Title: Oral therapy for the treatment of allergies and method of manufacture
United States Patent: 6,174,529
Inventors: Michael; J. Gabriel (Cincinnati, OH); Litwin; Allen (Cincinnati, OH)Assignee: University of Cincinnati (Cincinnati, OH)
Appl. No.: 947551Filed: October 11, 1997
An orally administrable therapeutic protein is provided by combining the therapeutic protein with a stabilizing agent in an aqueous solution. The solution is coated onto nonpareils and microencapsulated with a water emulsifiable enteric coating composition. The microcapsules are orally administered. The coating protects the protein as it passes through the stomach. Upon reaching the small intestines, the basic pH of the intestinal juices will dissolve the coating, allowing the protein to be released and induce antigen specific immune response which has the specificity of the native molecule. The stabilizing agent protects the therapeutic protein from denaturation during the encapsulation process. In addition to being immunogenic, when administered orally, encapsulated allergen has a therapeutic effect in the treatment of human allergies.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, an orally
administrable therapeutic agent such as a protein or protein containing
virus or bacteria is formed by microencapsulating the therapeutic protein
with an enteric~ coating. This is generally referred to as the therapeutic
protein.
The therapeutic agents are dispersed in an aqueous solution. The aqueous
solution is then sprayed onto nonpareils. Subsequently the microspheres
are coated with a water emulsion of a polymer which upon solidification is
acid resistant. This protects the therapeutic protein as it passes through
the stomach and releases it into the small intestines where it can act
upon the lymphoid tissue.
For purposes of the present invention, "therapeutic protein"
will include allergenic proteins and digested fragments thereof. These
include pollen allergens from ragweed, rye, Junegrass, orchard grass,
sweet vernal grass, redtop grass, timothy grass, yellow dock, wheat, corn,
sagebrush, bluegrass, California annual grass, pigweed, bernuda grass,
Russian thistle, mountain cedar, oak, box elder, sycamore, maple, elm,
etc., dust and mites, bee venom, food allergens, animal dander, and insect
venoms.
Further, any of these allergens digested according to the method disclosed
in U.S. Pat. No. 4,469,677, the disclosure of which is incorporated herein
by reference, are also suitable for use in the present invention. This
basically proteolytic enzymatic digestion of allergens. Accordingly,
polypepfides formed by such proteolytic enzymatic digestion are also
suitable for use as therapeutic proteins for use in the present invention.
Therapeutic proteins include microbial vaccines which include viral,
bacterial and protozoal vaccines and their various components such as
surface antigens. These include vaccines which contain glycoproteins,
proteins or peptides derived from these proteins. Such vaccines are
prepared from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus
pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Salmonellae
species, Shigellae species, Escherichia coli, Klebsiellae species, Proteus
species, Vibrio cholerae, Helicobacter pylori, Pseudomonas aeruginosa,
Haemophilus influenzae, Bordetella pertussis, Branhamella catarrhalis,
Mycobacterium tuberculosis, Legionella pneumophila, Pneumocystis carinjii
Treponema pallidum and Chlamydiae species, tetanus toxoid, diphtheria
toxoid, influenza viruses, adenoviruses, paramyxoviruses, rubella viruses,
polioviruses, hepatitis viruses, herpesviruses, rabies viruses, HIV-1
viruses, HIV-2 viruses, and papilloma viruses. Other therapeutic proteins
include those used for the treatment of autoimmune disease and to prevent
transplant rejection.
In obtaining bacteria preparations, it is preferable to employ lyophilized
bacteria which can be purchased or obtained by growing the bacteria,
killing them with heat, washing them, followed by lyophilization.
Autxoimmune disease is a disease in which the body produces an immunogenic
response to some constituent of its own tissue. An autoimmune disease can
be classified into those which predominantly affect one organ, such as
hemolytic anemia and chronic thyroiditis, and those in which the
autoimmune disease process is diffused through many tissues, such as
multiple sclerosis, systemic lupus erythematosus, and arthritis. Exemplary
autoimmune diseases and corresponding auto antigens include:
Autoimmune disease Therapeutic Protein
Multiple Sclerosis Myelin basic protein
Myasthenia Gravis Acetyl choline receptor
Rheumatoid Arthritis Type II collagen
Diabetes Mellitus Insulin
Juvenile Diabetes Mellitus Insulin
Autoimmune Thyroiditis Thyroid proteins
One of the primary problems with transplanting organs is rejection of the organs. The immune system of the recipient can be treated to reduce rejection by use of a therapeutic protein. The therapeutic protein itself is the major histocompatibility complex (MHC) protein. MHC proteins are divided into two major groups: MHC I and MHC II. Either or both may serve as a therapeutic protein, as well as peptides derived therefrom, i.e., fragments or synthetic peptides derived from known amino acid sequences of the protein.
A second component which can be added to the therapeutic protein is a stabilizing agent. Stabilizing agents provide physical protection for the protein. Generally these stabilizing agents are therapeutically inactive water soluble sugars such as lactose, mannitol and trehalose. These act to protect the therapeutic antigen during the coating process and passage through the gastrointestinal tract.
To form orally administrable microcapsules for use in the present invention, an aqueous solution of the therapeutic protein and the optional stabilizing agent is formed. The aqueous solution will include generally from about 0.5 to about 10% by weight of the therapeutic protein with about 1% being preferred, and from about 1% to about 10% by weight of the stabilizing agent with about 5% being preferred. It is desirable to add 1-10% of polyvinylpyrrolidone to bind the therapeutic protein to the nonpareil and act as a bioadhesive agent for the protein during the passage through the gastrointestinal tract.
Nonpareils are small, round particles of pharmaceutically inert materials. Generally nonpareils formed from the combination of sucrose and starch are preferred. One such brand is Nupareils which is sold by Ingredient Technology Corporation. The preferred size is 30-35 mesh.
The nonpareils are coated with the aqueous solution of the therapeutic protein, the stabilizing agent, and bioadhesive agent to provide a coating of 1-30% by weight on a solids basis. Glatt brand powder coater granulators such as the GPCG-1, GPCG-5, or GPCG-60 fluid bed coaters are suitable for use in this application. Coating conditions and times will vary depending on the apparatus and coating viscosity. But, generally all coating steps must be conducted at less than 50oC., preferably less than 35oC. to avoid denaturing the protein.
The protein coated microspheres are dried and subsequently coated with an acid stable polymer (enteric coating). Generally, the coating will be applied in the same manner as the protein with the same equipment.
The coating composition used in the present invention is preferably a water based emulsion polymer. The preferred coating is an ethylacrylate methacrylic acid copolymer sold under the trademark Eudragit L 30D manufactured by Rhom Pharma. This has a molecular weight of about 250,000 and is generally applied as a 30% aqueous solution. An alternate coating is hydroxypropylmethyl cellulose acetate succinate.
The coating composition can be combined with a plasticizer to improve the continuity of the coating. There are several well known plasticizers typically used. Triethyl-citrate (TEC) sold by Morfley Inc. is preferred. This can form about 1-30% of coating composition. Although plasticizers can be liquid, they are not considered to be solvents since they lodge within the coating altering its physical characteristics. They do not act to dissolve the protein. Any plasticizer which dissolves or denatures the protein would be unacceptable.
Talc (3.0% of coating composition) can also be added to prevent sticking between the particles if desired. Also, an antifoaming agent (0.0025 % of coating composition) such as sorbitan sesquioleate (Nikko Chemicals Company Limited) or silicone can be added. Both the talc and antifoaming agent are added only if needed.
The microspheres coated with the therapeutic protein and optional stabilizing and bioadhesive agents, are dried and are then coated with the enteric coating as previously described. The coating solution is about 30% polymer, 0-30% plasticizer, 0 to 3% talc and 0 to 0.0025% antifoaming agent and water. It is important that there be no organic solvents including alcohols and even glycols present in the coating composition. The presence of these solvents during coating application can denature the therapeutic protein. The coating is conducted in the same equipment used to coat the nonpareils with therapeutic protein. The temperature for this coating should be about 30oC. but less than 50oC.
In an alternate embodiment of the present invention, a therapeutically acceptable water dispersible aluminum compound such as aluminum sulfate or aluminum hydroxide are added to the aqueous dispersion or solution of protein prior to coating onto the nonpareil. This acts to increase immunogenicity of the proteins. Generally 1% to 10% of aluminum compound is added.
The enteric coated microspheres then can be placed in gel capsules for oral administration to humans. Dosage will depend on the individual and the course of the therapy. Generally, the dosages will be the same as dosages used for treatment when administered by injection. With transplant rejection, the dosage may vary greatly, depending on the patient's immune system. Generally, the dosage will be 0.1 to 100 mg administered daily starting about two weeks prior to transplant in order to induce a state of tolerance to a foreign graft (organ tissue or cell) and may continue post-transplant in order to maintain the tolerant state. Thereafter a lower maintenance dose can be administered daily.
For autoimmune treatment, the autoantigen, fragment, or analog is introduced orally in an amount of from 0.1 to 1000 mg per day, and may be administered in single dose form or multiple dose form. Preferably, the autoantigen, fragment or analog is administered in an amount of from 0.1 mg to 500 mg per day. As is understood by one skilled in the art, the exact dosage is a function of the autoantigen, the age, sex and physical condition of the patient, as well as other concurrent treatments being administered. Such preparations may be administered to an animal in need of treatment for such autoimmune disease so as to ameliorate, relieve, alleviate, reverse, or lessen the severity of the disease. Such preparations may also be administered to an animal who is predisposed to developing such autoimmune disease so as to prevent the onset of such disease or to lessen the severity of such disease when it does emerge.
The bacteria and viral dosage, again, is the same as the injected dosage--generally 10 .mu.g to 10 mg. A single dosage should be effective, however repeated lower dosages may be preferred to slowly build up the immunity.
The invention will be further appreciated in light of these following examples. In many of these examples OVA is tested in mice as a model. Human study with allergens has shown this to be quite indicative of human response. The mouse model is, of course, generally accepted in the study of infectious disease.
Claim 1 of 29 Claims
The preceding has been a description of the present
invention along with the preferred method currently known of practicing
the invention. While there are many minor modifications that can be made
without departing from the scope of the present invention, the scope of
the present invention should be defined by the appended claims wherein we
claim:
1. A method of administering an immunogen for activating the immune system
of a warm-blooded animal comprising orally administering to said animal an
amount of immunogenic composition effective for activating the immune
system of said animal, wherein said immunogenic composition
microencapsulated with a water-based enteric coating said enteric coating
including a solvent consisting essentally of water to thereby avoid
denaturing said immunogenic composition.
____________________________________________
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