A pharmaceutical formulation for parenteral or mucosal administration of antigens and/or vaccines to humans and animals, comprising monoglyceride preparations having at least 80% monoglyceride content and where the acyl group contains from 6 to 24 carbon atoms, together with fatty acids where the number of carbon atoms may be varied between 4 and 22.

The present invention relates to a novel pharmaceutical formulation for administration of antigens and/or vaccines. The preferred route of administration is via the mucosal membranes, however parenteral administration may also be used. The invention also relates to the use of certain compounds (as defined below) as adjuvants or vehicles in such formulation.

BACKGROUND OF THE INVENTION

An increasing number of specific antigens from different types of organisms (e.g. tumor cells, bacteria, virus and parasites) has been produced using cloning techniques over the last years. However, these antigens are frequently weak immunogens despite their high specificity.

To obtain good protection after vaccination, immune stimulating systems are needed that can enhance and activate the immune system against these weak antigens. Such immune stimulating systems are called adjuvants.

Adjuvants, presently mainly used in animal experiments, includes a highly heterogeneous group of substances; inorganic substances, oil emulsions, charged polymers, neutral substances or substances from bacteria.

There are presently large efforts in research and development in order to obtain a safe adjuvant with high efficacy to be used in humans. However, today there is presently no general adjuvant for this purpose.

Alum hydroxides and alum phosphates were the first two inorganic substances that were used in humans. The immune response obtained is a result of slow desorption of the precipitated antigen on the surface of the particle. Later it was shown that phagocyting cells were attracted by these alum salts leading to further enhancement of the immune response. However, these salts are not safe since granuloma formation has been reported (Slater et al, Br. J. Dermatol. (1982) Vol. 107, page. 103 108.). Furthermore, the alum salts can not be used for all antigens since all antigens are not adsorb on the surface.

In 1944 Freund introduced his adjuvant consisting of a mixture of vegetable oil, mineral oil, detergents and killed bacteria. The enhancement obtained was partly due to slow release of the antigen from the oil emulsion. Freunds adjuvant can however not be used in humans due to granuloma formation, induction of auto-immune reactions and the non-biodegradable mineral oil. Furthermore, the effect is difficult to control. The active substance in Freunds adjuvant has been isolated and its structure determined and shown to be N-acetyl muramyl-L-alaninisoglutamate, often called muramyl-dipeptide (MDP).

The adjuvant effect dependent of the particle size of polymetacrylate and polystyrene particles was examined on mice (Kreuter et al, Vaccine, (1986) vol 4, 125 129) by the use of ovalbumin (adsorbed on the particles) as a model antigen with subsequent assay of the immune response. The size of the particles was varied between 62 and 306 nm. The result was that smaller particles enhanced the immune response better than larger. The smaller particles gave a better effect than 0.2% Al(OH).sub.3. All preparations elicited a higher response as compared to fluid preparations. Similar experiments where particulate systems with smaller size results in a higher immune response as compared to larger particles are known in the scientific literature.

Almost all systems used today for enhancement of the immune response against antigens are particles or is forming particles together with the antigen. In the book "Vaccine Design--the subunit and adjuvant approach" (Ed: Powell & Newman, Plenum Press, 1995) all known adjuvants are described both regarding their immunological activity as well as regarding their chemical characteristics. As described in the book more than 80% of the adjuvants tested today are particles or polymers that together with the antigens (in most cases proteins) are forming particles. The type of adjuvants that not are forming particles are a group of substances that are acting as immunological signal substances and which under normal conditions consists of the substances that are formed by the immune system as a consequence of the immunological activation after administration of particulate adjuvant systems.

Using particulate systems as adjuvants, the antigens are associated or mixed with or to a matrix which has the characteristics of being slowly biodegradable. Of great importance using such matrix systems are that the matrix does not form toxic metabolites. Choosing from this point of view, the main kind of matrices that can be used are mainly substances originating from a body. With this background there are only a few systems available that fulfils these demands: lactic acid polymers, poly-amino acids (proteins), carbohydrates, lipids and biocompatible polymers with low toxicity. Combinations of these groups of substances originating from a body or combinations of substances originating from a body and biocompatible polymers can also be used. Lipids are the preferred substances since they display structures that make them biodegradable as well as the fact that they are the most important part in all biological membranes.

Lipids are characterized as polar or non-polar. The lipids that are of most importance in the present invention are the polar lipids since they have the capacity to form particulate systems in water. Another way of defining these lipids are as amphifilic due to their chemical structure with one hydrophobic and one hydrophilic part in the molecule thereby being useable as surface active substances. Examples of main groups of polar lipids are mono-glycerides, fatty acids, phospholipids and glycosphingolipids. These main groups can be further characterized depending on the length of the acyl chain and the degree of saturation of the acyl chain. Since the number of carbon atoms in the acyl chain can be in the range of 6 to 24 and the number of unsaturated bonds can be varied there are an almost infinite number of combinations regarding the chemical composition of the lipid.

Particulate lipid systems can be further divided into the different groups as discussed in the scientific literature such as liposomes, emulsions, cubosomes, cochleates, micelles and the like.

In a number of systems the lipids may spontaneously form, or can be forced to form, stabile systems. However, under certain circumstances other surface active substances has to be introduced in order to achieve stability. Such surface active systems can be of non- lipid character but possess the characteristics of the polar lipids having hydrophobic and hydrophilic parts in their molecular structure.

Another factor that has been shown to be of importance is that lipids exhibit different physical chemical phases, these phases has in different test systems been shown to enhance uptake of biological substances after administration to mucosal membranes.

In the classical immunology and in combination with vaccination against different types of infectious agents e.g. bacteria, virus or parasites the prevailing dogma has been to administrate the vaccine subcutanously or intramuscularly. However, research has during the last years shown that the body has a very effective immunological system that resides in the mucosa. It has been shown that you can administrate vaccines orally, nasally, rectally and vaginally. In the same way as for the classical immunization it has been shown that by mucosal vaccination there is also a need for enhancement of the immunological response by the addition of adjuvants.

In the same way as within the classical immunology where vaccines (antigens) are administrated parenterally, there is within mucosal immunization a great interest in directing the immunological response towards development of humoral and/or cellular response. If you obtain a humoral response it would be important to direct the response in a way that a certain class of antibodies would be obtained. In order to obtain such a goal, specific immune stimulating agents can be added to the formulation of antigens and adjuvants.

Different types of immune stimulating substances are available. One type is represented by proteins e.g. PHA, Con A, SEA or different types of interferons or interleukines. Another type of substance is represented by MDP, as mentioned above. Additional groups can be characterized as lipid derivatives since they show molecular structures which are amphiphilic. One example of such a substance is called MPL. Another similar substance is Quil A. A number of substances that can be classified within these categories are described in the book "Vaccine Design--the subunit and adjuvant approach" as discussed above.

It would be extremely valuable to be able to make the immunization procedures more effective directing the immunological response towards a certain class or subclass of antibodies and/or to be able to induce a strong T-cell response against the antigens.

DESCRIPTION OF THE INVENTION

It has now surprisingly been found that parenteral or mucosal administration of a pharmaceutical formulation containing one or two of the following adjuvants with admixed antigens and/or vaccines improves the immune response against the admixed antigens/vaccines. Said pharmaceutical formulation for parenteral or mucosal administration of antigens and/or vaccines to an animal comprise one or more substances selected from a) monoglycerides of the general formula

##STR00001## wherein R.sub.1 and R.sub.2 is H and R.sub.3 is one acyl group containing from 6 to 24 carbon atoms, preferably 8 to 20 carbon atoms, even more preferably 14 20 carbon atoms and where the acyl chain may contain unsaturated bonds. In a monoglyceride the acyl chain is normally in the R.sub.1 or R.sub.3 position. However there is normally a acyl migration between the 1 and 2 carbons in the glycerol molecule resulting in approximately 90% is in the R.sub.3 position and 10% in the R.sub.2 position. Thus, in the present invention distilled 1-monoglycerides from Danisco Ingredients (Denmark) with a purity of more than 80% preferably more than 90%, more preferably over 95% is used. The diglyceride content is maximum 3% and triglycerides and fatty acid content is less than 1.0%. The monoglycerides according to the invention normally contains more than more than 80% of a specific fatty acid, preferably over 90%. and b) fatty acids of the general formula CH.sub.3 --(CH.sub.2).sub.n--COOH where "n" may be varied between 4 and 22, preferably 8 to 18 and where the acyl chain may contain one or more unsaturated bonds.

The formulation according to the invention may comprise additional pharmaceutical excipients selected from the one or several of the following groups; preservatives and osmotic pressure controlling agents, pH-controlling agents, organic solvents, hydrophobic agents, enzyme inhibitors, water absorbing polymers, surfactants and absorption promoters, anti-oxidative agents, and the like.

The formulation according to the invention may comprise any antigen and/or vaccine selected among all the antigen and/or vaccines relevant to humans or animals, including marine animals.

This invention discuss lipids which, when mixed with antigens, enhance the immune activity against the antigens thereby functioning as an adjuvant in various vaccine formulations. Especially the invention comprise the use of a formulation for vaccination of the mucosa which can be immunologically activated by nasal, oral, vaginal or rectal administration. The invention also comprise the use of the lipid system for parenteral administration. The use of an adjuvant such as described in the present invention, which can be used both for parenteral as well as for mucosal administration is not limited to humans. Equally important is the use within the veterinary field for the immunization of e.g. cattle, pigs, chickens and the like. Furthermore, there is a large and growing interest in applying both parenteral as well as mucosal vaccines in the field of fish farming. In this area the administration can be performed by incorporation of the formulation in the food. Furthermore, the fish may be allowed to swim for a limited period of time in the vaccine formulation containing the antigens and the adjuvants thus being immunized by the mucosal route via the gills.

In the scientific literature there are reports showing how to enhance the uptake of a biologically active substance after administration to the mucosa together with certain lipids.

As an example Li & Mitra (Pharm. Res. vol 13:1, 1996) describes the administration of insulin mixed with phospholipids in the form of liposomes to the lung. They show that the effect is dependent on the length of the acyl chain and the charge of the particle. Optimal length was 10 carbon atoms and the charge preferably positive. Even negatively charged particles were effective but neutral system were inferior.

In the same way de Haan et al (Vaccine, 13:2, 155 62, 1995) describes a mixture of liposomes and the antigen hemeagglutinin. The mixture was administrated nasally to rats whereafter a positive immunological response could be detected. Gupta et al (Vaccine, 14:3, 219 25, 1995) describes that a mixture of diphtheria toxoid together with a non-phospholipid based liposom system administrated parenterally to rabbits results in an immune response which was at the same level as the marketed product which was Alum-adsorbed diphtheria toxoid.

A number of scientific reports also show that good immunological reponses are obtained after administration of liposomes to the mucosa where the antigen is entrapped or adsorbed to liposomes.

Studies in vitro on a human cell line obtained from a colon cancer (Caco-2) shows that the best penetrating effect, tested with the model substance mannitol, can be seen with a chain length of 10 carbon atoms. In this case the lipids consisted of the salts of fatty acids. The obtained mixture of these lipids forms together with water micelles (Lindmark et al, J.Pharm.Exp.Ther. 275, 958 65, 1995).

Liposomes consists of phospholipids and are formulated by a relatively lengthy and cumbersome process which i.a. involves organic solvents. Furthermore, the phospholipids are expensive.

As described below in the present invention, a similar immunological response can be obtained only by mixing the antigen with a lipid formulation which contains less complicated lipids having a substantially lower price and which can be formulated on a commercial basis in a very simple way.

Another systems that to some extent are similar to the present invention are formulations based on triglycerides. However, these systems are scientifically defined as emulsions of triglycerides where surfactants are used for stabilization. As stabilizers phospholipids or any other type of amphiphilic molecules such as Tween.RTM. are normally used. Furthermore, the appearance of such emulsions are normally milky, indicating a size of the oil droplets of about 1 .mu.m. It is well-known for the person skilled in the art that these surfactants are excellent adjuvants. Thus, the adjuvant properties of oil emulsions are primarily due to the characteristics of the surfactant and not of the triglyceride composition.

In PCT/DK94/00062 is disclosed a formulation for the topical administration of antigens and/or vaccines to mammals via the mucosal membranes. Said application disclose in the examples that the only formulation that enhances the immune response is a combination of caprylic/capric acid glycerides with polyoxyethylene sorbitan monoester (Tween 20.RTM.).

As exemplified in the present invention it is shown that a combination between a monoglyceride and a fatty acid can stimulate the immune system to produce antibodies and induce protective immunity. Furthermore the present invention shows that the disclosed formulation is able to produce high antibody titers by parenteral administration.

Thus, it was surprisingly found that the administration of antigens and/or vaccines to an animal either via the mucosal route or parenterally using a formulation comprising monoglycerides and/or fatty acids as a particulate lipid system can improve the immunological response towards the admixed antigens and/or vaccines. The monoglycerides are selected from a group with the general formula of 1-acyl-glyceride, wherein the number of carbon in the acyl chain may be varied between 8 and 24, preferably between 12 and 18. The acyl chain may be either saturated or unsaturated. The concentration of the monoglyceride may be in the range of 0.1 50 g per 100 ml of water, preferably in the range of 1 20 g per 100 ml of water. The fatty acid concentration may be in the range of 0.1 50 g per 100 ml formulation, preferably in the range of 1- 20 g per 100 ml water. When monoglycerides and fatty acids are formulated together the percent ratio of monoglyceride in fatty acid may be varied between 1 to 99%, preferably between 10 to 90%.

An enhancement of the immunological response after administration of monoglycerides and/or fatty acids together with antigens and/or vaccines has not been suggested anywhere in the prior art.

The present invention describes that mixtures of antigens with relevant lipids stimulates the body to generate protective immunity. Another advantage of the present invention is the simple formulation process and as compared to entrapment no material (antigen) is lost in the process. As an example can be mentioned that in the process of entrapment in liposomes the recovery is normally 10 20%. The rest is lost in the process.

Reports in the literature as discussed above, shows that by mixing liposomes and antigen an immune response is detected after administration to the mucosa.

However, the examples in this invention as described below shows that the system can be even more simplified by the use of lipids that are more stable, cheaper and which can be formulated to particles in a more convenient and simplified way.

The invention is exemplified by the following examples showing that the principle of co-administration of antigens, immune stimulating substances associated or in combination with particles function as an adjuvant.
 

Claim 1 of 7 Claims

1. A method of immunizing a human or animal, the method comprising administering to a human or animal by mucosal administration a vaccine composition comprising an adjuvant consisting essentially of: i) monoolein ii) oleic acid and iii) water wherein the concentration of i) is from 0.1 g to 50 g per 100 ml of water, and the concentration of ii) is from 1 g to 50 g per 100 ml of water, and with the proviso that the percent weight ratio of i) in ii) is between 10 to 90, and an immunogenic quantity of an antigen component selected from the group consisting of diphtheria toxoid, influenza and rota virus antigen.

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