United States Patent:  6,017,513

Assignee:   Biovector Therapeutics, S.A. (Labege Cedex, FR)

Filed:  December 9, 1997

A novel method for the mucosal administration of a substance to a mammal is provided. The method comprises contacting a mucosal surface of the mammal with the substance in combination with a Biovector. The Biovector has a core that comprises a natural polymer, or a derivative or a hydrolysate of a natural polymer, or a mixture thereof. A preferred natural polymer is a polysaccharide or an oligosaccharide. The core is optionally coated with an amphiphilic compound, such as a lipid.

DETAILED DESCRIPTION OF THE INVENTION

In the description of the invention below, the following interpretations will apply. The word "comprise" followed by an element of the invention used in describing an embodiment of the invention means that the embodiment includes, but is not necessarily limited to, that element. The embodiment may include other members of the same element or other elements as well. An element disclosed in the singular, i.e. "substance," does not preclude the presence of more than one element, i.e. "substances." All numbers are approximate, unless the language of the specification or its context indicates otherwise.

It has unexpectedly been discovered that Biovectors, as described in International PCT Application WO 94/23701, WO 94/20078, and WO 96/06638, are particularly well suited for the mucosal administration of substances to mammals, including farm animals, pet animals, laboratory animals, and humans. The mucosa refers to the epithelial tissue that lines the internal cavities of the body. For example, the mucosa comprises the alimentary canal, including the mouth, esophagus, stomach, intestines, and anus; the respiratory tract, including the nasal passages, trachea, bronchi, and lungs; and the genitalia. For the purpose of this specification, the mucosa will also include the external surface of the eye, i.e. the cornea.

The substance in combination with the Biovector may be added to any mucosal surface. Some particularly suitable mucosal surfaces include, for example, the nasal, buccal, oral, vaginal, ocular, auditory, pulmonary tract, urethral, digestive tract, or rectal surface.

The cross-linked polysaccharide or oligosaccharide preferably binds non-specifically to the mucosal surface. Applicants have unexpectedly discovered that non-specifically binding polysaccharides and oligosaccharides in accordance with the invention make superior carriers for delivering substances to mucosal surfaces. This discovery is surprising since, as mentioned above, European Patent 352 295 of Access Pharmaceuticals reported the requirement for a multivalent binding agent specific for endothelial surface determinants in carriers for drugs and diagnostic agents.

Properties of Biovectors

The Biovector comprises a core of a natural hydrophilic polymer, such as, for example, a cross-linked polysaccharide or a cross-linked oligosaccharide, or a derivative or hydrolysate of a cross-linked polysaccharide or a cross-linked oligosaccharide, or a mixture thereof. The polysaccharide or oligosaccharide may be naturally cross-linked or may be chemically cross-linked by methods known in the art. Some suitable chemical cross-linking methods include, for example, contacting the polysaccharide or oligosaccharide with a multi-functional agent, such as epichlorohydrin or phosphorous oxychloride. The minimum molar ratio of cross-linking agent to glucose residue may be, for example, 1:15, 1:12, or 1:10 in the case of phosphorous oxychloride and 1:50, 1:40, or 1:30 in the case of epichlorohydrin. The maximum molar ratio of cross-linking agent to glucose residue may be, for example, 1:0.5, 1:0.7, or 1:1 in the case of phosphorous oxychloride and 1:2, 1:3, or 1:5 in the case of epichlorohydrin. For epichlorohydrin, a preferred range of ratios of cross-linking agent to glucose residue is 1:15 to 1:7. For phosphorous oxychloride, a preferred range of ratios of cross-linking agent to glucose residue is 1:7 to 1:2. When phosphorous oxychloride is used as the multi-functional agent, the cross-linked product preferably comprises approximately 0.1 to 3.0 mmole phosphate/gram, preferably 0.4 to 1.0 mmole phosphate/gram, of final product.

Some suitable examples of naturally cross-linked polysaccharides include, for example, cellulose and its derivatives. Some suitable examples of chemically cross-linked polysaccharides include, for example, epichlorohydrin cross-linked starch, i.e. degradable starch microspheres (DSM), and epichlorohydrin cross-linked dextran, i.e. Sephadex.

The polysaccharides or oligosaccharides useful in the present invention may be derived from any saccharide monomer. Glucose is the preferred monosaccharide. The polymers or oligomers may be formed from the monomers in either the .alpha. or .beta. orientation, and may be linked at the 1-4 or 1-6 positions of each saccharide unit The polysaccharides or oligosaccharides preferably have a molecular weight between 1,000 to 2,000,000 daltons, preferably 2,000 to 100,000 daltons, and most preferably 3,000 to 10,000 daltons.

The preferred polysaccharides are starch (glucose .alpha. 14 polymers) and dextran (glucose .alpha. 1-6 polymers derived from bacteria). Starch is especially preferred. Starch from any of the well known sources of starch is suitable. Some suitable sources of starch include, for example, potato, wheat, corn, etc. Other suitable polysaccharides include, for example, pectins, amylopectins, chitosan, and glycosaminoglycan.

The cross-linked polysaccharides or oligosaccharides may also be derivatives of hydrolysates of the cross-linked polysaccharides or oligosaccharides mentioned above. Some preferred hydrolysates of starch include, for example, acid hydrolyzed starch, such as dextrins, or enzyme hydrolyzed starch, such as maltodextrins. The hydrolysis degree of the polysaccharide or oligosaccharide is determined by the reducing power of the hydrolysate, commonly expressed as the Dextrose Equivalent (DE) . The DE range preferably varies between 2 to 20, preferably 2 to 12.

An ionic group (0 to 3 milliequivalents, preferably 0 to 2 milliequivalents, of ionic charge per gram) is optionally grafted to the cross-linked polysaccharide or oligosaccharide. The ionic group may be an anionic group or a cationic group. The Biovectors preferably have a minimum of 0.2, 0.4, 0.6, or 0.8 milliequivalents of ionic charge per gram of polysaccharide core, and a maximum of 1.2, 1.4, 1.6, or 1.8 milliequivalents of ionic charge per gram of polysaccharide core. Methods are known in the art for grafting ionic groups to polysaccharides and oligosaccharides.

The cross-linked polysaccharide or oligosaccharide may be made anionic by grafting a negatively charged or acidic group. Some suitable anionic groups grafted to the polysaccharide or oligosaccharide include, for example, phosphate, sulfate, or carboxylate. The anionic group may be grafted by treating the polysaccharide or oligosaccharide with an activated derivative of a polyhydric acid, such as phosphoric acid, sulfuric acid, succinic acid, or citric acid. Activated derivatives of polyhydric acids include, for example, acyl halides, anhydrides, and activated esters. The preferred anionic group is phosphate grafted via treatment with phosphorous oxychloride. A Biovector to which a phosphate group is grafted is referred to as SMBV-P.

The polysaccharide or oligosaccharide may be made cationic by grafting a ligand that comprises a positively charged or basic group. Some suitable cationic groups grafted to the polysaccharide or oligosaccharide include, for example, quaternary ammonium ions, and primary, secondary, or tertiary amines. Some suitable ligands that can be grafted to the polysaccharide or oligosaccharide include, for example, choline, 2-hydroxypropyltrimethylammonium, 2-dimethylaminoethanol, 2-diethylaminoethanol, 2-dimethylaminoethylamine, and 2-diethylaminoethylamine. These ligands may be conveniently grafted to the polysaccharide or oligosaccharide by methods known in the art, such as, for example, by contacting the polysaccharide or oligosaccharide with a suitable derivative of the respective alkyl group, such as a chloride, bromide, iodide, or epoxide.

Another suitable method for grafting cationic groups to the polysaccharide or oligosaccharide includes grafting a polyhydric acid, as described above, and then using a free acid group, such as a free carboxylate group, to graft the basic ligand via, for example, an amide or ester bond. Amino acids are conveniently grafted this way. Some suitable examples of amino acids include, for example, glycine, alanine, glutamic acid or aspartic acid.

The preferred cationic group is quaternary ammonium. A Biovector to which a quaternary ammonium group is grafted is referred to as SMBV-Q.

It should be noted that Illum et al., International Journal of Pharmaceutics 39, 189-199 (1987), have reported finding no detectable amount of model drugs released from a cationic dextran microsphere, DEAE-Sephadex. Illum et al. attribute this lack of release to binding of the model drug to the cationic binding sites in the microsphere matrix. Applicants have, however, unexpectedly found efficient release of substances from polysaccharides, to which cationic groups have been grafted.

Optionally, the polysaccharide or oligosaccharide core of the Biovector is covalently bonded to a layer of lipid compounds. The layer of lipid compounds may coat the polysaccharide or oligosaccharide core either partially or completely. The lipid layer preferably comprises natural fatty acids, as described in International PCT Application WO 94/23701.

The cross-linked polysaccharide or oligosaccharide, either with or without a lipid layer, may also optionally be partially or completely coated with an outer layer one or more amphiphilic compounds. Such Biovectors are referred to as light Biovectors or L-SMBV. Biovectors consisting only of a core of cross-linked polysaccharide or oligosaccharide are referred to as core Biovectors.

The amphiphilic coating preferably adheres to the cross-linked polysaccharide or oligosaccharide, or to the optional lipid layer, by means of non-covalent bonds, such as by means of ionic or hydrogen bonds. The amphiphilic compounds suitable for the coating are selected to confer a physico-chemical environment appropriate to the substance, the mode of mucosal administration, and the desired effect.

The amphiphilic coating may comprise any amphiphilic compound that can be adsorbed on the surface of the core of the Biovector. Preferably, the amphiphilic coating comprises mainly a natural or synthetic phospholipid or ceramide, or a mixture thereof.

The phosphate group of the phospholipid may optionally be grafted to ionic or neutral groups. Some suitable phospholipids include, for example, phosphatidyl choline, phosphatidyl hydroxycholine, phosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl glycerol. A preferred phospholipid is dipalmitoyl phosphatidylcholine (DPPC).

The amphiphilic coating may also comprise a derivative of a phospholipid or ceramide. Some suitable derivatives of phospholipids include PEG-phospholipids, and phospholipids grafted to other molecules or polymers.

The amphiphilic coating may also comprise other amphiphilic compounds, either by themselves or in combination with the phospholipids, ceramides, or derivatives described above. Some suitable examples of such other amphiphilic compounds include poloxamers, modified polyoxyethylene, and other detergents and surface active compounds.

Additional compounds and mixtures thereof may be added to the phospholipids or ceramides in the amphiphilic coating. Some examples of such additional compounds include fatty acids, steroids (such as cholesterol), triglycerides, lipoproteins, glycolipids, vitamins, detergents, and surface active agents.

The preparation of Biovectors may normally be conveniently carried out, either as a simple one-step process (in case of a core Biovector) or a as a two step process: the core is first prepared and then is coated with an amphiphilic compound to create a light Biovector.

The size of the Biovector is an important element of the present invention. For example, Illum et al. have emphasized the importance of microspheres having a size larger than 10 .mu.m for nasal delivery. See Illum et al., International Journal of Pharmaceutics 39, 189-199 (1987).

Applicants have, however, unexpectedly found that Biovectors much smaller than 10 um are highly efficient carriers for administering substances to the nasal mucosa, as well as to other mucosa. The Biovectors of the present invention preferably have a minimum diameter of about 20 nm, more preferably about 30 nm, and most preferably about 40 nm. The maximum size of the Biovectors is about 200 nm, more preferably about 150 nm, and most preferably about 100 nm. The optimal size of the Biovector is between 60-90 nm, and most optimally about 80 nm.

The relatively small size of the Biovectors confers various advantages, making the Biovectors even more suitable for administration to the mucosa. For example, the Biovectors have larger relative surfaces and volumes than larger nanospheres and microspheres. In addition, the small size of the Biovectors permit convenient sterilization by microfiltration, thereby avoiding the need for preservatives.

The Biovectors can be administered in various forms. For example, the Biovectors can be administered in dispersed form, such as suspensions or gels. The Biovectors can also be produced in dry form by methods known in the art, and administered in a suitable metered-dosing device.

For example, a suspension or gel of dispersed Biovectors can be dried by lyophilization or spray drying. All light Biovectors, such as anionic and cationic light Biovectors, as well as all core Biovectors, such as anionic and cationic core Biovectors, can be dried. The Biovectors may be administered in dry form, or may be resuspended (i.e. rehydrated) in a suitable medium, preferably a pharmaceutically acceptable aqueous liquid or gel, and administered. For the purposes of this application, resuspended Biovectors mean Biovectors that have been dried and resuspended in a suitable medium.

Substances for Mucosal Administration

The substance administered to a mammal in combination with a Biovector in accordance with the present invention may be any substance that is administered to a mammal. Some suitable substances include, for example, therapeutic agents, prophylactic agents, and diagnostic agents. A substance may be introduced for more than one purpose, such as, for example, as combination therapeutic and prophylactic agents, prophylactic and diagnostic agents, and therapeutic and diagnostic agents.

The therapeutic agent may be any composition of matter used in the treatment of diseases and conditions that afflict mammals. Some suitable examples of therapeutic agents include a radiopharmaceutical, an analgesic drug, an anesthetic agent, an anorectic agent, an anti-anemia agent, an anti-asthma agent, an anti-diabetic agent, an antihistamine, an anti-inflammatory drug, an antibiotic drug, an antimuscarinic drug, an anti-neoplastic drug, an antiviral drug, a cardiovascular drug, a central nervous system stimulator, a central nervous system depressant, an anti-depressant, an anti-epileptic, an anxyolitic agent, a hypnotic agent, a sedative, an anti-psychotic drug, a beta blocker, a hemostatic agent, a hormone, a vasodilator, a vasoconstrictor, a vitamin, etc.

The prophylactic agent that is administered to a mammal in combination with a Biovector according to the invention may be any prophylactic agent used for preventing or reducing the effect of any disease or condition that afflicts mammals by any mechanism. For example, the prophylactic agent may be an antigen used in a vaccine against a pathogen. The pathogen may, for example, be a virus or a microorganism, such as a bacterium, a yeast, or a fungus. The virus may, for example, be an influenza virus, such as Haemophilus influenzae; a cytomegalovirus; HIV; a papilloma virus; a respiratory syncytial virus; a poliomyelitis virus; a pox virus, such as chicken pox virus (i.e. varicella zoster virus); a measles virus; an arbor virus; a Coxsackie virus; a herpes virus, such as herpes simplex virus; a hantavirus; a hepatitis virus, such as hepatitis A, B, C, D, E, or G virus; a lyme disease virus, such as Borrelia burgdorferi; a mumps virus, such as Paramyxovirus; or a rotavirus, such as A, B, or C rotavirus. Particularly good results have been obtained with vaccines against influenza virus and HIV.

A bacterium against which a vaccine according to the present invention is effective may be any bacterium capable of causing disease in mammals. For example, the bacterium may be a member of the genus Neisseria, such as N. gonorrhoeae and N. meningitidis; Aerobacter; Pseudomonas; Porphyromonas, such as P. gingivalis; Salmonella; Escherichia, such as E. coli; Pasteurella; Shigella; Bacillus; Helibacter, such as H. pylori; Corynebacterium, such as C. diphteriae; Clostridium, such as C. tetanii; Mycobacterium, such as M. tuberculosis and M. leprae; Yersinia, such as Y. pestis; Staphylococcus; Bordetella, such as B. pertussis; Brucella, such as B. abortus; Vibrio, such as V. cholerae; and Streptococcus, such as mutants Streptococci.

Other pathogens against which a vaccine according to the present invention is effective include, for example, a member of the genus Plasmodium, such as the species that causes malaria; a member of the genus Schisostoma, such as the species that causes Schisostomiasis or Bilharzia; and a member of the genus Candida, such as C. albicans.

The substance that can be combined with a Biovector may be a diagnostic agent. The diagnostic agent may be any composition of matter that is introduced into a mammal for the purpose of detecting any disease or condition, or to detect the concentration of a different substance added to the mammal, such as a drug or a vaccine. For example, the diagnostic agent may be a contrast agent or an imaging agent, including a magnetic imaging agent, that is capable of detecting an organ or other internal part of the body of the mammal. Alternatively, the diagnostic agent may be capable of detecting irregularities within the mammal, such as irregularities of the cornea, the respiratory tract, the digestive tract, the auditory canal, the urethra, the rectum, or any other part of a mammal containing a mucosal membrane.

For the above purposes, the diagnostic agent is advantageously labeled with a detectable group. The detectable group may, for example, be a radioactive group; a fluorescent group, such as, for example, fluorescene; a visible group, such as, for example, a marker dye; or a magnetic group, preferably suitable for magnetic resonance imaging.

The substance to be delivered in combination with a Biovector may, for example, be a small chemical molecule or a biological molecule. A small chemical molecule is usually a non-polymeric molecule that may or may not occur naturally in the mammal to which it is administered. The small chemical molecule may, for example, be an organic molecule, an inorganic molecule, or an organo-metallic molecule. Some examples of small chemical molecules include steroids, porphyrins, nucleotides, nucleosides, etc. as well as mixtures, and derivatives thereof.

Biovectors are particularly effective in delivering biological molecules to the mucosa For the purposes of this specification, a biological molecule is a polymer of a type that occurs in nature, or a monomer or moiety thereof. Such polymers typically comprise monomers such as amino acids, nucleosides, nucleotides, and saccharides, and mixtures thereof. Some structural classes of biological molecules include, for example, amino acids, peptides, proteins, glycoproteins, and lipoproteins; proteoglycans; monosaccharides, oligosaccharides, polysaccharides, and lipopolysaccharides; fatty acids, including eicosanoids; lipids, including triglycerides, phospholipids, and glycolipids.

Additional biological molecules that can be delivered to the mucosa by means of Biovectors include nucleotides, nucleosides, and nucleic acid molecules, including DNA and RNA polymers and oligomers. The nucleic acids may be, for example, ribozymes and antisense oligonucleotides. Nucleic acids may be administered for their own diagnostic or therapeutic potential, or for their ability to be expressed in connection with gene therapy.

Some functional classes of biological molecules include, for example, cytokines, growth factors, enzymes, antigens, (including epitopes of antigens and haptens), antibodies, hormones (including both natural and synthetic hormones and their derivatives), co-factors, receptors, enkephalins, endorphins, neurotransmitters, and nutrients. Some specific examples of biological molecules include, for example, insulin, an interferon, such as an .alpha.-, .beta.-, or .gamma.-interferon; an interleukin, such as any of IL-1 to IL-15; any of the interleukin receptors, such as IL-1 receptor; calcitonin; growth factors, such as erythropoietin, thrombopoietin, epidermal growth factor, and insulin-like growth factor-1.

Administration of the substance in accordance with the present invention may be accompanied by one or more supplementary compound for enhancing the activity, properties, or marketability of the substance. For example, adjuvants that enhance the absorption efficiency of the mucosa are known in the art. Some examples of such mucosa absorption enhancers include, for example, bile salts, such as sodium glycocholate, and surfactants, such as polyoxyethylene-9-lauryl ether. Adjuvants for enhancing the immunogenicity of antigens are also known. Some examples of immunogenicity enhancers include, for example, MPL, Quil A, QS 21, LPS, endotoxins, CTB, and BCG. Some additional supplementary compounds include, for example, disinfectants, preservatives, surfactants, stabilizing agents, chelating agents, and coloring agents.

Another important feature of the present invention is the flexibility in administering substances to the mucosa. For example, unlike most other pharmaceutical carriers, the present invention provides for the delivery of more than one substance per Biovector to be delivered to a mucosal surface.

There is also flexibility in where the one, or more than one, substance is located in the Biovector. For example, the one, or more than one, substance may be located in the inner core of the cross-linked polysaccharide or oligosaccharide. Alternatively, the one, or more than one, substance may be located at the outer surface of the cross-linked polysaccharide or oligosaccharide.

If the cross-linked polysaccharide or oligosaccharide is coated with an amphiphilic layer, the one, or more than one, substance may be located in the inner core of the amphiphilic compound layer. Alternatively, the one, or more than one, substance may be located at the outer surface of the amphiphilic compound layer.

If more than one substance per Biovector is administered to a mammal, some or all of the substances may be located in the same part of the Biovector. Alternatively, some or all of the substances may be located in the different parts of the Biovector.

Methods are known for directing substances to various parts of Biovectors. See International PCT Application WO94/20078.

As with other carriers, the substance may be pre-loaded in a Biovector, and the loaded Biovector stored prior to administration to the mammal. Preferably, however, the substance is post-loaded on an empty Biovector just prior to packaging or, such as in the case of labile substances for example, the Biovector may be used as the dilution media for entraping the substance just prior to administration to the mammal. Methods are known for pre-loading and post-loading Biovectors. See, for example, International PCT Applications WO 94/20078, WO 94/23701, and WO 96/06638 of Biovector Therapeutics S.A.

Advantages of Mucosal Administration with Biovectors

Some of the advantages of the mucosal administration of substances to mammals may be seen by reference to the examples below. These advantages are described for illustrative reasons only. The present invention is not, however, in any way limited by the examples.

As shown in the experiment described in Example II, for example, the ionic groups permit the mode of administration of Biovectors to be varied according to the requirements of a particular case. The protocol is described in detail in Example II. Briefly, three cationic formulations and three anionic formulations of ¹⁴ C-labeled Biovectors were administered intranasally to rats. At various times, the rats were sacrificed, and the percent of the label remaining in the nasal cavity and in the plasma was determined.

The results of this experiment, which are shown in FIG. 1, demonstrated that approximately 30% of the dose of three cationic Biovectors administered intranasally to rats remained in the nasal cavity five minutes after administration, and was still present after twelve hours.

The good mucoadhesion of the cationic Biovectors increased the residence time of the Biovector in the target mucosa. The increased residence time is important where increased bioavailability or a local effect of the administered substance is desired. A local effect of the administered substance is desired under a variety of circumstances.

For example, a local effect is desired when an antibiotic or antiviral drug is administered to treat a local bacterial or viral infection. Alternatively, a local effect is desired when a vaccine is administered to protect a mammal against a mucosal infection by a microorganism or virus. A third example of a situation where one desires a local effect is the administration of a diagnostic agent to image an organ that contains a mucosal membrane.

By contrast, the anionic Biovectors (SMBV-P1, SMBV-P2, and SMBV-P3), which exhibit comparable initial mucoadhesion (five minutes), have a more rapid clearance from the nasal mucosa than the cationic Biovectors. With the anionic Biovectors, less than 10% of the dose remaining five minutes after administration was found in the nasal cavities three hours after administration. There was no significant variation for the three anionic formulations tested.

A significant amount of labeled anionic Biovectors was, however, found in the plasma three hours and, to a lesser extent, six hours after nasal administration of SMBV-P1, SMBV-P2, and SMBV-P3, respectively. See Example II and FIG. 2. Therefore, anionic Biovectors are of particular use when a systemic response is desired.

In general, there are advantages in using positively charged Biovectors for administering Biovectors that have enhanced mucosal residency times. There are advantages in administering negatively charged Biovectors that have enhanced ability to pass through the mucosa to the blood stream. The advantages of both charge types of Biovectors can be combined by administering a mixture of a positively charged Biovector and a negatively charged Biovector.

The results of Example III confirm that in-vivo behavior of anionic Biovectors (SMBV-P1, SMBV-P2, and SMBV-P3) is different from that of cationic Biovectors (SMBV-Q1, SMBV-Q2, and SMBV-Q3). In this experiment, rats treated in accordance with the protocol of Example 2 were sacrificed after twelve hours, and the ¹⁴ C remaining in various organs was measured.

As expected, the relatively large amounts of ¹⁴ C from cationic Biovectors found in the nasal cavities, nasal cavity washings, and bronchi indicate an increased residence time of cationic Biovectors in the mucosa in which, or near which, the Biovectors are administered. For the anionic Biovectors, the significant amount of ¹⁴ C found in the liver and kidney demonstrates the increased trans-mucosal passage of the Biovectors into the bloodstream.

The large amount of ¹⁴ C from both cationic and anionic Biovectors found in the small and large intestine indicates that elimination of Biovectors following nasal administration occurred through the digestive tract. The increase in the residence time of Biovectors in the digestive tract is especially significant for the oral administration of antigens associated with Biovectors in the case of oral vaccination.

Further evidence for the good mucoadhesion of the cationic Biovectors is demonstrated by the results shown in Example IV. In this experiment, fluorescein-labeled cationic light Biovectors as either dispersed or resuspended suspensions were administered intranasally to rats. Approximately 20% of the resuspended Biovectors adhere to the mucosa upon administration, and the same amount remains for at least twelve hours. The dispersed Biovectors do not adhere to the nasal mucosa after three hours, except at low levels. Approximately one third of the administered fluorescent Biovectors are still found in suspension in the nasal washing five minutes after administration, but none is found six hours later.

Example V provides important evidence of the superiority of Biovectors in the mucosal administration of vaccines. In this experiment, a comparison was made between the intranasal (i.n.) administration of a monovalent split antigen of hemagglutinin (HA) and neuraminidase (N) prepared from viral membranes in cationic light Biovectors with the intranasal and subcutaneous (s.c.) administration of antigen alone. The experiment demonstrates that the antigen administered i.n. in a Biovector is able to elicit a superior mucosal and seric response.

Thus, the total IgG, specific IgG and inhibitory hemagglutination were at the same order of magnitude when the antigen was administered i.n. in a Biovector compared to antigen administered s.c. alone. However, the antigen/Biovector formulation induces the production of circulating and secretory IgA, while the antigen alone administered s.c. or i.n., for practical purposes, did not.

Moreover, the ratio of specific IgG to total IgG in the nasal washing was twice as high when the antigen was administered i.n. in a Biovector than when the antigen was administered alone s.c. A higher ratio means that the immune response is expected to be more specific and more protective. While not wishing to be bound by any theory, applicants believe that membrane antigens such as those used in this experiment are presented by the outer layer of the Biovector, creating a lipid surrounding favorable for presenting the antigen to the immune system.

The experiment described in Example VI compares the effect of different formulations of the gp160 protein of HIV on the mucosal immune response of rabbits. The protein was administered with two formulations of a positively charged light Biovector, a dispersed formulation and an resuspended formulation. As a control, the protein was administered in combination with a potent mucosal adjuvant, subunit B of cholera toxin (CTB). In each of the three cases, a series of immunizations were made at thirty day intervals. The first two immunizations were vaginal, the second two immunizations were oral, and the final immunization was intramuscular.

The results showed that the Biovectors were at least as efficient as CTB in inducing specific IgA secretions in the vagina and in saliva ten days after the second vaginal administration, (D₄₀). The resuspended SMBVs induced a 50% increase of the IgAs when compared to formulations of the antigen with CTB or in dispersed SMBV.

It should be noted that vaginal administration of the antigen induced secretion of :specific IgAs in the saliva as well as in the vagina. Thus, the antigen, which entered the MALT (mucosal-associated lymphoid tissue) at the vaginal level, induced the secretion of IgAs in situ. In addition, the Biovector formulations were able to stimulate a robust IgA response in the saliva by entering the so-called "common mucosal immune system."

The experiment described in Example VII compares the intranasal immunization of mice with influenza hemagglutinin in a control formulation with that of four formulations of light Biovectors: dispersed and positively charged, dispersed and negatively charged, resuspended and positively charged, and resuspended and negatively charged. The effect of pre-loading and post-loading each Biovector formulation on the relative serum IgG titer after 28 days was measured. In addition, a comparison of the relative titer obtained by administering the pre-loaded Biovectors to animals that were awake with that obtained by administering the pre-loaded Biovectors to animals that were anesthetized was made.

As expected, the control subunit antigen without any carrier or adjuvant is not very immunogenic when administered intranasally to mice, either anesthetized or awake. Of the SMBV subgroups, the positively charged and dispersed Biovectors showed a significant improvement (by more than an order of magnitude) of the titer over those obtained with the antigen alone or other Biovector formulations. Both the pre-loaded and post-loaded Biovectors have generally comparable effects. This versatility of the Biovector can be of particular interest, allowing either a mixing of the active substance with the Biovector upon administration, or integration of the active substance with the Biovector prior to its use.

Surprisingly, the anesthetized animals did not show a significant increase in antibody titers, suggesting that the deposition, if any, of the antigen in the lower respiratory tract or the lung had little biological effect.

Claim 1 of 48 Claims

1. A method for the mucosal administration of a substance to a mammal, the method comprising contacting a mucosal surface of the mammal with the substance in combination with a Biovector core, wherein the Biovector core comprises a natural polymer or a hydrolysate of a natural polymer, or a mixture thereof, and wherein the core is uncoated; or is partially or completely coated with no more than one layer, the layer comprising a lipid compound covalently bonded to the core, or an amphiphilic compound.

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