method according to claim 16, wherein said metal cation is selected from the group consisting of Ca', Na 4 Mni+, Col-% Mg** and Fe'.
11. 18. The method according to claim 15, wherein two cations are added.
12. 19. The method of claim 18, wherein said two cations are Ca4-' and Mg'. COMS ID No: SBMIk01257069 Received by IP Australia: Time 14:50 Date 2005-05-20 5-05: 2:37PM:DAVIES COLLISON CAVE 7/ 11 Ile method of claim 1, substantially as hereinbefore described with reference to the examples. DATED this day of May, 2005 Nidaria Technology, Ltd. by DAVIES COLLISON CAVE Patent Attorneys for the Applicant a a. a a. a a a a. COMS IDNo: SBMI-01257069 Received by IP Australia: Time 14:50 Date 2005-05-20