Link:  Pharm/Biotech Resources

Title:  Complexes of immunoglobulins polysaccharides for oral and transmucosal absorption

United States Patent:  6,913,746

Issued:  July 5, 2005

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

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

Appl. No.:  081081

Filed:  February 20, 2002

The present invention relates to complexes consisting of immunoglobulins and polysaccharides for oral and transmucosal use. The polysaccharides comprised in the complexes according to the invention form an envelope which protects and carries immunoglobulins allowing their systemic absorption through the gastric and mucosal district. Immunoglobulins have a different specificity depending on the required therapeutic effect. They are used in passive immunoprophylaxis for the prevention or therapy of infections caused by pathogenic agents such as virus, bacteria, parasites, or they are used in the modulation of endogenous bio-chemical balances, or in the detoxification from drugs of abuse, medicines, toxins.

SUMMARY OF THE INVENTION

The main object of the present invention are complexes of immunoglobulins and polysaccharides for pharmaceutical use. In the complexes according to the invention polysaccharides are chosen among: chitosane, chitosane having a low molecular weight and a high degree of deacetylation, methylglycolchitosane, alginic acid, polymanuronic acid and their salts or derivatives. In the complexes according to the invention immunoglobulins and polysaccharides are associated by means of non-covalent links, preferably ionic links.

The immunoglobulins of the complexes according to the invention are chosen among IgG, IgA, or their fragments F(ab′)2 or F(ab). Immunoglobulins are specific for exogenous agents such as external pathogenic agents, virus, bacteria, parasites or their antigenic fragments, or for toxins of mycotic origin, drugs, medicines; they can also be specific for endogenous bioactive substances, consisting of hormones, enzymes and proenzymes, bioactive peptides, metabolites, physiological precursors. They can be useful if it is necessary to modify endogenous levels of said substances both in pathologic situations and in normofunctional situations. Immunoglobulins having different specificities can also be associated in one complex to obtain a unique or synergic therapeutic effect.

The present invention considers as particularly preferred those complexes in which immunoglobulins are specific for: toxins of mycotic origin, or for medicines such as: monensin, corticosteroids, antibiotics, etc., or for virus, or for bacteria such as: Listeria monocytogenes, Salmonella thipy, s. entheriditis or for their antigenic components, which are typical cases of passive immunoprophylaxis. Complexes are also preferred, in which immunoglobulins are specific for hormones such as: chorionic gonadotropin, parathormone, glucagon, or for endogenous proenzyme prothrombin, and also for drugs of abuse such as: cocaine, heroine, lysergic acid and their salts and derivatives.

In such complexes the polysaccharides form a protective envelope around immunoglobulins and thus enable the oral and transmucosal absorption of the latter and their use beyond parenteral administration only.

Another object of the present invention is the use of said complexes of polysaccharides and immunoglobulins in the preparation of detoxifying medicines, of medicines for curing syndromes caused by drug overdose, of anti-ulcer medicines, of medicines for curing growth problems.

A further object of the present invention are pharmaceutical compositions containing as active agent the complexes according to the invention, associated or not with suitable excipients and adjuvants, among which the preferred one is the delipidated fraction of C. granulosum.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is represented by complexes of immunoglobulins (Ig) incorporated into polysaccharidic polymers wherein the polysaccharide is not chemically cross-linked to immunoglobulins and allows the systemic delivery of immunoglobulins. Immunoglobulins are in the form of fragments F(ab) with a molecular weight of about 50 kD, or of fragments F(ab′)₂ with a molecular weight of about 100 kD, or in the 150 kD form (Ig).

The authors have found that-and this represents the main object of the present invention-absorption through the enteral (oral) and/or transmucosal way allowing a systemic delivery of protein macromolecules, is efficiently achieved by coating such macromolecules with polysaccharides preferably derivatives of chitosane and/or alginic acid, with no limitations on molecular weight of the polysaccharide chosen.

The possibility to make immunoglobulins bioavailable (absorbable) by the oral and transmucosal way (perlingual, nasal, vaginal, rectal) opens up a series of new applications for the pharmaceutical use of these macromolecules.

Oral or transmucosal absorption is above all easier than parenteral administration: as a matter of fact, such kind of administration does not require the intervention of medical and/or skilled personnel.

According to a first embodiment the present invention is represented by complexes comprising immunoglobulins incorporated into polysaccharides, wherein the polysaccharides externally coat the immunoglobulins, carrying the latter, protecting their proteic structure and allowing a systemic delivery in an active form through the oral and transmucosal way. In the complexes of the invention polysaccharides and immunoglobulins are not linked through covalent links, but are rather associated through nonspecific interactions, such as van der Waals forces or ionic interactions.

The oral (enteral) or/and transmucosal administration presents a series of advantages with respect to parenteral administration even though providing a systemic way of action. In the present case of immunoglobulins, the enteral and transmucosal administration allows a slower and more gradual absorption of Ig and allows to control to a higher extent the dosage and the distribution in the bloodstream of said proteins without altering their efficacy. On the contrary, expecially in the case of repeated treatments with heterologous proteins, parenteral administration determines a fast increase in the level of heterologous macromolecules in the bloodstream which can result in phenomena of immuno-incompatibility, or even in anaphylactic shock, thus eliminating the possibility of medium/long-term repeated treatments. These adverse effects are eliminated by the enteral or transmucosal way of administration.

Oral or transmucosal absorption of the immunoglobulins complexes according to the present invention presents the following advantages: it allows the protected heterologous immunoglobulins to enter the bloodstream slower to achieve a final systemic effect more gradually. These conditions optimize the interaction between the heterologous Ig and the antigen or the target molecule in the bloodstream. Also the conditions of interaction between the immunocomplex (target molecule or antigen and Immunoglobulin) and the immune system for the clearance of immuno complex are optimized and this reduces immunoreactivity towards the heterologous immunoglobulin. A further advantage is represented by the possibility to rationalize during time the treatment by keeping the levels of the circulating product constant, thus globally enabling a better systemic distribution of immunoglobulins.

The incorporation of Ig into polysaccharides is carried out by using preparations of polysaccharides having different chemico-physical characteristics and with a different degree of derivatization. Polysaccharides are preferably chosen among chitosans with different substituents and their derivatives and polymers and derivatives of alginic acid. Chitosans comprises chitosane with low molecular weight (150,000), chitosane with medium molecular weight (400,000) and with a high degree of deacetylation, glycolchitosane, methylglycolchitosane, Protasan™. Particularly preferred polysaccharides are methylglycolchitosane, chitosane with low molecular weight and high degree of deacetylation and polymannuronic acid (MW 5-10 kD), or the alginate derivatives obtained for instance by enzymatic hydrolysis of alginic acid with alginate-lyase enzyme, and their derivatives or salts. Such polysaccharides or their derivatives are chosen among those which can form around the structure to be incorporated (in the specific case immunoglobulins) a polymeric "film" resistant to enzymatic activity and to chemico-physical variations in the digestive tract, and also enable the possibility to direct the incorporated substance towards mucosal cells, thus enhancing their absorption.

The absence of cross-linking between polysaccaride and Ig is a further advantage of the complexes according to the invention, since the method used for their preparation is easier and the final product does not contain any potentially toxic residues of the chemical cross-linking.

A characteristic of the complexes according to the invention is that polysaccharides coat immunoglobulins without being linked to the latter by means of covalent bonds, but rather forming a sort of surface envelope i.e. in a gel form as in the case of alginic acid.

The immunoglobulins which are incorporated into the polysaccharides are IgG or IgA, or their fragments F(ab′)₂ or F(ab). They are preferably IgG or their fragments F(ab′)₂ or F(ab) or biologically active fragments derived for instance by cloning the vanable heavy and light chains of the aforesaid Ig, such as scFv. IgG are prepared by methods known at the state of the art, for instance by immunization of mammals, such as mice, rabbits, etc. as polyclonal immunoglobulins (Johnstone A. & Thorpe, in "Immunochemistry in Practice", 1982, 27-31, Blackwell Sci. Publ. Oxford) or using the technique described for instance in Kohler G. & Milstein C., Nature 256:495-497, as monoclonal antibodies. Complexes containing antibodies produced in rabbits, sheep or horses are particularly preferred. Methods for the preparation of the fragments F(ab) or F(ab′)₂ are known in the art and are for instance described in WO 97/49732. Moreover, immunoglobulins can be also of commercial origin.

The antibodies which are used for the preparation of the complexes according to the invention have different specificities, chosen depending on the desired therapeutic effect. It is understood however that beyond the particular applications mentioned in the present description any complex of immunoglobulins and polysaccharides, in particular chitosane and alginate, their derivatives or substituents, for oral and/or transmucosal use allowing a systemic effect, falls within the frame of the present invention. Immunoglobulins with different specificities can also be combined in one complex so as to obtain a unique or synergic therapeutic effect. According to a particular application in the field of infectious diseases, the complexes according to the invention allow to keep and to improve the use of immunoglobulins in all cases requiring passive immunization, i.e. either in cases when the infective antigen is already present in the organism and an immediate immunoprotection till the development of endogenous antibodies as a consequence of active vaccination is required, or in cases of immunoprophylaxis to prevent infections.

A further and innovative application of the complexes according to the invention is represented by their use in the regulation of physiological biofunctional balances of a given subject by acting on endogenous molecules comprised in the group of: hormones, peptides, obtaining as a result metabolic variations which correct the functional or pathological state of the organism. A further embodiment of the present invention is represented by the possibility to correct functional and metabolic imbalances caused by organic degenerations of various nature and by the possibility to antagonize the accumulation medicine and drug of abuse having as a result the neutralization of toxic effects resulting thereof.

In the field of infectious diseases, said complexes comprises immunoglobulins with a specificity for the following viral agents: Herpes simplex, cytomegalovirus (CMV), chickenpox virus, rubella virus, syncytial virus, respiratory virus, influenza(flue) virus, Epstein-Barr virus, or for their antigenic components, or for the following bacterial agents: Listeria monocytogenes, Salmonella thipy, S. paratiphy, S. thiphymurium, S. choleraensis, Clostridium tetani, C. botulinum or Shigella etc., or for mycetes, such as Candida albicans, or for parasites such as Toxoplasma gondii; all other cases where passive immunization is required because of an existing or possible infection are however comprised in the aim of the present invention. Complexes containing anti-Listeria monocytogenes and anti-Salmonella enteriditis IgG are particularly preferred.

According to this embodiment, the complexes optionally comprise an immunomodulator preferably BVV, represented by the delipidated fraction of Corynebacterium granulosum. Alternatively polysaccharidic complexes containing the delipideted fraction of Corynebacterium are prepared on their own and administered in combination with the complexes comprising the immunoglobulin. The complexes consisting of polysaccharides and of the delipidated fraction of Corynebacterium granulosum are produced in the same way as complexes containing immunoglobulins. The delipidated fraction of Corynebacterium granulosum is prepared by growing bacteria in conditions of strict anaerobiosis in mediums and temperature, agitation and time conditions known at the state of the art, such as Bactonutrient broth dehydrated and yeast extract (Difco), in presence of NaCl and glucose. Bacteria are grown for about 30 hours, deactivated by a treatment at high temperature, for instance 30′ at 60° C., and then concentrated e.g. by centrifugation. The bacterial mass then undergoes delipidation by a series of extractions in organic solvents, such as for instance 1 extraction in acetone of about 24 hours, followed by an extraction in chloroform (24 hours) and then an extraction in a mixture of methanol-ether in a ratio 1:2 (vol:vol). The sediment of delipidated bacteria then undergoes mechanical breaking by means of a waring Blendor homogenizer, and is then centrifuged at low speed. The supernatant liquor, made of broken bacteria, is further centrifuged at high speed (e.g. 10,000 rpm, for 15-30′). The sediment obtained from this last passage is the BVV particle fraction.

According to a further embodiment immunoglobulins specific for toxins of mycotic origin such as ochratoxin and aflatoxin are used when food intoxication has to be prevented, allowing the clearance of such substances from the bloodstream. Particularly in the zootechnical field it can be necessary to detoxify animals from medicines used for increasing growth (progestinic agents, estrogens, thyrostatic agents, corticosteroids, sympathomimetic agents) or to prevent and/or cure infectious diseases (antibiotics such as oxytetracycline, ampicillin or fungistatics or coccidiostatics such as monensin), so as to ensure the decontamination of the final product (meat, milk, eggs).

According a further embodiment the complexes of the present invention are used to detoxify the organism from: drug of abuse (such as cocaine, LSD, heroine), medicines or hormones.

According to the different embodiments of the invention, the complexes according to the invention for the prevention or the therapy of intoxications, infections or any pathologic states due to exogenous substances or agents, are particularly preferred when the immunoglobulins therein are specific for: ochratoxin, aflatoxin and progesterone, Listeria monocytogenes and Salmonella entheriditis, for medicines such as monensin and for drugs of abuse, in particular for cocaine.

In case of use of the complexes according to the present invention for passive immunization i.e. for the clearance of the infectious agent or in the prevention of infection, the effect can be strengthened by simultaneous administration of immunomodulators incorporated into the same polysaccharide structures as those used for immunoglobulins together with immunoglobulin or separately. Such immunomodulators are derived from the insoluble fraction consisting of glycoprotein and peptidoglycane or Corynebacterium granulosum and are characterized by a significant specific and adjuvating immunostimulating activity. The simultaneous administration of complexes containing a class of immunoglobulins directed towards a given microbic antigen during or after the administration of a non-specific immunomodulator allows to activate the cells involved in the non-specific defense of the organism, also having a synergic effect in the complexing process (formation of circulating immunocomplexes) and in the phagocytosis process of the antigen in combination with specific immunoglobulins.

The complexes according to the invention, which can be absorbed by oral or transmucosal way, are used not only when it is necessary to reduce the hematic level of an external agent (as for the treatment of infectious diseases, or for detoxification from drugs, medicines, toxins, etc.), but also when it is necessary to regulate the endogenous levels of hormones, enzymes and proenzymes, bioactive peptides, precursors and/or metabolites of various nature produced by the organism itself, or any endogenous substances involved in cell biochemistry or in the biochemistry of the whole organism. Said regulation is necessary either to correct pathologic situations due to an alteration in the endogenous balance of said substances, in particular chronic, possibly chronic or degenerative diseases, or to change the biochemical balance of normofunctional subjects, for instance: growth in animals, physical efforts for athletes, pregnancy induction or block, increase of attention threshold, elimination of metabolites deriving from degenerative processes, etc.

In these cases the slow and gradual absorption of immunoglobulins by oral or transmucosal way due to the protective effect of polysaccharides carrying the immunoglobulin, as disclosed in the present invention, allows to carry out therapeutic interventions without parenteral administration.

The complexes according to the present invention are used according to the latter embodiment re-balance the levels of endogenous hormones or enzymes or proenzymes or bioactive peptides involved in the regulation of organic functions. For instance, if it is necessary to vary the levels of endogenous calcium, the complexes according to the invention will contain antibodies or their fragments having a specificity for calcitonin or parathormone, both involved in calcium homeostasis. If it is necessary to intervene in problems related to fat accumulation, for instance in obesity pathogenesis, immunoglobulins will be chosen among those having a specificity for lipases. In other cases related to an imbalance in amino sugar metabolism, Ig will be chosen for example among those having a specificity for β-D-N-acetyl-glucosaminidase enzyme.

For the regulation of the levels of endogenous substances are particularly preferred complexes containing immunoglobulins or their fragments having a specificity for: somatostatin, glucagon, cholecystoquinine, growth hormone (GH) for growth-related problems; calcitonin and parathormone for problems related to calcium homeostasis. Other preferred complexes are those containing antibodies or their fragments specific for prothrombin (PTT) as anti-thrombosis agents, or for chorionic gonadotropin (ChCG) as anti-pregnancy medicines, or for pentagastrin as anti-ulcer medicines.

Another object of the present invention is the use of the complexes of immunoglobulins and polysaccharides for the preparation of detoxifying medicines with oral and/or transmucosal absorption, with anti-ulcer effects, for the treatment of thromboses and of obesity and their use for the preparation of medicines for the treatment of overdoses in drug addictions, preferably addictions caused by cocaine, heroine or lysergic acid (LSD).

The complexes according to the present invention also consist of immunoglobulins with different specificity, so as to obtain multifunctional complexes, or such to contain also the immunoadjuvant, such as BVV as previously described.

The use of the complexes according to the invention is particularly useful in the zootechnical field for the preparation of food additives to detoxify animals used for the production of meat or milk.

A further object of the present invention consists in compositions for oral use containing as active agent the complexes of immunoglobulins and polysaccharides in combination with suitable adjuvants and excipients, such as for instance those used at the state of the art for the preparation of food granulates for humans and animals (maize starch etc.).

A further object of the present invention consists in compositions for transmucosal use, for instance by perlingual, enteric, nasal, vaginal or rectal way, containing as active agent the complexes according to the invention, which also consist of immunoglobulins having only one or more specificities, in combination with suitable excipients, diluents or solvents; a further object of the invention consists in compositions where adjuvants consist of the immunomodulator BVV, which is present both on its own within the complex and in combination with the immunoglobulins.

The preferred regimen according to the various embodiments, is comprised between 1-100 mg/kg of weight, preferably 5-20 mg/kg, once a day for 7 to 15 days.

For detoxification purposes the regimen may be varied and lowered to 1 to 5 days depending on the degree of intoxication and on the general conditions of the subject.

For the correction of dismetabolism or for the treatment of chronic diseases the treatment may follow the following scheme: once a day for 4 to 7 days with a 2-5 days interruption, then repetition of the treatment.

For the intervention in non-pathological conditions the regimen is preferably comprise between 0,1-100 mg/kg preferably 5-20 mg/kg, once a day for 3 to 10 days, preferably 4 days.

A further embodiment of the present invention is the process for the preparation of the complexes of immunoglobulins and polysaccharides, in particular alginic acid, polymannuronic acid, methylglycolchitosane, chitosane with low molecular weight and high degree of deacetylation, comprising the mixing of a concentrated solution of immunoglobulins (5-50 mg/ml) in Na₂SO₄, brought to a temperature between 50 and 60° C. with a solution containing the polysaccharides in a concentration between 0.1 and 10% by weight/volume and mixing by mechanical agitation at maximum speed.

Claim 1 of 17 Claims

1. An isolated composition consisting of immunoglobulins as the active ingredient, a polysaccharide selected from the group consisting of chitosanes and alginates, wherein the polysaccharidic molecules are neither chemically cross-linked to the immunoglobulins, nor to each other, in combination with suitable excipients and diluents.

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