Use of microparticles having a protein and an antibody adsorbed thereon for preparing a pharmaceutical composition for intranasal administration.

The present invention relates to use of a microparticle having a protein and an antibody adsorbed thereon for preparing a pharmaceutical composition for intranasal administration.

In the present description and the following claims, the term "protein" comprises any compound of condensation of two or more amino acids. The term therefore comprises, but is not limited to, biologically active peptides, polypeptides and proteins.

It is known that in various animal species, including human beings, absorption of proteins administered by nasal route is higher than 40% for peptides having from 3 to 6 amino acids (AA), about 10-15% for polypeptides having from 9 to 27 AA, and less than 1% for polypeptides having greater molecular weight (for example, for insulin (51 AA), the absorption is almost zero), although there are appreciable differences for the same peptide from species to species, or even from individual to individual of the same species [Lee W. A., Longenecker J. P. "Biopharm. Manufact." April pp 1-7 (1988)].

This is why at the end of the 80s only 3 nonapeptides, as such, (desmopressin, lypressin, oxytocin) were administered by the nasal route, and protease inhibitors and enhancers of transit through the nasal mucous membrane were studied from the early 80s. Generally, these enhancers are surfactants which increase passive permeability of nasal mucous membrane. Due to which it has been possible to have formulations for intranasal administration of calcitonin (32 AA), insulin (51 AA), growth hormone (191 AA) and other proteins and high molecular weight polypeptides [Lee W. A., Longenecker J. P. "Biopharm. Manufact." April pp. 1-7 (1988); Verhoef J. C. et al. "Eur. J. of Drug Metab. and Pharmacokin." 15, 83 (1990); Mishima M. et al. "J. Pharmacobio-Dyn." 10, s.69 (1987); Mishima M. et al. "J. Pharmacobio-Dyn." 12 32 (1989); Watanabe Y et al. "Chem. Pharm. Bull." 40, 3100 (1992); Schipper N. G. M. et al "Pharmaceutical Res. 10, 682 (1993); Shao Z. et al. "Pharmaceutical Res." 11, 1174 (1994)].

The enhancers, however, since they are essentially surfactants, have the disadvantage of damaging mucous membrane more or less profoundly and reversibly, thus increasing the passive permeability of the same.

In order to attempt to favour absorption, nasal transit retarders have also been used, such as viscous agents, adhesive polymers, and the like, and the results obtained have been moderate. Retardants, however, also have toxic effects on mucous membrane and cilia.

To obviate these disadvantages, administration of drugs incorporated in microspheres of starch which are not absorbed because they are too large but which are partially hydrated in the nasal lumen and slowly release the previously incorporated peptides has been proposed [Illum L. et al. "Int. J. Pharm." 39, 189 (1987); Bjork E., Edman P. "Int. J. Pharm." 47, 233 (1988)]. In this manner a retarding effect was obtained together with an improvement of absorption of small and medium-sized molecules. However, the absorption of large molecules does not improve because the mucous membrane is not impaired.

Finally, PCT patent application WO 94/28879 discloses a pharmaceutical composition for oral administration comprising a biologically active material, an antibody which specifically binds to the said biologically active material and a plurality of microparticles of polymeric material. In particular, the biologically active material belongs to the family of the peptides, polypeptides and proteins. Preferably, the microparticles are microspheres of polystyrene.

The biologically active material and antibodies are adsorbed on the said microparticles and the latter are endocytosed by the epithelium coating the follicles of the Peyer's patches in the mouse. It is therefore possible to calculate in the laboratory animal both the quantity of microparticles entering the carrier cells (uptake) and the quantity of microparticles which effectively passes transmurally and reaches the lymph in the mesenteric duct, which collects all the transported material.

In the above-mentioned application, the most efficient transport has been obtained by using bovine growth hormone as the protein and the specific antibody of the same, bGH-Ab, as the antibody.

Uptake measurements were carried out by inserting in vivo in the jejunum and ileum of the rat 3.6.times.10.sup.11 coated microparticles, fixing the tissue after 90 minutes and carrying out measurement (6 endocytotic cycles in 90 minutes were taken into consideration in the calculation).

The results obtained were as follows: a) total uptake in 90 minutes through 40 cm.sup.2: 8,400,000 microparticles [yield=0.023.degree./oo (=8,400,000/3.6.times.10.sup.11)]; b) total uptake per unit of area in 90 minutes through 40 cm.sup.2: 210,000 microparticles/cm.sup.2; [yield/cm.sup.2=0.00058.degree./oo (=210,000/3.6.times.10.sup.11).

In turn, transmural flow measurements were carried out by inserting in vivo in the jejunum+ileum of the rat 3.6.times.10.sup.11 coated microparticles and collecting the lymph from the cannulated mesenteric duct every 5 minutes.

The results were as follows: a) transmural transport on 40 cm.sup.2 in 90 minutes: 65,000 microparticles: [yield on 40 cm.sup.2=65,000/3.6.times.10.sup.11 (=0.00018.degree./oo)]; b) material transported transmurally in 90 minutes/cm.sup.2: 1625 microparticles/cm.sup.2; [yield/cm.sup.2=4.4.times.10.sup.-9 (=0.0000044.degree./oo)].

These data show that yield of endocytosis in the intestine is 130 times (2.3.times.10.sup.-51.8.times.10.sup.-7) higher than yield of transmural transport. This means that out of 130 endocytosed particles, 129 remain trapped in the said lymphoid tissue of the Peyer's patches and only 1 passes into the lymph.

It has now surprisingly been found that yield of active transport in nasal mucous membrane of a protein and the specific antibody of the said substance adsorbed on microparticles of polymeric substance is 400 thousand times higher than that of the intestine.

More particularly, since the absorption data in experiments on the intestine were obtained in vivo, these data relate to an incoming flow (lumen-lymph) comprising both active and passive paracellular transport which increases yield. Based on this initial premise, the ratio of yield of nasal and intestinal transport is without doubt higher than the previously mentioned 400 thousand times.

In fact, nasal mucous membrane was studied isolated in vitro, and it was possible to measure the two opposite, unidirectional flows of coated microparticles, and net absorption was calculated as the difference of these flows. Net absorption of nasal mucous membrane therefore does not include the incoming passive component but, unlike what has been reported for the intestine, is solely the result of active absorption.

It is further worth noting that, in addition to the extremely favourable yield ratio, in general the nasal route is also more advantageous in comparison with the oral route in that the absorbed substance does not have to pass through the taxing digestive system of the gastrointestinal tract and having then entered the circulation it does not suddenly have to pass through the liver.

Therefore, it is a first object of the present invention to provide an use of a polymeric of a microparticle having a protein and an antibody adsorbed thereon for preparing a pharmaceutical composition for intranasal administration.

The protein is preferably selected from the group comprising BSA (bovine serum albumin), insulin, enkephalin, hormones, growth factors, cytokines, coagulation factors, neuropeptides, antimicrobial agents and fragments thereof. The antibody, in turn, is an immunoglobulin selected from the group comprising IgM, IgA and IgG. The immunoglobulin is preferably specific for the protein. The microparticles preferably are microspheres of non-immunogenic polymeric materials such as polystyrene, latex or other polymers. Optionally, the polymeric material is of biodegradable type.

Preferably, the pharmaceutical composition according to the present invention is prepared in a suitable dosage form comprising an effective dose of a protein and an antibody adsorbed on microparticles of polymeric material together with a pharmaceutically acceptable inert ingredient.

Examples of suitable dosage forms for administration by the intranasal route are creams, ointments, aerosols, sprays and drops.

The dosage forms may also contain other conventional ingredients such as preservatives, stabilisers, buffers, salts for adjusting the osmotic pressure, emulsifiers, flavourings, and the like.

The quantity of protein and antibody in the pharmaceutical composition according to the present invention may vary within a wide range in relation to known factors such as, e.g., the stage and seriousness of the disease, the patient's body weight, the number of daily doses and the activity of the selected protein. The optimum quantity can nevertheless easily and routinely be determined by a person skilled in the art.

Generally, the protein/immunoglobulin ratio is of from 1 to 15,000 mols of protein for each mole of immunoglobulin. Preferably of from 1 to 5,000, even more preferably of from 1 to 100 mols of protein for each mole of immunoglobulin.

In turn, the quantity by weight of protein in the pharmaceutical composition according to the present invention will be easily determined in the individual case by a person skilled in the art on the basis of the known activity of the protein used.

The dosage forms of the pharmaceutical composition according to the present invention may be prepared by methods well known to the pharmaceutical chemist comprising mixing, granulation, compression, dissolution, sterilization, and the like.

Claim 1 of 8 Claims

1. A method for intranasally administering a composition comprising a microparticle and an antibody adsorbed thereon, wherein said administering comprises having a protein and an antibody thereon with the nasal mucosa of a patient in need thereof, and wherein the transepithelial transport obtained with 3.2.times.10.sup.11 microparticles/ml is 1.7%, wherein said antibody is an immunoglobulin specific for the protein.

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