AU731383B2 - Compounds for the prevention and treatment of helminth infections - Google Patents

Description

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S. a Invention Title: COMPOUNDS FOR THE PREVENTION AND TREATMENT OF HELMINTH

INFECTIONS

The following statement Is a full description of this Invention, Including the best method of performing it known to us '.1 1 COMPOUNDS FOR THE PREVENTION AND TREATMENT OF HELMINTH INFECTIONS Field of the Invention The present invention relates to compositions capable of eliciting an immune response in vertebrates against helminth infections. In particular, the present invention is go directed to vaccines which may be used to protect a vertebrate against infection from parasitic helminths.

Helminthic infections are a major cause of morbidity and mortality in both domesticated animals and human populations. Speaking generally, helminths refer to parasitic and non-parasitic species belonging to the phyla platyhelminthes (for example, flukes, tapeworms, and other flatworms) and nematahelminthes (for example, roundworms and e e their relatives). The following is illustrative of how helminthic infections occur. A helminth species enters the body of a host in the form of eggs or invasive larvae, for

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example, as a result of the ingestion by the host organism of food containing the eggs or larvae. The helminths then develop, moving slowly through different tissues, blood and/or lymph. Finally, they reach their "preferred" organ, and grow and mature. Eventually, the organ in which they reside is affected adversely.

Control measures rely to a large extent on improvements in hygiene, reduction in vector populations, and chemotherapy. Control measures which focus on hygiene and reduction of vector population have proved to be problematic especially in developing countries where such infections are %oe 15 most prevalent and such control measures are most difficult to S implement. Current chemotherapeutic medications all have e drawbacks such as high toxicity, the requirement that treatments be repeated and immunosuppressive side-effects.

Furthermore, the use of these preparations often results in the parasites' developing resistance to the chemotherapeutic medication. Indeed, nearly every country has documented cases of antihelminthic resistance. For these reasons, as well as the expense of repeated administration of chemotherapeutic compounds, a compound which would not have these drawbacks has been highly sought after.

Since a large variety of helminth species may be found in infected populations, it would desirable to produce a vaccine having a broad spectrum of prophylactic activity. To 3 date, the induction of strong protective host immunity following infection by helminths has been uncommon. The long co-evolutionary experience these parasites have had with their hosts has driven the host-parasite relationship to a level of accommodation that results in chronic or persistent conditions rather than acute infections that typically yield strong specific immunity. For this reason, unlike the situation with most microbial diseases of animals, few vaccines have been available for helminth control, and those which do exist have significant drawbacks.

Of the known methods of producing helminth vaccines, inactivated and living vaccines have the drawback that they are labor-intensive and only weakly immunogenic and they induce side-effects such as a localized inflammation, allergic II. 15 response, and fever. Furthermore, often the attenuated form used in the vaccine causes a disease similar to that induced by the virulent (wild) forms of the parasite. In addition, o• S* the macromolecular carrier proteins used in these vaccine preparations cause a number of immunopathologic side-effects in the vaccinated organism.

The other available class of vaccines comprises genetically engineered antigens. Yet these too are only S" weakly immunogenic.

In summary, the available types of vaccines are 25 generally ineffective and possess a narrow specificity, being directed against a single parasite.

4 Reported Developments A detailed description of helminths in whose life-cycles tissue-migration plays an important part, as well as a discussion of the pathological conditions they cause can be found for example in E.J. Soulsby, Helminth, Arthropods and Protozoa of Domesticated Animals, 7th Edition, Lea and Febiger, Philadelphia (1982) and in G.M. Urquhart, "Veterinary Parasitology, Longman Scientific and Technical, United Kingdom (1987).

All parasites elicit immune responses, but for many reasons are able to present a moving and sometimes invisible target to the host's immune response, to such an extent that the normal control mechanisms fail and immunological damage instead of immunity often occurs. This in turn frequently *e 15 leads the host to switch off its ineffective and often counterproductive immune response, thereby resulting in gross pathological changes and immunosuppression.

The structural and antigenic diversity of the parasitic helminths is reflected in the heterogeneity of the specific immune responses they elicit. Parasitic helminths often evade the immune system by masking and shedding their surface antigens and by varying their antigens during their residence in vertebrate hosts. This ability to mask, shed and vary surface antigens is a primary cause of the difficulty experienced heretofore in producing efficacious vaccines against helminths infection.

A review of modern vaccines used in the treatment of parasitic diseases is provided in, J.H.L. Playfair. et al., 5 The Lancet 335 (1990): 1263-1266, while a more general discussion of the nature of the immunological response of hosts to parasitic helminths can be found in the article by S.

Lloyd and E. J. L. Soulsby in "Parasitology in Focus: Facts and Trends', Ed. H. Mehlhorn, Springer-Verlag (1988) pp.

619-650. As indicated in the Playfair et al. review article and as mentioned hereinabove, since existing helminth control measures are expensive and difficult to implement on a wide scale, there is a strong need for vaccines capable of reducing the intensity and prevalence of helminth infection in host populations.

Prior to the present invention, it was thought unlikely that one antigen alone could confer adequate protection against a wide range of helminth infections based 15 on the difficulties referred to above encountered in producing *effective anti-helminth vaccines against even specific species. For an overall review of medical and scientific 9 challenges provided by helminths, see A.A.F. Mahmoud, Science .9* 246 (1989): 1015- 1021. (In addition see also the entries "Parasites, Escape from Immunity", by D.J. Mclaren, and "Parasites, Immunity to" by F.E.G. Cox, in the Encyclopedia of 9.

Immunology, eds. I.M. Roitt and P.J. Delves, Academic Press, 1 9 1992.) 0* 9 o E 9 6 Summary of the Invention According to the present invention, a polymer (hereinafter referred to as "synpol") is a copolymer of ethylenepiperazine N-oxide and N-(carboxymethyl)ethylenepiperazinium. This polymer can function as an immunostimulating agent, adjuvant or carrier. It may be combined with or conjugated to an antigen, and such a combination or conjugate may be used in a vaccine composition. By way of example, the polymer may be coupled to hyaluronidase, for use in protecting a vertebrate against infection by helminth, as described in more detail in EP-B-0789586.

Brief Description of the Drawings Figure 1 illustrates the general formula for synpol.

Figure 2 constitutes the structural formula of synpol produced as described S in Example 1.

15 Figure 3 constitutes the structural formula of synpol produced as described in Example 2.

Description of the Invention A preferred embodiment of "synpol" is a copolymer of ethylenepiperazine 20 N-oxide and N-(carboxymethyl)-ethylenepiperazinium bromide, as shown by the formula in Figure 1.

Synpol, unlike most other carriers and adjuvants, is non-immunogenic. It is thought that synpol has no recognizable antigenic determinants and, accordingly, does not provoke an immune response thereby avoiding undesirable side effects observed with most other adjuvants and carriers used in vaccine preparations.

In order to identify in a convenient way the various species of synpol one from another, the term "synpol" is used in combination and sequentially with values for each of the aforementioned letters and For example, ethylenepiperazine N-oxide and N-(carboxymethyl)ethylene-piperazinium bromide ith n 1000, q 0.35, z 0.60, m 0.05 is referred to as "synpol 1000-35/60".

An example of a specific synpol species copolymer used successfully as an immunostimulating carrier in the vaccine embodiments of the present invention will be referred to herein as "synpol 1000-20/50". Synpol having a molecular weight of at least about 15 kDa or greater is preferred in the practice of the present invention with synpol having a molecular weight greater then at least about 30 kDa being especially preferred.

As the term is used herein, "vaccine" refers to a composition which contains the compound of the present invention and which is in a form that is capable of being administered to a vertebrate. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the compound of the present invention is suspended or dissolved. In this form, the compound of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat a helminth infection.

The present invention also includes within its scope the use of the compound comprising hyaluronidase covalently bound to synpol in these contexts.

In addition, the invention includes within its scope synpol-antigen conjugate vaccines in which the antigen comprises proteins similar to hyaluronidase, for example, vaccines containing as the antigen other enzymes 20 used by pathogens to digest/degrade tissue (including collagenases and proteinases of different specificities).

The invention also includes within its scope the use of synpol coupled with 0 an allergen in order to abolish allergic reactions within a host to a given allergen.

Extensive investigation has shown that administering such an antigen-synpol conjugate induces preferentially the production of non-allergic antibody isotypes against the allergen in question. These normal non-pathogenic isotypes compete with the previously existing allergenic one IgE immunoglobulins) and specifically abolish the allergy. This method of specific desensitization has been clearly demonstrated and is now in the first stage of clinical trials.

_Soi The invention also includes within its scope the use of synpol coupled with Sth following antigens to enhance the immunogenicity of the coupled antigens, thereby serving to promote the induction of an effective prophylactic immune response: beta-subunit of cholera toxin, hemagglutinin from envelope of types A and B influenza viruses, p. 90 toxin from B. anthracis, the Vi antigen from salmonella, porin protein from the cell wall of E. coli and salmonellae, synthetic fragments of the gpl60 env-protein of HIV-1, F(ab)2 fragments of immunoglobulins (in order to induce an anti-idiotype response).

Examples The first two examples are illustrative of the preparation of two species of synpol, as identified in the examples.

Example 1 Preparation of Synpol 1000-20/50 A three step procedure was used to synthesize a copolymer of ethylenepiperazine N-oxide and N-(carboxymethyl)ethylene-piperazinium bromide.

15 The initial polymer, 1,4-ethylenepiperazine, was synthesized in the first step. For this purpose, the living chain polymerization of 1,4diazabicyclo[2.2.2]octane was performed according to the following protocol.

g of the preliminarily sublimed monomer and 0.05 g of ammonium bromide were sealed in a 10 ml glass ampule. A vacuum of residual pressure 5 x 20 10 3 mm Hg was produced in the ampule using a vacuum pump. The ampule was exposed for 25 hours at 2000C in a thermostat. Polymer yield was about 100%, M.W. 120,000 (estimated by LALLS low angle laser light scattering).

The second step was performed to product the N-oxide of poly-1,4ethylenepiperazine.

5 g of poly-1,4-ethylenepiperazine 120,000, n 1000) were dissolved in 250 ml of 1% acetic acid solution. Then, 4 ml of 30% H 2 0 2 were added, and oxidation lasted for 36 hours. After ultrafiltration and lyophilization, the N-oxide of poly-1,4-ethylenepiperazine 110,000, z 0.5n) was obtained.

The alkylation of the above poly-N-oxide was performed during the IN third step.

9 Poly-1,4-ethylenepiperazine N-oxide produced during the second step was dissolved in 125 ml of methanol and 16.5 g of bromoacetic acid were added. The alkylation reaction was carried out for 10 hrs. at 250C. The solvent was evaporated in a vacuum and the deposit dissolved in water, dialyzed against water for 24 hrs. and dried using lyophilization. Finally, the copolymer of ethylenepiperazine N-oxide and N-(carboxymethyl)ethylene-piperazinium bromide of the following formula was obtained (see Figure 2).

The yield was 95%. The oxidation ratio was estimated by the chromometric (or titanometric titration) method and by the ratio of integral intensities of PMR-spectrum bands in region 2.5-4.5 m.d. The chromometric or titanometric titration method refers to the method of quantitative determination of N-oxide groups reduced by salts of bivalent chrome or trivalent titanium (Brooks, R. T. and P. D. Sternglanz, Anal. Chem., 1959, v. 31, N4, p. 561-565). Oxidation S ratio amounted to z 0.5n. Alkylation ratio was determined by IR-spectra 15 (1735 cm band) and PMR-spectra (2.5-4.5 m.d. region) and accounted q 0.2n.

Example 2 Preparation of Synpol 200-35/65 S: A copolymer of ethylenepiperazine N-oxide and N- (carboxymethyl)ethylene-piperazinium bromide with M.W. 25,000 (n 200, q 0.35n, z 0.65n) was synthesized using a three step procedure, similar to the o° 20 one of Example 1.

In the first step, 10 g of the preliminarily sublimed monomer and 0.11 g of ammonium bromide were sealed in a 10 ml glass ampule. Then a vacuum (5 x 10 3 mm Hg) was produced in the ampule by a vacuum pump, and the ampule was kept at 2000C for 15 hours. The yield of poly-1,4-ethylenepiperazine was about 100%, M.W. 80,000 (measured by LALLS).

In the second step, the N-oxidation of poly-1,4-ethylenepiperazine was carried out as follows.

g of poly-1,4-ethylenepiperazine obtained in the first step were dissolved in 250 ml of 1% acetic acid solution. Then 4.6 ml of 30% H 2 0 2 was added at 2-4oC using gentle agitation. The oxidation lasted for 48 hours. Then after ultrafilter cleaning and lyophilization, the N-oxide of poly-1,4-ethylenepiperazine 50,000, z 0.65n) was recovered.

The quantity of poly-1,4-ethylenepiperazine N-oxide produced in the step above was dissolved in 125 ml of methanol and then 16.5 g of bromoacetic acid were added. The reaction of alkylation was carried out at for 24 hours. The solvent was evaporated in a vacuum and the resulting deposit obtained was dissolved in water, dialyzed for 24 hours against water, and lyophilized. There was produced a copolymer of ethylenepiperazine N-oxide and N-(carboxymethyl)ethylene-piperazinium bromide having the following formula (see Figure 3).

The yield was 95%. The oxidation and alkylation ratio, both estimated as in Example 1, were z 0.65n and q 0.35n respectively.

The conjugation of synpol to hyaluronidase, and the properties and use of the conjugates, are described in WO-A-95/07100.

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Claims (11)

1. 2. A polymer having the formula shown in Figure 1.
2. 3. A polymer having the formula shown in Figure 2.
3. 4. A polymer having the formula shown in Figure 3.
4. 5. A polymer according to any of claims 1 to 4, having a molecular weight of at least 15 kD.
5. 6. A polymer according to claim 5, having a molecular weight of at least kD.
6. 7. A polymer according to any of claims 1 to 6, for therapeutic use as an immunostimulating agent.
7. 8. A polymer according to any of claims 1 to 6, for therapeutic use as an immunostimulating adjuvant.
8. 9. A polymer according to any of claims 1 to 6, for therapeutic use as an immunostimulating carrier. The combination of an antigen and a polymer according to any of claims 1 to 6.
9. 11. A conjugate of an antigen and a polymer according to any of claims 1 to 6.
10. 12. A vaccine composition including a combination according to claim 10 or a conjugate according to claim 11.
11. 13. Use of a polymer, combination, conjugate or vaccine according to any of claims 1 to 12, for the manufacture of a medicament for use in stimulating the immune response. S S 0O S S@ S. DATED this 23rd day of January, 2001. PETROVAX, INC WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P14386AU00 CJH/JPF/MBL/SIG