Link:  Pharm/Biotech Resources

Title:  Vaccines absorbable by the transmucosal way

United States Patent:  6,936,250

Issued:  August 30, 2005

Inventors:  Wyss; Rolando (Vaduz-Liechtenstein, CH); Bizzini; Bernad (Albi, FR); Volpato; Ivo (San Mariano, IT)

Assignee:  Grisotech S.A. (Soazza, CH)

Appl. No.:  101034

Filed:  March 18, 2002

The main object of the present invention is to provide polysaccharide-coated antigens derivatized with lectins for pharmaceutical use. In the vaccines, polysaccharides are preferably selected from the group consisting of chitosan, low-molecular-weight and high-deacetylation-degree chitosan, methyl glycol chitosan, alginic acid, polymannuronic acid and salts or derivatives thereof. In the vaccines of the invention, antigens are microorganisms, infectious agents or constituents thereof, hormones, enzymes, proenzymes, narcotics, bioactive peptides, metabolites, biological precursors, cell constituents, allergens, and the lectins are of vegetable origin.

SUMMARY OF THE INVENTION

It is the main object of the present invention to provide polysaccharide-coated antigens derivatised with lectins for pharmaceutical use. In said vaccines, polysaccharides are preferably selected from the group consisting of chitosan, low-molecular-weight and high-deacetylation-degree chitosan, methyl glycol chitosan, alginic acid, polymannuronic acid and salts or derivatives thereof. In the vaccines of the invention, antigens are microorganisms, infectious agents or constituents thereof, hormones, enzymes, proenzymes, narcotics, bioactive peptides, metabolites, biological precursors, cell constituents, allergens, and the lectins are of vegetable origin.

Also, this invention extends to the use of the claimed vaccines in the preparation of drugs for the prevention and treatment of growth-related disorders, and for the treatment of osteoporosis, ulcer, hypercholesteremia, obesity, infertility, lipoidosis, allergies.

The invention has for its object also the provision of a procedure for the preparation of said vaccines and of compositions containing, as active ingredient, polysaccharide-coated vaccines derivatised with lectins.

DETAILED DESCRIPTION OF THE INVENTION

The Applicants observed that when antigens, also of proteic nature, are coated with polysaccharides, they are absorbed by the mucosa and stimulate both local and humoral immunity. Furthermore, when antigens are conjugated or derivatised with lectins, they are more effectively directed towards the mucosal cells, in particular, those expressing gangliosides GM1 and GM2 (M cells) and which are involved in the stimulation of the local and systemic immune response. The incorporation in polysaccharides of antigens derivatised with lectins allows the exploitation of the directional effect of lectins that, being protected by the polysaccharide coating, do not produce concomitant undesired side effects. It follows that the exploitation of the lectin directional effect, of the polysaccharide protection and transport, and of the immunomodulating effect of some polysaccharides. e.g. chitosan, results in dosage control and elimination of side effects, if any, produced by lectins. The major advantage of the vaccines of the invention is that they are absorbed by the oral and by the transmucosal way. The vaccines according to the present invention are adsorbable either by direct local administration on external mucosae (nasal, pharingeal, rectal, vaginal, or intestinal mucosae) or by oral administration, the latter allowing the vaccines to reach the internal mucosae of the gastro-intestinal tract.

Furthermore, they are more efficacious than the corresponding coated- but non-conjugated vaccines, in that they are more efficiently directed towards the mucosal cells involved in the stimulation of the immune response.

It is, therefore, the main object of the present invention to provide polysaccharide-coated vaccines consisting of antigens derivatised with lectins, for pharmaceutical use.

According to the present invention polysaccharides form a protective coat surrounding the antigen-lectin conjugate and exert a triple action: they protect the antigen from the degradation due to proteolytic enzymes and gastric environment and, therefore, may be administered per os; isolate lectins from non-specific and potentially toxic absorption; and may have an immunostimulating effect. Therefore, polysaccharides carry the vaccines, while protecting their proteic structure and allowing their absorption by the oral and transmucosal ways in the active form.

The oral or transmucosal absorption has further advantages over the parenteral one. In the specific case of heterologous proteins, such as for example immunoglobulins, the oral or transmucosal absorption, which is slower and more gradual, allows better to control the immunoglobulins dosage and distribution in the blood, and does not alter their efficacy. The absorption by the oral and transmucosal ways of the vaccines according to the invention is slower and more gradual than that by the parenteral way: it follows that the conditions of antigen-immune system interaction may be better rationalised with time.

Derivatised antigens are incorporated in polysaccharides using polysaccharide preparations having different physicochemical characteristics and different derivatisation degrees.

Polysaccharides are preferably selected out of chitosan, alginate and derivatives thereof, such as for example low-molecular-weight (150,000) chitosan, medium-molecular-weight (400,000) and high-deacetylation-degree chitosan, glycol chitosan, methyl glycol chitosan, Protasan™. Particularly preferred are methyl glycol chitosan, low-molecular-weight and high-deacetylation-degree chitosan, and polymannuronic acid (MW 5-10 kD), obtained e.g. by alginic acid enzymatic hydrolysis with the alginate-lyase enzyme, and derivatives thereof. Said polysaccharides or derivatives thereof are selected from the group capable of surrounding the structure to be incorporated (in the specific case, antigens derivatised with lectins), with a polymeric "thin layer" resistant to the enzymatic action and to the physicochemical variations of the digestive system. They are also capable of directing the incorporated structure towards mucous cells of the intestinal tract, thus facilitating their absorption and the immuno-stimulating properties.

Non-cross-linked polysaccharides are preferably used for immunoglobulins incorporation. Further advantages result from the absence of cross-linking: the process for the preparation of the complexes of the invention is simpler and the final product does not contain potentially toxic residues derived from chemical cross-linking.

A feature of the vaccines of the present invention is that the coat-forming polysaccharides and the antigens derivatised with lectins are not covalently bound. Said coat is something like a superficial envelope in the form of a gel, as is the case e.g. of alginic acid. The bonds between polysaccharides and derivatised antigens are not covalent, but are preferably non-specific, weak interactions or ionic bonds.

In the vaccines of the invention, antigens may be of an endogenous or exogenous origin. They are selected from the group consisting of: infectious agents or their constituents thereof, allergens or antigenic constituents or their epitopes thereof, hormones, enzymes and proenzymes, narcotics, bioactive peptides, metabolites, biological precursors, cell constituents.

According to a preferred feature of the invention, antigens of exogenous origin derive from infectious agents or are inactivated infectious agents. According to a preferred feature, said infectious agents are selected from the group of: Herpes simplex, cytomegalovirus (CMV), chicken pox virus, rubella virus, syncytial virus, respiratory virus, influenza virus, Epstein-Barr virus, chicken rhinotracheitis virus, chicken respiratory virus, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Salmonella choleraensis, Clostridium tetani, Clostridium botulinum, Shigella flexneri, Candida albicans, Toxoplasma gondii or antigenic components or soluble or insoluble fractions thereof. Particularly preferred are the vaccines consisting of candidin antigen extracted from Candida albicans, Listeria antigen, IRT (Infectious Rhinotracheitis) antigen and chicken respiratory virus antigen. According to the invention, said antigens are derivatised and/or inactivated by conjugation with lectins and incorporated in polysaccharides. According to a further preferred embodiment, exogenous substances are narcotics for which antibodies formation may be usefully induced, e.g. for the purpose of detoxification. According to this preferred embodiment, antigens are narcotics, particularly those selected from the group consisting of cocaine, heroin, lysergic acid (LSD) or salts or derivatives thereof, which are derivatised with lectins and incorporated in polysaccharides. Particularly preferred is cocaine.

A further and innovative application of the vaccines of the invention is the control of the subject physiological (endogenous) biofunctional equilibria. In fact, antibodies induction towards endogenous substances, such as peptides, hormones or cell constituents, results in a metabolic variation of the body functional state or even in the correction of some pathological conditions. By way of example, when the antigen is cholesterol, the production of specific antibodies induced by the vaccine of the invention allows a reduction in the circulating cholesterol level. According to this embodiment, it is possible to correct functional and metabolic disequilibria deriving from organic degenerations of different nature. Therefore, in this case, the vaccines of the invention preferably consist of glucagon, somatostatin, cholecystokinin, calcitonin and derivatives thereof and/or conjugates with lectins, in polysaccharides. The natural body homeostasis, maintained by all endogenous molecules and components, is implemented, at a physiological level, by natural antibodies and specific inhibitors. This equilibrium state is not impaired by the active vaccination with the vaccines of the present invention. Conversely, a vaccination of this type can restore the homeostasis in a number of pathological conditions, as for example those described in the present invention.

Particularly preferred are the vaccines in which the endogenous substance or endogenous cell component, hormone or bioactive peptide, is cholesterol or somatostatin, derivatised with lectins and incorporated in polysaccharides according to the invention.

In the vaccines according to the invention, lectins are chosen on the basis of their ability to direct vaccines towards mucosal cells, in particular M-cells, involved in the immune response.

Particularly preferred are the lectins exerting an agglutinating action and particularly those selected from the group consisting of proteins derived from: Lens culinaris, Glycine max, Ulex Europaeus (UEAI+UEAII), Arachis hypogaea, Phaseolus vulgaris. According to a preferred feature of the invention, lectins are chemically derivatised with the antigen according to methods known in the art and, preferably, are derivatised with the antigen in ratio of 1 antigen molecole to 1-10 lectin molecules. Alternatively, they may be directly mixed with the antigen by high-speed mechanical stirring at 4° C. to 20° C. for 1 min.

The applicants have surprisingly found that, when the vaccines of the invention are administered together with immuno-adjuvants derived from the lipid-free Corynebacterium granulosum fraction, and are incorporated in polysaccharides according to the procedure used for the incorporation of antigens derivatised with lectins, i.e. by mixing a concentrated solution of derivatised antigen or of a lipid-freed BVV fraction (0.5-50 mg/ml) in a suitable buffer, selected on the basis of criteria known in the prior art, heated from 25 to 65° C., with a polysaccharide-containing solution in a concentration of 0.1 to 10% by wt./vol. in a buffer having pH of 3.5 to 8.5, and by high-speed mechanical stirring for 30 sec to 2 min or occasionally for 3 to 5 times for 30 to 60 sec., the antibody response following the vaccine treatment is dramatically increased.

It is, therefore, a further object of the present invention to provide adjuvant complexes consisting of the lipid-freed Corynebacterium granulosum fraction (BVV), incorporated in polysaccharides selected in the group consisting of chitosan, alginate and derivatives thereof, such as for example, low-molecular-weight (150,000) chitosan, medium-molecular-weight (400,000) and high-deacetylation-degree chitosan, glycolchitosan, methylglycolchitosan, Protasan™.

Particularly preferred are methylglycolchitosan, low-molecular-weight and high-deacetylation-degree chitosan, and polymannuronic acid (MW 5-10 kD), obtained e.g. by enzymatic hydrolysis of alginic acid with alginate-lyase enzyme, and derivatives thereof. Said polysaccharides or derivatives thereof are selected from the group capable of forming, around the structure to be incorporated (in the specific case antigens derivatised with lectins), a polymeric "layer" resistant to the enzymatic activity and to the physicochemical variations of the digestive system. According to a therapeutic scheme, the administration of the vaccines of the invention consists in a primary vaccine dose for sensitisation, followed by booster doses, all administered by the oral way and/or transmucosal absorption. Alternatively, the oral or transmucosal administration of the vaccines of the invention is combined with a primary sensitisation dose administered by the parenteral way. In both cases, the oral or transmucosal dose consists of 1 to 50 mg/administration both in the primary sensitisation and booster doses. The number of sensitisation and booster administrations ranges from 3 to 10. According to the protocol, sensitisation by the parenteral way is effected with a single 0.5 to 5 mg dose.

According to a further embodiment of the invention, vaccines are administered by the oral and transmucosal ways. The phrase "transmucosal ways" refers to the buccal, perlingual, rectal, vaginal ways of administration.

According to a still further embodiment, the vaccines of the invention are used in the preparation of drugs for the treatment of narcotics overdose syndrome, for the prevention and treatment of growth-related disorders, osteoporosis, ulcer, hypercholesteremia, obesity, infertility and pregnancy and menopause-related syndromes, for increasing the physical resistance to stress, for the treatment of allergic disorders, coagulation diseases, as well as in the preparation of drugs for the prevention and treatment of bacterial, viral and mycotic infections.

The vaccines of the invention are for human and animal use and, being administered per os, are prepared in the form of additives to foodstuff and water. It follows that they may be administered more easily that the vaccines for parenteral use only. It is a further advantage of the invention that the claimed vaccines may be collected and/or concentrated by filtration; moreover, even when re-suspended in a physiological saline solution, such as PBS at 4° C., they remain active for a long time.

It is a further object of the present invention to provide compositions for oral administration containing, as the active ingredient, the vaccines of the invention combined with appropriate adjuvants and excipients, such as those used in the prior art for the preparation of granular foodstuff for humans and animals (cornstarch, etc.). Particularly preferred are the compositions containing, as active ingredient, the vaccines derivatised with lectins and incorporated in polysaccharides, combined with BVV immunostimulants complexes, i.e. consisting of the lipid-freed Corynebacterium granulosum fraction with suitable excipients and/or diluents.

It is a further object of the invention to provide a procedure for the preparation of the vaccines of the invention essentially consisting in the following steps: a) antigen derivatisation by formation of a covalent bond with lectins, in ratios of 1 antigen molecule to 1 to 10 lectin molecules, or a′) preparation of the lipid-freed Corynebacterium granulosum fraction; b) incorporation of the derivatised (or conjugated) antigen obtained in step a) or of the lipid-freed Corynebacterium granulosum BVV fraction obtained in step a′) in the selected polysaccharide, preferably alginic acid, chitosan or salts or derivatives thereof, with mechanical stirring at 25° C. to 65° C.

According to a preferred embodiment, the incorporation of derivatised antigens in polysaccharides (step b) is carried out by mixing a concentrated solution of antigen derivatised with lectins or of a lipid-freed BVV fraction (0.5-50 mg/ml) in a suitable buffer selected on the basis of criteria known in the prior art, heated to 25 to 65° C., with a polysaccharide-containing solution in a concentration of 0.1 to 10% by wt./vol. in a buffer having pH of 3.5 to 8.5, and with high-speed mechanical stirring for 30 sec to 2 min or occasionally for 3 to 5 times for 30 to 60 sec.
 

Claim 1 of 16 Claims

1. An immunogenic composition comprising as an active ingredient an antigen selected from the group consisting of:

vaccines, microorganisms, infectious agents, and microorganism and infectious agents soluble or unsoluble fractions wherein said antigen is covalently conjugated to a lectin, further comprising a polysaccharidic coating.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.