WO2000033810A1

WO2000033810A1 - Use of vitamin b12 in a nasal composition for delivering active agents to the central nervous system

Info

Publication number: WO2000033810A1
Application number: PCT/FR1999/003060
Authority: WO
Inventors: Didier Betbeder; Sandrine Sperandio
Original assignee: Biovector Therapeutics (S.A.)
Priority date: 1998-12-09
Filing date: 1999-12-08
Publication date: 2000-06-15
Also published as: FR2787029A1

Abstract

The invention concerns the use of vitamin B12 in a pharmaceutical composition to be administered by nasal pathway, to provide and/or increase the passage for an active agent, present in said composition to the CNS. The invention also concerns pharmaceutical compositions for nasal administration of active agents to the CNS, and the method for preparing them.

Description

USE OF VITAMIN B12 IN A NASAL COMPOSITION FOR THE DELIVERY OF ACTIVE AGENTS TO 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 interested in the passage and delivery of therapeutic or diagnostic active agents in the central nervous system. So raw cancer and cardiovascular problems are the main ones. Causes of death in developed countries, diseases linked to the nervous system are among the first factors that expose populations to health problems (morbidity). Advances in biology and neurology have, however, been difficult to materialize in the form of new diagnostic and therapeutic tools for the central nervous system (CNS), in particular because they are particularly difficult to administer effectively. There is indeed no diffusion of substances and nutrients from the blood capillaries at the level of the spinal cord and the brain as at the level of the other organs. Exchanges are limited at the level of the blood-brain barrier (BBB) and the choroid plexus, where cerebrospinal fluid (CSF) is formed. This brings nutrients, hormones and leukocytes 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 by rejecting others.

The delivery of exogenous substances to the CNS cannot be done by conventional systemic therapeutic routes, with the exception of extremely lipophilic compounds of low molecular weight or of compounds capable of selective transport linked to the presence of a transporter or endocytosis phenomena (Ikumi Tamai et al. Drug Delivery through the BBB, (1996) Advanced Drug Delivery Reviews 19: 401-424). Most drugs used in neuropsychiatry today as for example codeine, benzodiazepines, phenytoin, etc. are liposoluble pharmaceutical products which cross the BBB after oral administration (Oldendorf, .H. 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 used 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 Flo Metab. 11: 581-586) entry of drugs to the brain.

The BBB in particular 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 therapeutic potential such as peptides, ion channels, neuronal receptors, neurotrophic factors and genetic determinants are to date limited. in their medical applications due to their difficulty of administration to the CNS as xenobiotics.

To overcome these drawbacks, it has been proposed to make a given molecule liposoluble by chemical transformation (lipidization). Lipidization is used for the production of pro-drugs, liposoluble analogs of hydrophilic pharmacological compounds capable of crossing biological membranes. However, the BBB expels this type of compound to the blood and their half-life is extremely short. For this, tissue sequestration systems, or even inhibition of P-glycoprotein (Ikumi Tamai et al. Drug Delivery through the BBB, (1996) Advanced Drug Delivery Reviews 19: 401-424) are being developed in parallel. Chemical changes to the molecule are delicate and can compromise the affinity for the receptor responsible for biological activity. Lipidization can only be used for molecules of relatively low molecular weight (less than 1,000, or even 600-800), ie at best tetra-peptides and penta-peptides (Pardridge, WM Peptide lipidization and liposomes. In Peptide Drug Delivery to the Brain pp 129-140 (1991) Raven Press, New York). Small liposomes (25-50 nm) could be of interest for the delivery of biologically active products at the central level. However, the larger 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, in particular due to 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 pp 140-148 (1991) Raven Press, New York.

In addition, intracerebro-ventricular implants of several types are being tested, such as:

- an implant of genetically modified cells for the expression of protein factors, encapsulated in a hollow fiber (BioWorld Today, American Health Consultants Vol 8 35: 1,4, 1997), a micro-pump loaded with drugs for a few months (Winn , SR et al. Polymer-encapsulated cells genetically modified to secret human nerve growth factor promote the survival of axotomized septal cholinergic functions. PNAS (1994) Vol 19: 2324-2328), or controlled release formulations using lipids, where drugs are encapsulated in sponge-structured microvesicles (Chatelut E. et al

Sustained-release methotrexate for intracavitary chemotherapy. J. Pharm. Se (1994) Vol 83, 3: 429-432). But this type of administration is extremely cumbersome because it involves surgical intervention.

Formulations of active substances which penetrate the CNS without having to be implanted are sought, in particular by exploiting the endogenous transporters of the BBB. These transporters contain 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 nervous tissue. This can be obtained, for example, by fusion of the active protein with a protein having a cellular receptor at the central level, as described in the international patent application PCT published under the number WO95 / 02421.

Other recent studies have shown the passage of a hexapeptide, dalargine, through the BBB of mice, after intravenous administration of a formulation in nanoparticles (230 nm) of butyl cyanoacrylate, provided that these particles are covered with surfactant, polysorbate 80. These peptide-charged particles are said to penetrate 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 particles Brain Research (1995) 674: 171-174).

The first administration of a recombinant peptide at the level of the CNS was carried out by intra-cerebroventricular infusion, directly into the CSF. This method allows delivery at therapeutic concentrations and close to the ventricular walls, the CSF not penetrating into 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 delicate to use, has been used to administer, for example, methotrexate to treat meningeoid leukemia, penicillin for bacterial meningitis, immunoglobulins for viral meningitis and morphine for chronic pain.

The nasal cavity represents a natural route of administration of active substances which offers many potential advantages including the relative surface of the nasal mucosa, the richly vascularized epithelium, the permeability to lipophilic substances and the relative permeability to hydrophilic. In addition, the blood supply to this mucosa, linked to the pulmonary circulation, avoids the liver (Harris, AS Review: Clinical opportunities provided by the nasal administration of peptides. Journal of Drug targetting Vol.l, pp 101-116 (1993 )).

The cells making up the epithelium of the nasal mucosa are joined by tight junctions

(tight junctions), which limit the passage of molecules between the lumen of the cavity and the blood vessels. On the other hand, the basal pole of the olfactory neurosensory cells of the nasal epithelium lengthens in an axon which ends in the olfactory bulb of the brain. This axon crosses the basement membrane of the mucosa and the submucosal space. The olfactory neuronal pathway could thus constitute a gateway to the brain. In addition, the subarachnoid space extends at the level of the axons of the neurosensory cells in digitations which come into contact with the nasal submucosa. There is therefore a flow of CSF coming from the olfactory lobe towards the space under mucosal. The particular anatomy of the nasal mucosa therefore confers an unusual neighborhood with the CSF.

Furthermore, at the end of the 19th century, researchers postulated that the olfactory nerve could allow the transport of tracer substances and various organisms from the nasal mucosa to the olfactory bulb and to the CNS.

In 1970 De Lorenzo (De Lorenzo Taste and Smell in vertebrates J & A Churchill ltd (1970), 151-176) intranasally administered gold particles to the monkey and estimated the speed 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 like horseradish peroxidase and albumin, a movement of the nasal mucosa towards the subarachnoid space.

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 liposoluble substances, such as progesterone allows to reach directly the cerebrospinal fluid without entering the peripheral blood compartment. The bioavailability of progesterone being greater after nasal insufflation than after intravenous infusion. Peptides, highly hydrophilic substances penetrate the membrane diffusion barriers between the nose and the subarachnoid olfactory space at considerably lower speeds than progesterone.

The PCT patent application published under the number WO91 / 07947 describes pharmaceutical compositions for the nasal administration of active substances which use this neural pathway. The compositions associate the biologically active product with a transporter capable of inducing absorption, the drug would then undergo retrograde and / or anterograde neural transport.

Sakane (Sakane & Al STPPharma Sciences (1997), 7, (1), 98-106) recently published a systematic study on the direct transport of the nasal cavity to cerebrospinal fluid: the study of cephalexin, hydrophilic antibiotic, shows clearly direct transport from the nasal cavity to the cerebrospinal fluid. the study of different sulfonamides with variable lipophilicity shows that transport is dependent on lipophilicity and on the non-ionized fraction of the active principle. the study of dextrans of different molecular weights has shown that the active ingredients can be transported up to 20KDa of molecular weight. finally a study of peptide transport was carried out. The molecule chosen is an oligopeptide analogous to 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 methods of nasal instillation in rats, aimed at minimizing the leakage of the product into the pharynx, the amounts of peptide obtained in the cerebrospinal fluid are very low, 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 now discovered that vitamin B12 is capable of activating the passage of hydrophilic substances towards the central nervous system (CNS).

Vitamin B12 is the name generally used for all cobalamins. Cyanocobalamin and hydroxocobalamin are the main forms in clinical use. Vitamin B12 preparations are used primarily in the treatment and prevention of B12 deficiency. They are generally administered intramuscularly, at doses of 250 to 1000 μg at the start of treatment.

In order to overcome the drawbacks of this mode of administration, the prior art has proposed the administration by the nasal route of vitamin B12 (Van Asselt DZ in Br. J. Clin. Pharmacol. (1998), 45, 1, p. 83-6; Slot WB in Gascroenterology (1997), 113, 2, p. 430). These studies report that the nasal absorption of hydroxocobalamin is rapid, strong and well tolerated and that it leads to a significant increase in cobalamin. in the plasma. The administration of vitamin B12 by the nasal route at doses of 50 to 1000 μg per spray for the treatment of deficiencies has also been described in the international PCT patent applications published under No. WO86 / 05987 and WO86 / 05988.

Vitamin B12 is also known for its analgesic properties. In combination with other B vitamins (B1, B6), by injection, it contributes to the antinociceptive effect of the whole, this effect being largely due to B6. (Jurna I. in Klin. Wochenschr. 1990, 68, 2, p.129-35).

Canadian Patent No. 2,034,576 reports the use of vitamin B12 alone, in humans as an analgesic and to reduce or prevent inflammation, at very high doses, between 5-100 mg / d.

Furthermore, European patent application No. 108,017 describes the use of vitamin B12 in combination with central analgesics of the morphine type or its derivatives. In this case, it is its own analgesic properties that are sought in order to allow the reduction of 2 to 4 times the dose of morphine used in order to reduce the undesirable effects. Thus, in the injectable solutes described, the amounts of B12 or analogs used are of the order of mg or ten mg.

In order to assess the properties of vitamin B12 on the passage from the nose to the brain of active substances, several drugs of central antinociceptive interest have been studied: buprenorphine, DAGO and morphine. The opiate-type compounds that have been tested are molecules of low molecular weight, between 50 and 10,000, relatively water-soluble with the exception of buprenorphine. Their passage through the CNS is measured by their antinociceptive activity. Opiates produce their effects by interaction with three types of receptors: mu, delta and kappa. The selected compounds, including in particular the derivatives of the endogenous enkephalin, have a specific action on the opiate receptors mu (Pasternak GW et al Morphine-6-glucuronide, a potent mu agonist, Life Sci. 41: 2845-2849, (1987); Kosterlitz HW, et al. Br. J. Phar acol 73, 299 (nineteen eighty one)). These receptors bathe in the CSF, therefore the observation of a biological activity indicates the passage of the substance up to this level. These compounds are easily degraded and are unlikely to reach the brain systemically when given in low doses. DAGO is a pentapeptide with the formula:

Tyr, D-Ala, Gly, N-Me-Phe, Gly-ol, and molecular weight 513. It is an analogue of the amino terminus of beta endorphin, known to be more resistant to proteases than 1 ' endogenous enkephalin. Morphine is the prototype of the opioid analgesic and narcotic, the main alkaloid of opium. Used as hydrochloride, its molecular weight is 376. In medical applications, it can be administered parenterally for postoperative pain and orally for cancer-related pain.

Buprenorphine is an oripavine, more lipophilic than morphine, derived from thebaine. Used in the form of hydrochloride, its molar mass is 467. It is a powerful analgesic, 25 to 50 times more than morphine, which is endowed with a partial agonist-antagonist activity of opiate receptors. It is used in the treatment of acute or chronic pain by parenteral or sublingual administration.

The work carried out in the context of the present invention with these compounds has made it possible to demonstrate new properties of vitamin B12 as an activator for the passage of active agents from the nose to the central nervous system. More particularly, vitamin B12 has the following properties:

- It allows the passage from the nose to the brain of active substances in doses where the substance alone is not active by nasal administration. - It increases the effect of a dose of active substance by nasal administration compared to the drug alone administered by the same route

It prolongs the duration of action of the active substance administered by the nasal route when this substance alone is already active by this route.

These observations tend to suggest that vitamin B12 does not act by simple passive effect but that it induces an active phenomenon responsible for the penetration of the substance. Indeed, vitamin B12, used alone at doses of 0.1 to 10 μg does not induce any antinociceptive activity. We are therefore very far from the massive doses that must be administered to obtain analgesic activity. The work carried out by the Applicant suggests that vitamin B12 interacts with the nasal mucosa by promoting direct passage from the nose to CSF. However, a passage from the nose to the blood and then from the blood to the brain through the BBB cannot be excluded at least as one of the components of the effects observed.

Consequently, the invention relates to the use of vitamin B12, in an aqueous pharmaceutical composition administered by the nasal route to a human or animal subject, to obtain and / or increase the passage of an active agent present in said composition at the level of the SNC.

The new use according to the invention is remarkable in that vitamin B12 makes it possible to obtain and / or promote the passage of the nose to the CNS of active substances for the diagnosis or the treatment of a CNS affection, and more particularly small molecules and / or fragile molecules such as peptides or proteins.

Obtaining and / or increasing the passage of an active agent administered via the nasal route to the CNS results in the case where the active agent is a pharmacologically active substance at the level of the CNS by obtaining and / or increasing the effectiveness of said substance.

Quite surprisingly, the Applicant has found that the nasal administration of active substances at the level of the CNS in the presence of vitamin B12 at infrali inal doses makes it possible to obtain an increase in the duration of action of the active substance and in its intensity.

The doses of vitamin B12 used are very low compared to those of the active agent, and the staggered administration of vitamin B12 and the active agent in the same dose ratio makes it possible to obtain the same effect.

More particularly, the Applicant has defined that the quantity, by weight, of vitamin B12 in the pharmaceutical composition for administration by the nasal route according to the invention is less and advantageously very less, by weight, than the quantity of active agent. Thus, in the use according to the invention, the amount of vitamin B12 in the pharmaceutical composition for administration by the nasal route is between 1 μg and 1 mg.

The invention therefore also relates to the use of an aqueous composition comprising vitamin B12 advantageously at the dose indicated above, and at least one active agent, for the preparation of a medicament for nasal administration to be used in diagnosis or therapy of an S duC condition in a human or animal subject.

Depending on the nature and properties of the active agent, the invention relates both to the field of diagnosis and to that of the treatment and prevention of a disease of SΝC.

As a therapeutic agent, mention may be made of those chosen from: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonians, antiepileptics, antimigraine drugs, or any other agent useful in the treatment of neurodegenerative, psychiatric or behavioral disorders, anticancer drugs , antivirals, antiprions, antibiotics, or even active agents such as growth factors, nucleic acids, "ita ines, 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. Vitamin B12 used for nasal administration according to the invention can be presented in a medicament which may or may not also contain the active agent. In case the drug containing vitamin B12 does not also contain the active agent, a delayed nasal administration of vitamin B12 and the active agent (s) may be considered, in order or in the other.

In the use according to the invention, the pharmaceutical composition for nasal administration can also contain any vehicle which is pharmaceutically compatible with vitamin B12 and / or the active agent, as well as optionally a permeabilization agent for the nasal mucosa.

The invention therefore also relates to an aqueous pharmaceutical composition for nasal administration of at least one active agent to be delivered to the CNS, characterized in that it comprises at least one active agent and vitamin

B12.

The pharmaceutical composition according to the invention is remarkable in that it makes it possible to obtain and / or increase the passage of the active agent administered via the nasal route to the CNS.

Obtaining and / or increasing the passage of an active agent administered via the nasal route to the CNS results in the case where the active agent is a pharmacologically active substance at the level of the CNS by obtaining and / or increasing the effectiveness of said substance, administered via the nasal route, at the level of the CNS.

In the composition of the invention, the active agents are simply mixed with vitamin B12. The amount of vitamin B12 in the pharmaceutical composition according to the invention is lower and advantageously very lower, by weight than the amount of active agent. It is particularly preferred to use a dose of vitamin B12, in the pharmaceutical composition for administration by nasal route according to the invention, of between approximately 1 μg and 1 mg. This dose of vitamin B12 is very much lower than that described in one prior art for the use of vitamin B12.

As indicated previously, the vitamin B12 used for the administration by nasal route according to the invention, can be presented in a pharmaceutical composition containing or not also the active agent. Thus, the nasal administration of vitamin B12 and of the active agent (s) can be carried out thanks to the preceding compositions comprising these two components, or consist in the successive taking of one then of the other of these constituents, each presented in a separate pharmaceutical composition, in respective doses in accordance with the doses indicated above for a composition containing the two components.

The invention therefore also relates to a pharmaceutical composition containing as active substance only vitamin B12 at the dose defined above, the active agent to be delivered at the level of the CNS being itself contained in a composition containing or not containing vitamin B12. The invention therefore relates to a pharmaceutical kit comprising: a first composition comprising vitamin B12 advantageously combined with a pharmaceutically acceptable vehicle and compatible with vitamin B12,

- A second composition comprising at least one active agent associated with a pharmaceutically acceptable vehicle and compatible with said active agent, One form of use, particular of this pharmaceutical kit, consists in grouping the two compositions on a common nasal applicator so as to deliver both said compositions simultaneously. In the case of a successive administration of the two compositions, the nasal administration of vitamin B12 can be carried out before or after the nasal administration of the active agent (s).

The pharmaceutical composition for nasal administration of the invention may of course also contain any vehicle which is pharmaceutically compatible with vitamin B12 and / or the active agent, as well as optionally a permeabilization agent for the nasal mucosa. These compositions can be presented in any form suitable for administration by the nasal route 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 principle is a therapeutic or diagnostic agent.

As previously, by way of therapeutic agent, there may be mentioned those chosen from: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonians, antiepileptics, antimigraine, or any other agent useful in the treatment of neurodegenerative, psychiatric or disorder disorders behavior, anticancer, antiviral, antiprion, antibiotics, or even active agents like 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. Pharmaceutical compositions according to

1 the invention may also include one or more agents permeabilizing the nasal mucosa, of the type of those defined above.

Finally, the invention relates to a process for the preparation of a pharmaceutical composition described above for the nasal administration of an active agent to be delivered to the CNS, consisting in mixing at least one active agent and a lower quantity and advantageously very lower by weight per relative to the quantity of said principle active, vitamin B12. It is more particularly preferred, in the process of the invention, to mix the active principle with 1 order of 1 μg and 1 mg of vitamin B12.

Other advantages and characteristics of

The invention will appear on reading the examples which follow concerning the study of the antinociceptive effect of morphine, DAGO and buprenorphine administered by the nasal route in the presence of vitamin B12 on a mouse model, and referring to the drawings in annex in which:

- Figures 1 and 2 illustrate the comparison of the antinociceptive effect obtained by nasal administration of morphine alone (500 μg) and morphine combined with increasing doses of vitamin B12 (0.1-10 μg). - Figures 2 and 4 illustrate the comparison of one antinociceptive effect obtained by nasal administration of DAGO alone (100 μg) and DAGO in the presence of 1 μg of vitamin B12.

- Figures 5 and 6 illustrate the comparison of the antinociceptive effect obtained by nasal administration of 8.5 μg of buprenorphine alone and buprenorphine in the presence of 1 μg of vitamin B12

1) Products used. - Morphine hydrochloride: Francopia - France

- DAGO: E7384 Sigma - France

- Vitamin B12 or cyanocobalamin: V2876 Sigma

France

- Buprenorphine hydrochloride: Sigma B9275 France

- PBS 150 mM (NaH ₂ PO ₄ / Na ₂ HPO ₄ 10mM, NaCll20mM, KC1 2.7mM): Sigma - France

2) Preparation of solutions. - Preparation of a vitamin B12 solution.

Vitamin B12 at 10 mg / ml: solubilization of 10 mg of vitamin B12 in 1 ml of water.

Vitamin B12 at 1 mg / ml: 100 μl of vitamin B12 at 10 mg / ml in 930 μl of water. Vitamin B12 at 0.2 mg / ml: 200 μl of vitamin B12 at 1 mg / ml in 800 μl of water.

- Preparation of a Morphine solution. Morphine at 60 mg / ml (Solution A): solubilization of 60 mg of morphine in 1 ml of water

- Preparation of a Buprenorphine solution. Buprenorphine 1 mg / ml: solubilization of 1 mg buprenorphine in 1 ml of water. We vortex well.

3) Preparation of samples.

The preparation of the samples is carried out extemporaneously, in venoject tubes by successive addition as indicated in Tables I to IV below:

Table I: Vitamin B12

Table II: Morphine in the presence of vitamin B12

Table IIII: DAGO in the presence of vitamin B12

Table IV: Buprenorphine in the presence of vitamin B12

4) Results. a) Measurement of antinociceptive activity (Ameur & Smith test).

The antinociceptive activities of the various preparations have been quantified as being the increase in the latency time of the 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 min, 30, 60, 90, 120, 240 minutes after administration. Each batch is made up of 10 male Swiss mice (Charles Rivers strains, USA) weighing 29-31 grams. A maximum threshold of 6 or 8 seconds of stimulation is set to prevent tissue burn. When the animal has still not responded to this cut-off, analgesia is considered to be 100%.

The latency times measured in seconds are reduced in% of antinociception:

% antinociception = 100 x (latency time - basic time) / (eut off - basic time)

An antinociceptive effect of 15% is considered minimal to be taken into account. b) Administration protocols.

For nasal administration (i.n.) 5 μl of sample is instilled using a P20 pipette into each nostril. Or a total administered volume of 10 μl. c) Effect of vitamin B12 alone.

The presence of 10 to 0.1 μg of vitamin B12 does not give any nasal antinociceptive activity. d) Nasal administration of morphine (dose of 500 μα) in the presence of vitamin B12.

The results are given in FIG. 1.

It is noted that the presence of 1 μg or 10 μg of vitamin B12 per 500 μg of morphine makes it possible to improve the antinociceptive activity of morphine by the nasal route.

At 0.1 μg of vitamin B12 per 500 μg of morphine, the antinociceptive activity obtained is similar to that obtained with morphine alone. e) Nasal administration of DAGO (dose of 100 uσ) in the presence of vitamin B12.

The results are given in FIG. 2.

The presence of 1 μg of vitamin B12 per 100 μg of DAGO makes it possible to improve the antinociceptive activity of the peptide by the nasal route f) Administration of buprenorphine (dose of 8.5 uσ) in the presence of vitamin B12.

The results are given in FIG. 3.

The presence of 1 μg of vitamin B12 for 8.5 μg of buprenorphine makes it possible to improve the antinociceptive activity of the active principle administered by the nasal route.

Claims

1) Use of vitamin B12 in an aqueous pharmaceutical composition administered by the nasal route in a human or animal subject, to obtain and / or increase the passage of an active agent present in said composition at the level of the CNS.

2) use according to claim 1 for obtaining and / or increasing the effectiveness of a pharmacologically active substance at the level of the CNS administered by the nasal route.

3) Use according to any one of claims 1 to 2, characterized in that the amount of vitamin B12 in the pharmaceutical composition for administration by nasal route is, by weight, less and advantageously much less than the amount of agent active administered nasally.

4) Use according to any one of claims 1 to 2, characterized in that the amount of vitamin B12 in the pharmaceutical composition for administration by the nasal route is between 0.1 μg and 1 mg.

5) Use according to any one of claims 1 to 4, characterized in that the active agent is a therapeutic agent, chosen in particular from: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonians, antiepileptics, antimigraine, or any other agent useful in the treatment of neurodegenerative, psychiatric or behavioral disorder, anticancer, antiviral, antiprion, antibiotic, or even active agents such as growth factors, nucleic acids, vitamins, hormones. 6) Use according to any one of claims 1 to 4, characterized in that the active agent is a diagnostic agent, chosen in particular from: antibodies and molecules making it possible to target CNS receptors, medical imaging agents used in MRI, scintigraphy, PET and ultrasound.

7) Aqueous pharmaceutical composition for nasal administration of at least one active agent to be delivered to the CNS, characterized in that it comprises at least one active agent and vitamin B12, the said agent (s) ( s) active being mixed (s) with vitamin B12 in said composition.

8) Pharmaceutical composition according to claim 7, characterized in that it comprises an effective amount of vitamin B12 to obtain and / or increase the effectiveness of a pharmacologically active substance at the level of the CNS.

9) Composition according to any one of claims 7 to 8, characterized in that the amount of vitamin B12 in the pharmaceutical composition for administration by the nasal route is between 0.1 μg and 1 mg.

10) Pharmaceutical composition according to any one of Claims 7 to 9, characterized in that it also contains any vehicle which is pharmaceutically compatible with vitamin B12 and / or the active agent, as well as optionally a mucosal permeabilization agent nasal.

11) Pharmaceutical composition according to any one of claims 7 to 10, characterized in that the active agent is a therapeutic agent, chosen in particular from: analgesics, anesthetics, analgesics, antispasmodics, antiparkinsonians, antiepileptics, antimigraine, or any other useful agent in treatment neurodegenerative, psychiatric or behavioral disorder, anticancer, antiviral, antiprion, antibiotic, or even active agents such as growth factors, nucleic acids, vitamins, hormones.

12) Pharmaceutical composition according to any one of claims 7 to 10, characterized in that the active agent is a diagnostic agent, chosen in particular from: antibodies and molecules making it possible to target CNS receptors, imaging agents used in MRI, scintigraphy, PET and ultrasound.

13) Process for preparing a pharmaceutical composition according to any one of claims 7 to

12, characterized in that the active agent is mixed with a lower and advantageously very lower amount, by weight, relative to said active ingredient, of vitamin B12.