FR2771929A1 - USE IN A PHARMACEUTICAL COMPOSITION FOR NASAL ADMINISTRATION OF HYDROPHILIC PARTICLES FOR THE DELIVERY OF ACTIVE AGENTS TO THE CENTRAL NERVOUS SYSTEM - Google Patents

Abstract

The invention concerns the use in a pharmaceutical composition to be administered through the nose, of a hydrophilic particle whether or not bearing ionic ligands and optionally coated with a layer of amphiphilic compounds, having a size ranging between about 20 and 200 nm and advantageously between 50 and 100 nm to ensure and/or enhance the passage of an active agent administered through the nose up to the central nervous system. The invention also concerns pharmaceutical compositions to be administered through the nose of active agents to be delivered to the central nervous system, and their preparation method.

Description

USE IN A COMPOSITION
PHARMACEUTICAL FOR NASAL ADMINISTRATION OF
HYDROPHILIC PARTICLES FOR THE DELIVERY OF AGENTS
ACTIVE IN THE CENTRAL NERVOUS SYSTEM.

 The present invention relates to the field of treatment and diagnosis of diseases of the central nervous system, and is more particularly concerned with the passage and delivery of therapeutic or diagnostic active agents in the central nervous system.

 While cancer and cardiovascular disease are the leading causes of death in developed countries, nervous system diseases are among the primary factors that expose people to health problems (morbidity).

Advances in biology and neurology, however, have difficulties to materialize in the form of new diagnostic tools and therapeutics of the central nervous system (CNS), particularly because they are particularly difficult to administer effectively.

 There is indeed no diffusion of substances and nutrients from the blood capillaries in the spinal cord and the brain as in the other organs. Exchanges are limited at the level of the blood-brain barrier (BBB) and the choroid plexus, where the cerebrospinal fluid (CSF) is formed. It brings nutrients, hormones and leucocytes to CNS cells. The BBB strictly controls the composition of the extracellular environment of neurons by limiting the entry of most substances present in the blood and rejecting others.

The delivery of exogenous substances to the CNS can not be done through conventional systemic routes of therapy, except for extremely low molecular weight lipophilic compounds or compounds susceptible to selective transport linked to the presence of a carrier or to endocytosis phenomena (Ikumi Tamai et al.
Delivery through the BBB, (1996) Advanced Drug Delivery
Reviews 19: 401-424). Most of the drugs currently used in neuropsychiatry, such as codeine, benzodiazepines, phenytoin, etc., are lipid-soluble pharmaceutical products that cross the BBB after oral administration (Oldendorf,
WH Lipid solubility and drug penetration of the Blood
Brain-barrier (1974) Proc. Soc. Exp. Biol. Med. 147: 813-816). The entry of glucose and lactic acid, which are polar substances, or L-Dopa (Nutt, JG et al., The "on-off" phenomenon in parkinson's disease.

Relation to levodopa absorption and transport. (1984) N.

Engl. J. Med. 310: 483-488), a neutral amino acid for the treatment of Parkinson's disease, are linked to specific transporters. Some transporters, such as P-glycoprotein, are bidirectional (Knudsen, GM et al., Kinetic analysis of the human BBB transport of lactate and its influence by hypercapnia (1991).
Cerebral Blood Flow Metab. 11: 581-586) and limit the entry of drugs to the brain.

In particular, the BBB excludes the entry of proteins and a large number of peptides from the plasma, so much so that the interstitial fluid of the brain contains less than 0.5% of the plasma proteins. Thus, the work carried out in neurobiotechnology on numerous hydrophilic and / or high molecular weight compounds having a very high potential in therapeutics such as peptides, ion channels, neuronal receptors, neurotrophic factors and genetic determinants are so far limited. in their medical applications because of their difficulty in CNS administration such as xenobiotics
To overcome these disadvantages, it has been proposed to make a given molecule fat soluble by chemical transformation (lipidization). Lipidization is used for the manufacture of pro-drugs, liposoluble analogues of hydrophilic pharmacological compounds capable of crossing biological membranes. However, the EHE represses this type of compound towards the blood and their half-life is extremely short.

For this, tissue sequestration systems, or even inhibitors of the P-glycoprotein (Ikumi Tamai et al., Drug Delivery through the BBB, (1996) Advanced Drug Delivery Reviews 19: 401-424) are developed in parallel. The chemical modifications made to the molecule are delicate and may compromise the affinity to the receptor responsible for the biological activity. Lipidization can only be used for molecules of relatively low molecular weight (less than 1,000 or even 600-800), at best tetrapeptides and pentapeptides (Pardridge, W.M.

Peptide lipidization and liposomes. In Peptide Drug
Delivery to the Brain pp 129-140 (1991) Raven Press, New
York).

Small liposomes (25-50 nm) may be of interest for the delivery of biologically active products at the central level. But the higher size liposomes (0.3-2 μm) are trapped in the cerebral vascular space and therefore present risks of embolism. However, liposomes are unstable in the brain, particularly because of the presence of the enzyme lecithin-transferase at the level of neuronal membranes and glial cells (Pardridge,
WM Peptide lipidization and liposomes. In Peptide Drug
Delivery to the Brain 140-148 (1991) Raven Press, New
York.

In addition, intracebroventricular implants of several types are being tested as
an implant of cells genetically modified for the expression of protein factors, encapsulated in a hollow fiber (BioWorld Today, American Health Consultants Vol. 35: 1.4, 1997),
a drug-laden micro-pump for a few months (Winn, SR et al., Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival of axotomized septal cholinergic functions PNAS (1994) Vol 19: 2324-2328) , or
lipid-controlled controlled release formulations in which the drugs are encapsulated in sponge-structured microvesicles (Chatelut
E. et al Sustained release methotrexate for intracavitary chemotherapy. J. Pharm. Sc (1994) Vol 83, 3: 429-432).

 But this type of administration is extremely cumbersome because it involves surgery.

 Formulations of active substances that would penetrate the CNS without having to be implanted are sought, in particular by exploiting the endogenous transporters of the BBB. These transporters comprise membrane receptors which, in the presence of their ligand, are internalized by the endothelial cell. The vesicles containing the ligand migrate to the abluminal wall and are released to the nerve tissue.

This can be achieved, for example, by fusing the active protein with a protein having a cellular receptor at the central level, as described in PCT International Patent Application Publication No. WO9502421.

 Other recent studies have shown the passage of a hexapeptide, dalargine, through the mouse BBB, after intravenous administration of a formulation in nanoparticles (230 nm) of butylcyanoacrylate, provided that these particles are covered with surfactant, polysorbate 80. These peptide-laden particles would enter by phagocytosis or endocytosis. In this experiment, the peptide must be associated with the nanoparticles, the injection of a simple mixture containing the drug, the microparticles and the surfactant does not cause any biological effect (Kreuter, J. et al Passage of peptides through the BBB with colloidal polymer Brain Research (1995) 674: 171-174).

 The first administration of a recombinant peptide in the CNS was performed by intra-cerebroventricular infusion directly into the CSF.

This method allows delivery at therapeutic concentrations and near the ventricular walls, with CSF not entering the cerebral parenchyma (Pardridge, WM Transnasal and Ventricular Delivery of Drugs.) In Peptide Drug Delivery to the Brain pp. 109-122 (1991) Raven Press, New York). This type of administration, invasive and extremely difficult to use, has been used to administer, for example, methotrexate for treating meningoid leukemia, penicillin for bacterial meningitis, immunoglobulins for viral meningitis and morphine for chronic pain. .

 The nasal cavity is a natural route of administration of active substances that offers many potential benefits including the relative surface of the nasal mucosa, richly vascularized epithelium, permeability to lipophilic substances and relative permeability to hydrophilic. In addition, the blood supply of this mucosa, linked to the pulmonary circulation, avoids the liver (Harris, A.S.

Clinical opportunities provided by the nasal administration of peptides. Journal of Drug Targetting Vol.l, pp 101-116 (1993)). Also, nasal administration has been extensively tested for its ability to deliver substances to the blood, including polypeptides such as insulin, GnRH (gonadotrophin releasing hormone) and GHRH (growth hormone releasing hormone), vasopressin and its analogues, etc. Formulas such as nasal spray, calcitonin, oxytocin, desmopressin and GnRH (gonadotropin-releasing hormone) analogues are available on the market. Most often, permeabilizing agents should be used in the formulations. These substances, such as bile salts, including sodium deoxycholate, saponin, nonionic surfactants, increase the absorption and bioavailability of the active product by altering the mucosal film or even the structure of the mucosal epithelium. Therefore, their use in pharmaceutical formulations poses problems of tolerance and safety, especially for chronic applications (Bjork E., Edman P.,
Microspheres as a nasal drug delivery system.

Pharmaceutical particulate carriers. Therapeutic applications, Edited by A. Rolland, Marcel Dekker Inc.

(1993) pp 21-31). Microspheres of various sizes (45-200 μm) and various polymers (starch, dextran, cellulose derivatives, etc.) administered in powder form have the capacity to increase the duration of contact with the mucosa and probably also to dehydrate the mucus. They have demonstrated their ability to increase drug absorption and passage through the blood, including insulin, with few side effects at the nasal level (WO 9107947).

 The cells forming the epithelium of the nasal mucosa are joined by tight junctions, which limit the passage of molecules between the lumen of the cavity and the blood vessels.

In contrast, the basal pole of the olfactory neurosensory cells of the nasal epithelium elongates into an axon that ends in the olfactory bulb of the brain. This axon crosses the basement membrane of the mucosa and the submucosal space. The olfactory neural pathway could thus be a gateway to the brain.

 In addition, the subarachnoid space is prolonged at the axons of the neurosensory cells in fingerings that come into contact with the nasal submucosa. There is therefore a flow of CSF from the olfactory lobe to the sub mucosal space.

The particular anatomy of the nasal mucosa thus confers an unusual neighborhood with the CSF.

 Moreover, by the end of the 19th century, researchers had postulated that the olfactory nerve could allow the transport of tracing substances and various organisms from the nasal mucosa to the olfactory bulb and the CNS.

 In 1970 De Lorenzo (De Lorenzo Taste and Smell in Vertebrates J & A Churchill Ltd. (1970), 151176) intranasally administered gold particles in the monkey and estimated the rate of progression between the nose and the olfactory bulb at 2.5mm / h.

In 1971, Kristensson & Al (Kristensson & Al
Acta Neuropathol. (1971), 19, 145-154) demonstrated that if sensitive methods are used to trace macromolecular substances such as horseradish peroxidase and albumin, movement of the nasal mucosa to the subarachnoid space was observed.

 Later Pardridge (Partridge Peptide drug delivery to the brain Raven Press (1991), 101), in his book on the delivery of peptides to the brain states that the nasal administration of highly fat-soluble substances, such as progesterone can directly reach the cerebrospinal fluid without entering the peripheral blood compartment. The bioavailability of progesterone is greater after nasal insufflation than after intravenous infusion. Peptides, highly hydrophilic substances penetrate the membrane diffusion barriers between the nose and the olfactory subarachnoid space at speeds considerably lower than progesterone.

 The PCT patent application published under the number WO9107947 describes pharmaceutical compositions for the nasal administration of active substances that use this neuronal pathway. The compositions associate the biologically active product with a transporter capable of inducing absorption, the drug would then undergo a retrograde and / or anterograde neuronal transport.

Sakane (Sakane & Al STPPharma Sciences (1997), 7, (l), 98-106) recently published a systematic study on the direct transport of the nasal cavity to the cerebrospinal fluid.
- The study of cephalexin, a hydrophilic antibiotic, clearly shows a direct transport from the nasal cavity to the cerebrospinal fluid.

 - The study of different sulfonamides with variable lipophilicity shows that the transport is dependent on the lipophilicity and the non-ionized fraction of the active principle.

 - The study of dextrans of different molecular weights showed that the active ingredients could be transported up to 20KDa of molecular weight.

 Finally a study of the transport of peptides was carried out. The chosen molecule is an oligopeptide analogue of the enkephalin: (D-Ala 2) -Methionine enkephalinamide, it is detected in the cerebrospinal fluid after nasal administration and its concentration is increased by the use of an absorption promoter: sodium glycocholate and by protease inhibitors puromycin (aminopeptidase inhibitor) and thiorphan (enkephalinase inhibitor).

 However, despite nasal instillation methods in the rat, aiming to minimize the leakage of the product into the pharynx, the amounts of peptide obtained in the cerebrospinal fluid are very small, even in the presence of an absorption promoter or protease inhibitors (of the order of one hundred ng / ml), not allowing a therapeutic effect.

The Applicant has developed its expertise in the preparation of synthetic particulate vectors, referred to by the general term of
Supramolecular Biovectors, hereinafter referred to as "BVSM" or "Biovectors", capable of associating, and advantageously of vectorizing, different substances or active principles, and therefore useful for the manufacture of pharmaceutical compositions.

A first type of Biovectors
Supramolecular, and its method of preparation, has been described in European Patent No. 344,040.

These are particles comprising from the inside to the outside
a non-liquid hydrophilic central nucleus consisting of a matrix of naturally or chemically cross-linked polysaccharides or oligosaccharides, which can be modified by various ionic groups;
a layer of fatty acids grafted by covalent bonds to the nucleus;
one or more lipid layers consisting in particular of phospholipids.

The developments of this first generation of Supramolecular Biovectors led the Applicant to design and prepare new
Supramolecular biovectors, with improved properties in particular depending on the transported active ingredients.

PCT international patent applications published under Numbers WO 94/23701, WO 94/20078 and WO 96/06638 describe these Biovectors, their manufacture, their association with various active principles and their use for the preparation of pharmaceutical compositions.

 The teaching of the above patent applications is incorporated herein by reference.

The know-how available today
Applicant on the preparation of the BVSM allows him to have a wide range of type of BVSM. These BVSMs are formed of a crosslinked hydrophilic polymer, preferably polysaccharides, in the form of nanoparticulate gel, these BVSM are said to be of the PSC type (or
NPS in French).

This polymer may optionally carry positive ionic ligands, BVSM said cationic, or negative, so-called anionic BVSM. This charged polymer or not is optionally covered with a layer of amphiphilic compounds, preferably phospholipids, BVSM said Light type described in the PCT patent application
W09420078.

 The size of the BVSM is between 20 and 200 nm, preferably between 50 and 100 nm and more preferably between 60 and 80 nm.

The BVSM can be sterilized by filtration and their combination with the active principles is done on the
Completely manufactured BVSM (Major & Al Biochem. &
Biophys. Acta (1997), 1327, 32-40). This makes it possible to work the biological molecules, particularly fragile, in optimal conditions.

 BVSMs protect biological molecules from degradation (Prieur R. et al., Vaccins (1996) Vol 14, N06 pp. 511-520 Combination of recombinant hCMV IE1 protein with 80 nm cationic biovectors: protection from proteolysis and potentiation of presentation to CD4 ) and showed that they increase the biological activity of peptides and proteins such as enzymes, antigens, etc.

 BVSMs are known to be used as transporters of active substances, for example peptides, antigens or oligonucleotides. In this use, there is formation of a real complex between the biovector and the active substance, such as ionic interactions between the cationic nucleus of the BVSM and the anionic oligonucleotide. Thus, a stable ionic conjugate is formed in a biological medium.

In this type of preparation, the oligonucleotide / BVSM ratio is 5 to 10% and the measured association yield is greater than 90%. Similarly, the French patent application published under the number 2,723,849 describes that the increase in immunogenicity is not obtained with a simple antigen / BVSM mixture, without prior interaction of the antigen and the particulate vector.

The antigen is also associated with the particulate vector by ionic and / or hydrophobic bonds. To obtain this result, for example, 1 mg of protein GST-e4 is incubated per 10 mg of BVSM, which makes it possible to obtain average association levels of 90% regardless of the type of BVSM used (Prieur R. et Vaccines (1996) Vol 14, N06 pp 511-520).

 The Applicant has now discovered that the BVSM are particularly suitable for nasal administration and have a prolonged retention time in the nasal mucosa when administered as a spray in animals and in humans. Such a result is quite exceptional for a liquid formulation, especially since the presence of the BVSM is still significant after 12 and detectable up to 24 hours.

 This mucosal adhesiveness has been used for the mucosal administration of active substances, including antigens, associated in formulations with BVSM. In these formulations, the amounts of BVSM used are adapted to the amount of active substance that is to be delivered, the formulation consisting of the combination of the active substance (s) with the BVSM, this one playing the role of a carrier.

 In addition, the BVSM do not show toxicity by nasal administration, the mucosal epithelium, in particular ciliary movement and tight junctions, is intact after chronic applications for one month.

 In vivo, when labeled anionic BVSM are administered intravenously in the rat, only a small amount passes fugally into the cerebral microcirculation: about 0.15% of the administered dose is present at 5 min. hour. This quantity is not sufficient to consider that the anionic BVSM crosses the BBB in vivo, the cationic BVSM being undetectable under the same conditions.

The research work carried out by the
Applicants in the context of the present invention have shown that, in vi trio, the cationic light BVSMs crossed a transocytotic blood-brain barrier model (Cechelli, R. et al., Journal of Controll Release 1992, 21, p. 81-92, Cechelli, R. et al., Journal of Neurochemistry 1990, 54, 5, pp. 17981801). But these same particles without phospholipidic membrane, as well as light anionic BVSM Light, do not cross little or not this same barrier. On the other hand, when administered nasally,
BVSM are not found at the cerebral level. The in vitro results can not be correlated with the results obtained in vivo.

 In order to evaluate the properties of BMSVs on the passage from the nose to the brain of active substances, several drugs of central antinociceptive interest were studied, the Ss endorphin, DAGO and morphine.

The opiate type compounds that have been tested are low molecular weight molecules, between 50 and 10,000, relatively water soluble.

Their passage in the CNS is measured by their antinociceptive activity. Opiates produce their effects through the interaction with three types of receptors: mu, delta and kappa. The selected compounds, including endogenous enkephalin derivatives, have a specific action on mu opiate receptors (Pasternak GW et al Morphine-6-glucuronide, a potent mu agonist, Life Sci., 41: 2845-2849, ( 1987)
Kosterlitz HW, et al. Br. J. Pharmacol 73, 299 (1981)). These receptors are immersed in the CSF, therefore the observation of a biological activity indicates the passage of the substance to this level.

These compounds are easily degraded and are unlikely to reach the brain systemically when administered at a low dose.

 - ss endorphin is an endogenous enkephalin of 31 aa. It is recognized as not crossing the BBB (Hougten RA, et al b endorphin: stability, clearance behavior and entry into the CNS after intravenous injection of the tritiated peptide in rats and rabbits, PNAS ASA 77 4588-4591 (1980). labile, its use is delicate in vivo and its analgesic power depends on the physiological state of the animals.

- DAGO a pentapeptide [Tyr, D-Ala, Gly,
N-Me-Phe, Gly-ol], an amino-terminal analogue of ss endorphin, known to be more resistant to proteases than endogenous enkephaline.

Molecular weight of 513.

 - Morphine is the prototype of the analgesic and opiate-type narcotic, the main alkaloid of opium. Its molecular weight is 285.

In medical applications, it can be administered parenterally for postoperative pain and orally for cancer pain.

 Initially, we tried to associate ss endorphin, DAGO, morphine with different types of BVSM (anionic, cationic, phospholipid nuclei or not). From this first series of formulations, it was verified that only the ss endorphin was capable of associating with an anionic BVMS in a satisfactory manner, whereas no other combination with the aforementioned drugs gave a satisafaisante association.

For ss endorphin formulation BVSM
Light anionic / endorphin was tested at a dose of 20 μg of BVSM for 20 μg of endorphin and at a dose of 2 μg of BVSM for 20 μg of endorphin. As a negative control, the cationic BVSM were used in doses where they do not associate the drug (2 μg of BVSM for 20 μg of endorphin)
- Ss endorphin alone nasally showed no antinociceptive effect
- A measurable effect at 30 minutes was observed with anionic BVSM formulations
- Amazingly, an effect with the
Cationic BVSM has also been observed
- Anionic BVSM also had a greater effect with the 2 μg dose of BVSM than with 20 μg.

 The BVSM designed as vehicles for the transport and presentation of active substances, as described in the patent applications mentioned above, no longer explain the results obtained.

 In order to consolidate this new concept, we used DAGO and morphine-based formulations for which none of the tested BVSMs could show a satisfactory association with endorphin.

The work carried out with these compounds made it possible to highlight new properties of
BVSM no longer as a transporter but as an activator of the passage of active agents from the nose to the central nervous system. More particularly, the BVSM have the following properties
- They allow the passage of the nose to the brain of active substances at doses where the substance alone is not active by nasal administration.

 - They allow to increase the effect of a dose of active substance by nasal administration compared to the drug alone administered by the same route or the drug administered with a permeabilizer.

- They allow by nasal administration obtaining effects quantitatively comparable to those obtained by the same dose in systemic administration
- They extend the duration of action of the active substance administered nasally when this substance alone is already active by this route (increase in the duration of action).

 The results obtained made it possible to observe that light and PSC type BVSM are effective.

However, light and PSC-type BVSM do not have the same efficacy at the same doses (in dry weight equivalent of the polysaccharide part).

 On the other hand, unlike the use of the BVSM as transporter, there is no new effect in the novel use according to the present invention because the anionic and cationic BVSM are effective.

 Finally, the new use of the BVSM according to the invention is therefore different from the previous uses as a transporter, in that the substances tested are no longer attached to the BVSM but simply mixed with the BVSM in weight ratios such as the excess of free drug does not allow to consider any association. Furthermore, remarkably, the Applicant has observed that the doses of BVSM useful according to the present invention have a strong impact on the properties reported above.

We found that there is an optimal window for the dose of BVSM. Too much or too little BVSM does not significantly alter the intrinsic behavior of the drug.

This observation tends to suggest that the BVSM do not act by a passive effect alone but that they induce an active phenomenon responsible for the penetration of the substance. The mechanism of action of
VBMS on the passage of active substances from the nasal mucosa to the brain is not clear, but it is already certain that, the so-called "carrier effect" of the
BVSM described in the prior art does not explain the above properties.

 The work carried out by the Applicant suggests that the BVSMs interact with the nasal mucosa by promoting the direct passage of the nose-CSF.

However, a passage from the nose to the blood then from the blood to the brain through the BRE can not be excluded at least as one of the components of the effects observed.

In the new use according to the invention, the BVSM are particularly suitable for obtaining and / or promoting the passage of the nose to cholesterol. Their effectiveness in promoting the delivery of active substances through the mucosa depends on their lipophilicity, as well as their ability to increase the stability of the active substances tested (such as insulin or enkephalins) by inhibiting proteolytic activity (Sayani & Al Crit Rev. in
Therap. Drug carrier Systems (1996), 13, (1 & 2), 85184). It has also been shown that this effect is related to their ability to alter the mucosa, which precludes use for purposes other than experimental ones.

 Dihydrofusidates, derived from fusidic acid, are less toxic (although irritating) than bile salts and are, for some, more effective as promoters of mucosal absorption.

Their effectiveness is dose-dependent. Acidic, basic and neutral derivatives have been tested, the acids being the most effective. They would act by inhibiting the proteolytic activity and inducing the formation of micelles that solubilizes the peptide / protein.

 Unlike these products, the BVSM are not irritating. In addition, unlike BVSM, the permeabilizing agent tested (sodium deoxycholate) does not have the same effects as the BVSM on the biological activity of the active substances tested.

 Illum and his collaborators first introduced the principle of using microspheres for the nasal delivery of poorly absorbed substances.

 Thus were tested: biodegradable microspheres of starch (DSM), microspheres of dextran, and more recently chitosan.

 DSMs were the first to be tested (Illum & Al J. Pharm (1987), 39, 189-99): In sheep, the bioavailability of insulin was increased by 10 Nasal administration of insulin alone. An optimal ratio insulin / DSM equal to 1 / 3-4 mg / kg DSM has been established, ratio for which one achieves a maximum reduction of the blood glucose.

 Dextran spheres crosslinked with epichlorohydrin (sephadex) were later used analogously to DSMs (Ryden et al., Int. J.).

Pharm. (1992), 83.1). It has been found that the effectiveness of insulin in sephadex particles is lower than that of insulin in equivalent dose DSM in lowering blood glucose levels.

 Later, Illum also tested chitosan in the nasal delivery of insulin (Illum et al., Pharm Res (1994), 11, (8), 1186-9). Chitosan is a high molecular weight cationic polysaccharide derived from chitin shellfish shells. It is a bioadhesive polymer. Used as a gel (0.5% w / v) with insulin, it has demonstrated its ability to deliver insulin for 3 to 4 hours.

 While permeabilizing agents have recognized irritating effects on the nasal mucosa, it seems that microspheres are better accepted.

However, 8-week DSM rabbit tests showed for some animals mild epithelial hyperplasia and increased calciform cells (Bjork & Al Int, J. Pharm.

 (1991), 75,73).

 In addition, the optimization of the therapeutic value of the insulin / microsphere formulations leads to the coadministration of permeabilizing agents.

Thus in sheep when DSM are combined with lysophosphatidylcholine (LPC), the bioavailability of insulin is increased threefold (Farraj & Al J.

Against. Rel. (1990) 13.253).

Likewise, desmopressin starch microsphere formulations with LPC show a significant improvement in the bioavailability of desmopressin compared to the microsphere delivery system alone or the LPC formulation alone. These results prove that there is a synergistic effect between the microsphere effect and the effect
LPC (Critchley & Al J. Pharm Pharmacol.

 (1994) 46, (8), 651-6). However, it should be noted that the LPC, although considered as a mild permeabilizer, has hemolytic effects on erythrocytes that vary according to the species (function of the lipid composition of the erythrocyte). The non-toxicity of the BVSM under the intended conditions of use has an advantage over this type of product.

The mechanism of action of the microspheres is not fully understood. The hypotheses are tightening however around 2 mechanisms of action
the bioadhesive properties of the microspheres allow a greater contact time between the therapeutic substance and the absorption sites of the nasal membrane.

 the microspheres allow transient expansion of the tight junctions between the cells, thus allowing the passage of larger hydrophilic molecules (Edman experiment on monolayers of Caco-2 cells) (Edman & Al J. Control.

Rel. (1992), 21.165-172).

 Accordingly, the present invention relates to the use in a pharmaceutical composition for the nasal administration of a hydrophilic polymer particle carrying or not ionic ligands and optionally coated with a layer of amphiphilic compounds, to obtain and / or increase the passage of an active agent administered nasally to the CNS.

 Obtaining and / or increasing the passage of an active agent administered nasally to the CNS results in the case where the active agent is a pharmacologically active substance in the CNS by obtaining and / or the increase of the efficacy of said substance, administered nasally, in the CNS.

 In the use according to the invention, the particles have a size of between approximately 20 and 200 nm and advantageously between approximately 50 and 100 nm.

 The amount, by weight, of hydrophilic particle in the pharmaceutical composition for nasal administration according to the invention is lower and advantageously much lower, by weight, than the amount of active agent. Thus, in the use according to the invention, the amount of hydrophilic polymer particle, expressed by weight of dry hydrophilic polymer, is advantageously at least 5 times and advantageously at least 10 times lower, by weight, than the amount of active agent. administered nasally.

 It is therefore most particularly preferred in the use according to the present invention a ratio, by weight, of at least 5 parts of active agent for a part of particle, and advantageously of at least 10 parts of active agent for a part. of particle.

This ratio is of course dependent on the amount of active agent administered. For exemple
- in the case of DAGO, the ratio, by weight, is 50 parts of active agent for one part of particle,
in the case of morphine, the ratio, by weight, is 2500 parts of active agent for one part of particle.

 This ratio is the reverse of that described in the prior art for the use of BVSM as active agent transporter.

 It is particularly preferred to use a particle dose in the pharmaceutical composition for nasal administration of from a few nanograms to a few milligrams.

 The BVSMs used for nasal administration according to the invention may be presented in a pharmaceutical composition which may or may not also contain the active agent. In the case where the composition containing the BVSM also does not contain the active agent, it is preferred to have a delayed nasal administration of the BVSM and the active agent (s), or vice versa.

 In the use according to the invention, the pharmaceutical composition for nasal administration may also contain any vehicle that is pharmaceutically compatible with the BVSM and / or the active agent, as well as optionally an agent for permeabilizing the nasal mucosa.

 In the use according to the invention, the active ingredient is a therapeutic or diagnostic agent.

 As a therapeutic agent, mention may be made of: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonians, antiepileptics, anti-migraine agents, or any other agent useful in the treatment of neurodegenerative, psychiatric or behavioral disorders, anti-cancer agents antivirals, antipirants, antibiotics, or active agents such as growth factors, nucleic acids, vitamins, hormones, etc.

 As a diagnostic agent, mention may be made of those chosen from: antibodies and molecules making it possible to target CNS receptors, medical imaging agents used in MRI, scintigraphy, PET, ultrasound.

 The invention also relates to a pharmaceutical composition for the nasal administration of an active agent to be delivered to the CNS, characterized in that it comprises said active ingredient and an effective amount of a hydrophilic polymer particle, with or without ligands. ionic and optionally coated with a layer of amphiphilic compounds, to obtain and / or increase the passage of said active agent administered nasally to the CNS.

 Obtaining and / or increasing the passage of an active agent administered nasally to the CNS results in the case where the active agent is a pharmacologically active substance in the CNS by obtaining and / or the increase of the efficacy of said substance, administered nasally, in the CNS.

 In the compositions of the invention, the particles have a size of between about 20 and 200 nm and advantageously between about 50 and 100 nm.

 As indicated above, the amount of particles in the pharmaceutical composition for nasal administration according to the invention is lower and advantageously much lower, by weight than the amount of active agent. The amount, by weight, of hydrophilic particle in the pharmaceutical composition for nasal administration according to the invention is lower and advantageously much lower, by weight, than the amount of active agent. Thus, in the composition of the invention, the amount of hydrophilic polymer particle, expressed by weight of dry hydrophilic polymer, is advantageously at least 5 times and advantageously at least 10 times lower, by weight, than the quantity of active agent administered. nasally.

 A ratio, by weight, of at least 5 parts of active agent for a part of a particle, and advantageously of at least 10 parts of active agent for a portion of the particle, is therefore particularly preferred in the composition according to the invention. particle.

This ratio is of course dependent on the amount of active agent administered. Thus, the ratio between the amount of active agent and particle is between 1 and 10 6 depending on the therapeutically effective dose of the active agent. For exemple
- in the case of DAGO, the ratio, by weight, is 50 parts of active agent for one part of particle,
in the case of morphine, the ratio, by weight, is 2500 parts of active agent for one part of particle.

 This ratio is the reverse of that described in the prior art for the use of BVSM as active agent transporter.

 It is particularly preferred to use a particle dose in the pharmaceutical composition for nasal administration of from a few nanograms to a few milligrams.

 The BVSMs used for nasal administration according to the invention may be presented in a pharmaceutical composition which may or may not also contain the active agent. In the case where the composition containing the BVSM also does not contain the active agent, nasal administration of the BVSM is preferred after the nasal administration of the active agent (s).

 The invention therefore also relates to a pharmaceutical composition containing as active substance only BVSM in the proportions defined above with respect to the active agent to be delivered at the CNS which is contained in a composition containing or not BVSM.

 The pharmaceutical composition for nasal administration of the invention may also contain any vehicle pharmaceutically compatible with the BVSM and / or the active agent, as well as possibly a permeabilizer for the nasal mucosa. These compositions may be in any form suitable for nasal administration such as creams, gels, sprays, powders coated or not on a support, etc.

 In the pharmaceutical composition for nasal administration of the invention, the active ingredient is a therapeutic or diagnostic agent.

 As a therapeutic agent, mention may be made of: analgesic, anesthetic, analgesic, antispasmodic, antiparkinsonian, antiepileptic, antimigraine, or any other agent useful in the treatment of neurodegenerative, psychiatric or behavioral disorders, anticancer disorders antivirals, antipirants, antibiotics, or active agents such as growth factors, nucleic acids, vitamins, hormones, etc.

 As a diagnostic agent, mention may be made of those chosen from: antibodies and molecules making it possible to target CNS receptors, medical imaging agents used in MRI, scintigraphy, PET, ultrasound.

 The pharmaceutical compositions according to the invention may also comprise one or more agents permeabilizing the nasal mucosa, of the type of those defined above.

The invention finally relates to a method for preparing a pharmaceutical composition for the nasal administration of an active agent to be delivered to the
CNS, said composition comprising said active ingredient and an effective amount of a hydrophilic polymer particle, with or without ionic ligands and optionally coated with a layer of amphiphilic compounds comprising mixing the active ingredient and a lower and advantageously much lower amount , by weight, of particles relative to said active ingredient. It is more particularly preferred in the process of the invention to mix the active ingredient and the particles in a ratio, by weight, of at least 5 parts of active agent for one part of particle, and advantageously of at least 10 parts by weight. parts of active agent for a particle part.

Other advantages and characteristics of the invention will appear on reading the following examples relating to the availability of BVSM in the nasal cavity, their toxicity, their preparation and their use in pharmaceutical compositions for the nasal administration of agents. assets, and referring to the annexed drawings in which
- Figure 1 shows the residence time of cationic BVSM light labeled with indium 111 in the human nasal cavity as a percentage of the administered dose and as a function of time.

 FIG. 2 represents a comparison of the effect of administering 500 μg of morphine nasally, alone or in the presence of cationic light BVSM at different doses: 0.2 μg, 2 μg, 10 μg.

 FIG. 3 represents a comparison of the effect of the administration of 500 μg of morphine nasally, alone or in the presence of cationic light BVSM at a dose of 2 μg.

 FIG. 4 represents a comparison of the effect of the administration of 500 μg of morphine nasally, alone or in the presence of 0.2 μg of cationic NSP NMBS.

 FIG. 5 represents a comparison of the effect of the administration of 500 μg of morphine nasally alone or in the presence of anionic light BVSM at a dose of 2 μg.

 FIG. 6 represents a comparison of the antinociceptive effect of 500 μg of morphine alone subcutaneously with the administration of morphine 500 μg in the presence of cationic BVSM Light 2 μg nasally.

- Figure 7 shows the effect of a permeabilizing agent of the nasal mucosa on the biological activity of morphine. Comparison of the effect of administering 500 μg of morphine with 1% NaDoc with the administration of morphine 500 μg in the presence of
Cationic Light BVSM 2 ug.

 - Figures 8 and 9 show a comparison of the areas under curves of the preceding figures.

 FIG. 10 is a comparison of intranasal administration of 100 μg DAGO alone and in the presence of 2 μg cationic light BVSM.

 FIG. 11 is a comparison of the administration of 100 μg of DAGO subcutaneously and in the presence of 2 μg cationic light BVSM by the intranasal route.

 FIG. 12 represents the effect of a permeabilizing agent on the intranasal administration of 100 μg of DAGO.

 FIG. 13 represents the effect of a permeabilizing agent and of the 2 μg cationic light BVSM on the intranasal administration of 100 μg of DAGO.

FIG. 14 represents a comparison of the administration of 100 μg of DAGO in the presence of BVSM
Cationic light 2 μg nasally with the administration of 0.05 μg DAGO by ICV.

FIG. 15 represents a comparison of the areas under curves of the preceding graphs (14,13,12,11,10)
FIG. 16 represents the effect of the administration of 500 μg of morphine nasally and the delayed administration of 2 g of cationic BVSM Light.

FIG. 17 represents the areas under the curve of the delayed administration of 500 g of nasal morphine followed by 2 g of cationic light BVSM compared to
the simultaneous administration of both,
the administration of morphine alone, nasally and subcutaneously,
the administration of morphine with 1% NaDoc nasally.

Example 1: Study of the residence time of
BVSM in the human nasal cavity.

 1) Preparation of a radiolabelled formulation (indium 111) of BVSM liaht cationioues.

A 111InCl3 solution (Cisbio, France) at 370
MBq / ml is mixed with an InC13 solution (Fluka, France) (15 mM InCl 3, 2 mM sodium citrate, pH 6.0). This solution is added to a suspension of BVSM corresponding to Example 3. Thus the final suspension has the following characteristics
BVSM 15mg / ml; 0.3 mM InCl3; LmM sodium citrate, pH 6.0.

 The concentration of free Indium is determined on a gamma counter after dilution to 1/10 in 1 mM citrate buffer pH 6.0 and ultrafiltration on 100 Kd microcon (Amicon).

 The suspension thus prepared is sterilized by filtration (0.2 Wm) stored at 40C until administration. For administration, 120 μl of suspension are introduced into monosprays (Pfeiffer, UK) allowing the administration of 100 μl / spray.

Table I summarizes the results obtained for the preparation of the batches used in the human pharmacokinetic study. Under these conditions, the labeling of the BVSM with InCl3 is practically quantitative with association yields of 87.5 ± 9.8%. In addition doses of radioactivity (0.39 t 0.03
MBeq / dose) are perfectly compatible with scintigraphic monitoring after nasal administration in men of the BVSM.

 Table I below shows the characteristics of the Indium 111 labeled cationic BVSM preparations used in the human pharmacokinetic study.

Table I

<tb><SEP> Average <SEP> SD
<tb> PSC <SEP> (mg / ml) <SEP> 12.7 <SEP> 1.7
<tb> DPPC <SEP> (mg / ml) <SEP> 2.3 <SEP> 0.2
<tb> Cholesterol <SEP> (mg / ml) <SEP> 0.67 <SEP> 0.06
<tb> Radioactivity <SEP> (MBq / dose) <SEP> 0.39 <SEP> 0.03
<Tb>
2) Biovector residence time in the human nasal cavity.

 Ten healthy, fasting, non-smoking males aged 18 to 35 years were given 1.5 mg of the cationic BVSM light formulation and 111In each nostril (100 Fl / nostril). This dose is equivalent to about 0.02 mg / kg for a weight of about 75 kg.

The study is a non-randomized study. After administration, scintigraphic images are collected with a gamma camera to monitor the radioactivity administered. A continuous series of lateral planes of the head is collected.

 The amount of radioactivity in the nasal cavity steadily declines during the duration of the study, 20% of the dose is still detected at 12 hours and 7% at 24 hours, which is a significant retention time compared to state of the prior art. The elimination half-life of the nasal cavity is about 2.3 hours, with a 2-compartment model.

These results confirm the potential of MSVB for nasal drug delivery.

 Figure 1 illustrates the average amounts of radioactivity in the nasal cavity (as a percentage of the administered dose) as a function of time.

 Example 2: Study of the toxicity of the BVSM administered to beaal dogs and rabbits.

1) Dog:
The objective of this study is to evaluate the potential toxicity of cationic light BVSMs and
BVSM Light anionic by intranasal administration 2 times daily for 4 weeks in beagle dogs.

 Classic tests: mortality, clinical signs, body weight, food consumption are carried out during the 4 weeks. In the same way laboratory tests: hematological analyzes, biochemical analysis of the blood and urine analysis are carried out before the beginning of the treatment period and in week 4. At the same time electrocardiographic, blood pressure and ophthalmological examinations are carried out.

 In addition, at week 4, one hour after the first daily administration, the mucociliary clearance rate is estimated by measuring the rate of transit of glucose particles from the nose to the pharynx. The distance from the opening of the nostril to the nasopharynx is measured at autopsy.

 Finally, all animals are subjected to an autopsy. The organs are weighed and preserved. Microscopic examination is performed on all macroscopic lesions and organ tissues.

 Mucociliary clearance measurements clearly show that the administration of light anionic BVSM light or cationic light BVSM does not alter the properties of the mucosa or the ciliary movement inside the nasal cavity.

 No significant biological changes can be attributed to the substances tested. No macroscopic or microscopic observations can be considered as toxicologically significant, in particular no macroscopic or microscopic abnormalities have been observed in the nasal cavities.

As a result, the "No Observable Adverse
Effect Level (NOAEL), in terms of clinical signs, body weight, food consumption, ophthalmology, electrocardiography, blood pressure and laboratory tests are greater than 3.68 mg BVSM light cationic / animal / day and 3.80 mg BVSM light anionic / animal / day intranasally.

 2) Rabbit.

 The aim of this study is to evaluate the local tolerance of 4 formulations of BVSM (light anionic and cationic BVSM, BVSM, cationic and anionic PSC) after intranasal administration twice daily for 7 consecutive days in rabbits.

 Standard toxicity tests for mortality, clinical signs, body weight, food consumption are performed daily during the study. At the end of the treatment period the animals are sacrificed. All macroscopic lesions and the pulmonary system and nasal cavities are preserved. Histological examination of nasal cavities (6 levels), trachea (2 levels), main bronchi and lungs are performed on all animals.

 General Toxicity: No evidence of a general reaction is noted during the study. In particular, body weight, feed consumption of treated animals is comparable to that of control animals.

 Microscopic examination: Histological examination of the nasal cavities reveals the absence of any inflammatory and / or hyperplastic modification related to treatment at any level of examination. Small events observed in microscopy in nasal cavities, tracheas, bronchi and lungs are those commonly seen in untreated laboratory rabbits and can not be considered to be of toxicological significance.

 Some clinical signs have been noted in control animals and treated animals and are therefore attributable to repeated nasal administration. The effects are comparable for all substances tested, whatever their nature. Histologically, these clinical signs are not correlated with microscopic observations.

 The substances tested are well tolerated by the rabbit when administered twice daily for 7 days nasally.

 Example 3: Synthesis of the BVSM.

 1) Synthesis of anionized BVSM - PSC.

500 g of maltodextrin Glucidex (ROQUETTE,
Lestrem, France) are introduced into a 10 liter reactor (TRIMIX) with 2 liters of demineralized water. After solubilization at 40C, 500 ml of 10M sodium hydroxide (NaOH) are added with mechanical stirring.

 When the temperature of the solution is stabilized at 40C, 1700 ml of 10 M NaOH and 283.3 ml of phosphorus oxychloride (POC13) are introduced under controlled flow rates. The crosslinking reaction proceeds with mechanical stirring for 20 hours. At the end of the addition of the reagents, the reaction mixture is stirred for 15 minutes. A volume of 5 liters of demineralized water is then added. The pH is brought back to 7.0 with glacial acetic acid.

 The hydrogel obtained is ground by means of a high-pressure homogenizer (RANNIE Lab). The pressure exerted is 500 bars. At the end of this step, the dispersed matrices have an average diameter of 60 nm.

 The purification is carried out by successive steps of (1) 0.45 μm microfiltration to remove too large particles, (2) constant volume diafiltration to remove small molecules (salts, polysaccharide fragments).

 Finally, the anionic polysaccharide [PSC] nuclei are concentrated and recovered in sterile flasks.

 2) Svnthesis of anionic BVSM-Liaht.

 The anionic polysaccharide nuclei are diluted with osmosis water in the proportion of 1 mg / ml (example: 250 mg PSC / 250 ml). The dispersion is subjected to magnetic stirring (5-10 minutes) and then homogenized (RANNIE Minilab) at 400 bars for 3 minutes. The PSC dispersion is placed in a bain-marie at 800C.

 The lipids (example: DPPC, DPPC mix and cholesterol), in powder form, are weighed such that the total mass represents, for example, 30% (w / w) of the mass of the PSCs (75 mg of lipids for 250 mg PSC). The constitutive lipids of the membrane are mixed and solubilized with 2 ml of 95% ethanol (v / v).

The homogenizer is brought to a temperature of 600C. Concomitantly, the PSCs dispersed at 800C are subjected to magnetic stirring and the ethanolic solution of lipid is injected into the dispersion of
PSC.

 The anionic light BVSM thus obtained are then stored under sterile conditions after filtration over 0.2 μm.

 3) Svnthesis of cationic PSCs.

 500 g of Glucidex maltodextrins (ROQUETTE) are solubilized with 0.880 liters of water at 200C with stirring. Then, 7 g of NaBH4 are introduced and the mixture is left stirring for 1 hour.

 220 ml of 10 M NaOH are added and then 30.25 ml of epichlorohydrin (Fluka). After 12 hours of reaction, 382.3 g of glycidyltrimethylammonium chloride (Fluka) are introduced and the mixture is stirred for 10 hours.

 The gel obtained is diluted with 8 liters of demineralized water and brought back to pH 7 with glacial acetic acid.

It is then milled through a high homogenizer
4) Vnthesis of BVSM Liaht cationiaues.

 The cationic polysaccharide rings are diluted with osmosis water in the proportion of 1 mg of PSC / 1 ml of water. The dispersion is placed firstly with magnetic stirring (5-10 minutes) and then homogenized (RANNIE Minilab) at 400 bars for 3 minutes. The PSC dispersion is placed in a Bainmarie at 80 C.

Lipids (example: DPPC, or mixture of
DPPC with cholesterol), in powder form, are weighed so that the total mass represents, for example 30% (w / w) of the mass of PSC (75 mg of lipids per 250 mg of PSC). The constitutive lipids of the membrane are mixed and solubilized with 2 ml of 95% ethanol (v / v).

 The homogenizer is brought to a minimum temperature of 600 ° C. by circulating water in a closed circuit.

Concomitantly, the PSCs dispersed at 800C are subjected to magnetic stirring and the ethanolic solution of lipid is injected into the dispersion of
PSC.

 The cationic light BVSMs thus obtained are then stored under sterile conditions after filtration over 0.2 μm.

 5) Synthesis of neutral BSCM PSC.

 500 g of Glucidex maltodextrins (ROQUETTE) are solubilized with 0.880 liters of water in a reactor thermostated at 200C with stirring. Introduce about 7 g of NaBH4 and leave stirring for 1 hour.

220 ml of 10M NaOH are added and then 30.25 ml of epichlorohydrin (Fluka). After 12 hours of reaction, dilute the gel with 8 liters of demineralized water and neutralize by addition of glacial acetic acid.

 The hydrogel obtained is ground by means of a high-pressure homogenizer (RANNIE Lab). The pressure exerted is 400 bars. At the end of this step, the dispersed matrices have an average diameter of 60 nm.

 The purification is carried out by successive steps of (1) 0.45 μm microfiltration to remove too large particles, (2) constant volume diafiltration to remove small molecules (salts, polysaccharide fragments).

 Finally, the PSCs are concentrated by sterile filtration and collected in sterile vials.

 6) Svththesis of light neutral BMSMs.

 Neutral polysaccharide cores are diluted with osmosis water in a glass receptacle (250 mg PSC / 250 ml). The dispersion is placed firstly with magnetic stirring (5-10 minutes) and then homogenized (RANNIE Minilab) at 400 bars for 3 minutes. The PSC dispersion is placed in a bain-marie at 800C.

 The lipids (eg DPPC), in powder form, are weighed so that the total mass represents, for example, 30% (w / w) of the mass of the PSCs.

The constitutive lipids of the membrane are solubilized with 2 ml of 95% ethanol (v / v).

 This new dispersion is homogenized at 450 bars for 25 minutes in closed circuit at 600C minimum. At the end of this step, the preparation is introduced into a glass flask then subjected to low pressure at 600C to remove the ethanol remained in solution.

The neutral light BMSMs thus obtained are then stored under sterile conditions after filtration over 0.2 μm.
Example 4: Preparation of the formulations of active substances.

 1) DAGO.

 150 mM PBS buffer: One sachet of PBS (Sigma 1000-3) is dissolved in 1 liter of distilled water.

10% NaDOC solution: 1 g of NaDOC (sodium deoxycholate, Sigma D6750) is dissolved in 10 ml of distilled water.
a) Witness DAGO (administration in)
- 100 μg of DAGO / dose / 10 p1
- packaging in 15 mM PBS
- Final volume for a dose = 10 ul
Preparation of the DAGO Control (300 μl total volume) 3 mg of DAGO (Sigma E7384) are dissolved in 270 μl of distilled water. 30 μl of 150 mM PBS are added.

The solution is then vortexed.
b) Nasal administration of DAGO and NaDOC 1%.

- 100 μg of DAGO / dose / 10 μl
0.1 μg of NaDOC (ie 1%)
- packaging in 15 mM PBS
Final volume = 10 ul = 1 dose.

 Preparation of the DAGO Control: 3 mg of DAGO (Sigma E7384) are solubilized in 270 μl of distilled water, 30 μl of 150 mM PBS. The solution is then vortexed.

 Preparation of the 1% NaDOC solution 50 μl of 10% NaDOC solution in 450 μl of water.

5 μl of NaDoc are then administered twice, then 2 to 3 minutes later, 5 μl of DAGO are administered twice.
c) Witness DAGO (SC administration)
- 100 pg of DAGO
- packaging in 15 mM PBS
- Final volume = 10 pl = 1 dose.

Preparation of a solution of DAGO at 5 mg / ml
3 μg of DAGO are solubilized in 600 μl of distilled water.

* Sample preparation to administer 400 μl of 5 mg / ml DAGO solution. 3.2 ml of distilled water and 400 μl of 150 mM PBS are added. The solution is then vortexed.
d) Formulation DAGO / BVSM (administration in)
- 100 pg of DAGO
- 2 pg of BVSM
- packaging in 15 mM PBS
- Final volume = 10 ul = 1 dose.

Preparation of Biovectors Typical SMBVs
Cationic light is diluted in osmosis water to obtain a concentration of 1.31 g of PSC per liter.

Preparation of the formulation (total volume of 300 μl): 3 mg of DAGO (Sigma E7384) are solubilized in 224.2 μl of distilled water, 45.8 μl of BVSM are added to 1.31 g of PSC / liter and 30 μl of 150 mM PBS. The solution is then vortexed.
e) DAGO 0.05 my ICV Administration.

0.05 mg DAGO / dose / 5 l (15mM PBS
Preparation of a solution of DAGO at 6 mg / ml: diluted to 10 th to obtain a concentration of 0.6 mg / ml
- 10 μl DAGO solution at 0.6 mg / ml
- 530 μl of water
- 60 ul PBS 150mM
2) Morphine.

 Preparation of the 150 mM PBS buffer: A sachet of PBS is dissolved in 1 liter of distilled water.

Preparation of a 10% solution of NaDOC 1 g of NaDOC (sodium deoxycholate, Sigma D6750) is dissolved in 10 ml of distilled water.
a) Morphine control (in administration)
- 500 μg of Morphine
- packaging in 15 mM PBS
- Final volume = 10 l = 1 dose.

Preparation of the Morphine 10 mg
Morphine hydrochloride (Francopia, France) are solubilized in 180 μl of distilled water and 20 μl of 150 mM PBS. The solution is then vortexed
b) NASAL Administration of Morphine and
NaDOC 1%
- 500 μg of Morphine / dose / 10 μl
0.1 μg of NaDOC (ie 1%)
- packaging in 15 mM PBS
- Final volume = 10 ul = 1 dose.

Preparation of the Morphine control: 10 mg of
Morphine are weighed and put in a Venoject tube. 180 μl of distilled water and 20 μl of 150 mM PBS are added. The solution is then vortexed.

 Preparation of the 1% NaDOC solution 50 l of 10% NaDOC solution in 450 l of water.

5 μl of NaDoc are then administered twice, followed by 2 to 3 minutes, after which 5 μl of morphine are administered twice.
c) Morphine control (sc administration)
- 500 μg of Morphine.

 packaging in 15 mM PBS.

 - Final volume = 10 pl = 1 dose.

Preparation of the sample to be administered 10 mg of Morphine are solubilized in 3.6 ml distilled water and 400 μl 150 mM PBS. The solution is then vortexed.
d) Morphine Formulation / BVSM (in administration):
- BVSM Lisht cationic 2 ua
500 μg of Morphine 2 2 μg of BVMS
final volume: 10p1 in 15mM PBS
Preparation of the BVSM The cationic light BVSM are diluted in osmosis water to obtain a concentration of 1.31 g of PSC per liter.

 Preparation of the formulation (total volume of 200 μl): 10 mg of Morphine are solubilized in 149.5 μl of distilled water, 30.5 μl of biovector diluted to 1.31 g / l and 20 μl of 150 mM PBS. The solution is then vortexed.

- Cationic light BVSM 0.2 g:. 500 g of morphine
. 0.2 g of cationic light BVSM
Final volume: 10 ul in 15mM PBS
Preparation of the BVSM: The cationic light BVSM are diluted in water to obtain a concentration of 0.131 g / l in PSC.

 Preparation of the formulation (total volume of 200 μl): same as previous example.

- BVSM Liaht cationic 10 ua. 500 g morphine
10 μg BVSM Light cationic. Final volume 10 l in 15mM PBS.

 Preparation of the BVSM The cationic Light BVSM are diluted to 13.1 g / l PSC.

 Preparation of the formulation (total volume 200 μl): 10 mg of morphine are solubilized in 164.7 μl of distilled water 15.3 μl of biovector at 13.1 g / l and 20 l of 150 mM PBS. The solution is then vortexed.

Cationic PSC 0.2 ua: 500 mg morphine. 0.2 g cationic PSC
Final volume 10 μl in 15 mM PBS
Preparation of the BVSM: The cationic PSC are diluted in water to obtain a concentration of PSC of 0.27 g / l
Preparation of the formulation (total volume 200 μl): 10 mg of morphine are solubilized 165.2 μl of distilled water, 14.8 μl of biovector at 0.27 g / l in PSC and 20 μl of 150 mM PBS.

- BVSM Light anionic (Phosphate) 2 uq. 500 μg morphine. 2 g BVSM Light anionic
Final volume 10 ul in 15mM PBS
Preparation of the BVSM The anionic light BVSM are diluted 10 th in water to obtain a concentration of 0.61 g / l in PSC
Preparation of the formulation (total volume 200 μl): 10 mg of morphine are solubilized in 114.4 μl of water, 65.6 μl of 0.61 g / l PSC and 20 μl 150 ml PBS.

 Example 5: Results.

 The antinociceptive activities of the different preparations were quantified as the increase in the lag time of withdrawal of the tail of the mouse subjected to nociceptive stimulation (light beam directed on the end of the tail).

The latency times are measured twice before the administration of the formulations and at 15 minutes, 30, 60, 90, 120, 240 minutes after administration. Each batch is composed of 10 Swiss mice of 29-31 grams. A maximum threshold of 6 or 8 seconds of stimulation is set to prevent tissue burn. When the animal has not responded to this "cut-off", analgesia is considered 100%.

 An antinociceptive effect of 15% is considered minimal for consideration.

1) Administration protocols.
a) Nasal administration (in).

 5 μl of sample are administered with a P20 pipette into each nostril. That is a total administered volume of 10 μl.

For the test Active ingredient + NaDOC
Administration of 2 μl 5 μl NaDOC 1% then 2 to 3 minutes after administration of 2 μl of active ingredient.
b) Subcutaneous administration (sc)
200 μl of sample are injected subcutaneously to each mouse.

 2) Results with the Morphine.

 Figures 2 and 3 show that morphine administered nasally has an antinociceptive effect.

This effect is maximal at 30 minutes and decreases rapidly one hour after administration.

 In the presence of cationic light BVSM we observed a maximal antinociceptive effect at 30 minutes and a duration of this effect greater than that of morphine alone, with a decrease from 2 hours.

 Figure 4 shows, in the presence of cationic BVSM-PSC, a maximal antinociceptive effect later than that of morphine alone, but its duration is as for LSSM Light cationic superior to that of morphine alone.

 The BVSM (Light type and PSC type) can increase the duration of action of morphine administered nasally.

Figure 5 shows, in the presence of BVSM
Light anionic, a superior effect and longer duration of action than those of morphine alone.

 In Figure 6, it is observed that morphine administered subcutaneously induces a rapid antinociceptive effect (about 90% after 15 minutes). The maximum effect decreases from 90 minutes. Morphine administered nasally in the presence of BVSM has a slightly lower maximal antinociceptive effect (approximately 80%). This effect decreases from 90 minutes, less rapidly than morphine alone systemically. 30 to 40% of the antinociceptive effect is still recorded after 4 hours in the presence of nasally administered BVSM, whereas the effect of morphine alone administered subcutaneously is no longer considered significant.

Figure 7 shows the effect of NaDOC at 1%, a substance known to promote the passage of active substances from the nose to the blood. By using
Na DOC during nasal administration of morphine, no increase in antinociceptive effect was observed compared with morphine alone. This observation suggests that the activity observed after nasal administration of morphine is not due to a passage of morphine from the nasal mucosa to the blood but rather to a direct passage from the nose to the brain (CSF in particular). This result confirms the results obtained by T. Sakane et al. (STP Pharma
Science 7 (1) 98-106 (1997)).

 As seen in Figure 3, there is a longer duration of effect than that of morphine in the presence of permeabilizer.

 Figures 8 and 9 show that in all cases where morphine is associated with a biovector, whatever its type, the area under curve is greater than that corresponding to morphine alone, phenomenon related to the change in duration. the antinociceptive effect in the case of the use of BVSM.

 Morphine in the presence of nasal BVSM appears to have an effect equivalent to that of morphine alone systemically. Bioequivalence of the routes of administration is observed using BVSM.

 3) Results with DAGO.

 Figure 10 shows that DAGO alone nasally is not active (antinociceptive effect below the threshold of 15%) the presence of BVSM allows the passage of DAGO to the brain and significant antinociceptive activity is observed.

 In FIG. 11, it is observed that systemically administered DAGO induces a peak of antinociception of 40% at half an hour after administration. This effect is fleeting and is below the 15% threshold in less than an hour. DAGO administered nasally in the presence of BVSM induces antinociceptive effect slightly delayed in time, but to reach the same intensity (40%) one hour after administration. However, this effect is still detectable 2 hours after administration. A given dose of DAGO nasally in the presence of BVSM is therefore more effective than the same dose systemically.

This observation argues for the hypothesis of a passage of DAGO from the nose to the brain that would be induced by the BVSM and that would not borrow the bloodstream.

 Figure 12 shows that NaDOC induces a mild antinociceptive effect of DAGO. It allows a passage of DAGO from the nasal mucosa to the brain.

Figure 13 indicates, however, that Na DOC allows the
DAGO to reach the brain and express antinociceptive activity. However, this activity is lower than that observed in the presence of BVSM. Na
DOC is a powerful permeabilizer (and toxic) mucous membrane that promotes the passage of substances in the blood. BVSM are not toxic at the mucosal level. However, they are more powerful than the Na DOC for the activation of the passage from the nose to the brain, either by taking the blood path or by taking another passage.

 Figure 14 shows that in the presence of BVSM, the antinociceptive effect is less rapid and a little less important, but again the duration of action is longer.

 This result is confirmed by FIG. 15 where it is noted that the area under the curve corresponding to the administration of DAGO and BVSM is greater than the areas corresponding to the administration (whatever the mode) of DAGO alone or in the presence of NaDOC permeabilizer.

 With regard to delayed administration, it can be seen in FIG. 16 that the shape of the curves corresponding to the administration of morphine, then of BVSM or of BVSM then of morphine is very similar to that corresponding to the joint administration of morphine and BVSM of Figure 4. This is confirmed in Figure 17, where there is an area under curve for the delayed administration of morphine close to that corresponding to the joint administration of morphine and BVSM. These results clearly show that the concept of carrier assigned in the prior art to the BVSM is not applicable here.

Claims (17)

 1) Use in a pharmaceutical composition for the nasal administration of a hydrophilic particle bearing or not ionic ligands and possibly coated with a layer of amphiphilic compounds, to obtain and / or increase the passage of an active agent administered nasally to the CNS.  2) Use according to claim 1 for obtaining and / or increasing the efficacy of a pharmacologically active substance in the CNS administered nasally.  3) Use according to one of claims 1 to 2, characterized in that the hydrophilic particle has a size between about 20 and 200 nm and preferably between about 50 and 100 nm.  4) Use according to any one of claims 1 to 3, characterized in that the amount of hydrophilic particle in the pharmaceutical composition for nasal administration is, by weight, less and advantageously much less than the amount of agent active administered nasally.  5) Use according to claim 4, characterized in that the amount of hydrophilic particle, expressed by weight of dry hydrophilic polymer, is at least 5 times and advantageously at least 10 times lower, by weight, relative to the amount of agent. active administered nasally.  6) Use according to any one of claims 1 to 5, characterized in that the active agent is a therapeutic agent, chosen in particular from: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonian, antiepileptic, antimigraine, or any other agent useful in the treatment of neurodegenerative diseases, psychiatric or behavioral disorders, anticancer, antiviral, antiprions, antibiotics, or active agents such as growth factors, nucleic acids, vitamins, hormones.  7) Use according to any one of claims 1 to 5, characterized in that the active agent is a diagnostic agent, chosen in particular from: antibodies and molecules for targeting CNS receptors, medical imaging agents used in MRI, scintigraphy, PET and ultrasound.  8) Pharmaceutical composition for the nasal administration of an active agent to be delivered to the CNS, characterized in that it comprises said active ingredient and an effective amount of a hydrophilic polymer particle, with or without ionic ligands and optionally coated with a layer of amphiphilic compounds, to obtain and / or increase the passage of said active agent administered nasally until CNS.  9) Pharmaceutical composition according to claim 8, characterized in that it comprises an effective amount of said hydrophilic particle to obtain and / or increase the effectiveness of a pharmacologically active substance in the CNS.  10) Pharmaceutical composition according to one of claims 8 to 9, characterized in that the hydrophilic particle has a size between about 20 and 200 nm and preferably between about 50 and 100 nm.  11) Composition according to any one of claims 8 to 10, characterized in that the amount of particles is, by weight, less and advantageously much less than the amount of active agent.  12) Composition according to claim 11, characterized in that the amount of hydrophilic particle, expressed by weight of dry hydrophilic polymer, is at least 5 times and advantageously at least 10 times lower, by weight, relative to the amount of agent. active administered nasally.  13) Pharmaceutical composition according to any one of claims 8 to 12, characterized in that it further contains any vehicle pharmaceutically compatible with the BVSM and / or the active agent, and optionally a permeabilizing agent of the nasal mucosa .  14) Pharmaceutical composition according to any one of claims 8 to 13, characterized in that the active agent is a therapeutic agent, chosen in particular from: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonian, antiepileptic, anti-migraine, or any other agent useful in the treatment of neurodegenerative diseases, psychiatric or behavioral disorders, anticancer, antiviral, antiprions, antibiotics, or active agents such as growth factors, nucleic acids, vitamins, hormones.  15) Pharmaceutical composition according to any one of claims 8 to 13, characterized in that the active agent is a diagnostic agent, chosen in particular from: antibodies and molecules for targeting CNS receptors, imaging agents medical devices used in MRI, scintigraphy, PET and ultrasound.  16) Process for the preparation of a pharmaceutical composition according to any one of Claims 8 to 15, characterized in that the active agent is mixed with a lower and advantageously much lower amount, by weight, relative to said active ingredient. of hydrophilic polymer particles, bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds.  17) Process according to claim 16, characterized in that the active ingredient and the particles are mixed in a ratio, by weight, of at least 5 parts of active agent for a part of particle, and advantageously of at least minus 10 parts of active agent for one part of particle.