Pharmaceutical formulation for administering paracetamol by buccal/gingival route

Abstract

The present invention concerns a pharmaceutical formulation for administering paracetamol by buccal/gingival route consisting of a hydroalcoholic solution comprising dissolved paracetamol, characterised in that: #the mass of paracetamol is between 95 mg and 190 mg, #the volume of said hydroalcoholic solution is between 1.0 ml and 2.0 ml, #the degree of alcohol of said hydroalcoholic solution is between 48.5° and 52.5°, and #the concentration of paracetamol in said hydroalcoholic solution is between 85 mg/ml and 110 mg/ml. The present invention also concerns said pharmaceutical formulation for the use of same for accelerating the speed at which paracetamol passes through the blood-brain barrier, and the use thereof as a drug, in particular for the symptomatic treatment of pain or fever.

Claims

1. A pharmaceutical formulation for the buccal/gingival administration of paracetamol consisting of a hydroalcoholic solution comprising dissolved paracetamol, wherein: the mass of paracetamol is between 120 mg and 170 mg, the volume of said hydroalcoholic solution is between 1.2 mL and 1.7 mL, the alcohol content of said hydroalcoholic solution is between 48.5° and 52.5°, and the concentration of paracetamol in said hydroalcoholic solution is between 90 mg/mL and 105 mg/mL, and the hydroalcoholic solution consists of a water/ethanol mixture having a viscosity greater than 1.5.Math.10.sup.−3 Pa.Math.s, and wherein the hydrodynamic radius at 40° C. of the paracetamol is less than 2.1 Å.

2. The pharmaceutical formulation according to claim 1, wherein the volume of ethanol represents 50% of the total volume of said hydroalcoholic solution.

3. The pharmaceutical formulation according to claim 1, wherein the hydroalcoholic solution comprises a flavoring and/or a sweetener.

4. A method for the treatment of pain and/or fever comprising the administration to a person in need thereof of a pharmaceutical formulation according to claim 1.

5. A method for accelerating the crossing of the blood-brain barrier by paracetamol comprising the buccal/gingival administration of a pharmaceutical formulation consisting of a hydroalcoholic solution comprising dissolved paracetamol, wherein: the mass of paracetamol is between 120 mg and 170 mg, the volume of said hydroalcoholic solution is between 1.2 mL and 1.7 mL, the alcohol content of said hydroalcoholic solution is between 48.5° and 52.5°, and the concentration of paracetamol in said hydroalcoholic solution is between 90 mg/mL and 105 mg/mL, and the hydroalcoholic solution consists of a water/ethanol mixture having a viscosity greater than 1.5.Math.10.sup.−3 Pa.Math.s, and wherein the hydrodynamic radius at 40° C. of the paracetamol is less than 2.1 Å.

6. The method according to claim 5, wherein the volume of ethanol represents 50% of the total volume of said hydroalcoholic solution.

Description

PRESENTATION OF THE FIGURES

(1) FIGS. 1A and 1B reproduce the results of a DOSY experiment performed on sample EU-95.

(2) FIG. 1A shows more precisely the plot of signal strength (CH.sub.3 of paracetamol) as a function of magnetic field gradient strength.

(3) FIG. 1B corresponds to the DOSY .sup.1H-NMR spectrum recorded at 400 MHz, at 25° C., 8 scans for 16 gradient increments, dl=2 s, LB=5 Hz. The horizontal dimension represents the chemical shifts of the protons of the compounds in the mixture, the vertical dimension the diffusion coefficients, after Laplace transformation.

(4) FIGS. 2A and 2B represent the hydrodynamic radius of water, of ethanol and viscosity during ethanol-in-water dilution experiments, at 25° C. and 40° C. respectively. R.sub.H (±0.1 Å) is calculated from the DOSY .sup.1H-NMR diffusion coefficient measurements. Viscosity values are taken from the literature [Khattab et al. (2012) Korean J. Chem. Eng. 29, 812-817]. The shaded area corresponds to the mole fractions studied in the presence of paracetamol.

(5) FIGS. 3A and 3B represent the hydrodynamic radius of water, of ethanol, of paracetamol and viscosity for samples EU-95, EU-95-30, EU-95-50 and EU-95-80, at 25° C. and 40° C. respectively. R.sub.H (±0.1 Å) is calculated from the DOSY .sup.1H-NMR diffusion coefficient measurements. Viscosity values are taken from the literature [Khattab et al. (2012) Korean J. Chem. Eng. 29, 812-817].

(6) FIG. 4 is a schematic representation of the device used for the cellular model mimicking the blood-brain barrier. It is a transwell system.

(7) FIG. 5 shows the flow of paracetamol across the in vitro blood-brain barrier model as a function of time in the following four situations: US: paracetamol comes from solution U 95, the culture medium contains 5% saline; U: paracetamol comes from solution U 95, the culture medium does not contain saline; PS: paracetamol comes from Perfalgan, the culture medium contains 5% saline; and P: paracetamol comes from Perfalgan, the culture medium does not contain saline.

(8) FIG. 6 shows the change in the abluminal concentration (i.e. in the lower compartment of the transwell) of paracetamol “U95” over time, for saline contents in the culture medium of 0%, 5%, 20% and 50%.

(9) FIG. 7 shows the change in the abluminal concentration (i.e. in the lower compartment of the transwell) of paracetamol “Perfalgan” over time, depending on whether saline is present in the culture medium (5%, curve PS), or not (curve P).

DETAILED DESCRIPTION OF THE INVENTION

(10) The present invention first relates to a pharmaceutical formulation for the buccal/gingival administration of paracetamol consisting of a hydroalcoholic solution comprising dissolved paracetamol, wherein: the mass of paracetamol is between 95 mg and 190 mg, the volume of said hydroalcoholic solution is between 1.0 mL and 2.0 mL, the alcohol content of said hydroalcoholic solution is between 48.5° and 52.5°, and the concentration of paracetamol in said hydroalcoholic solution is between 85 mg/mL and 110 mg/mL.

(11) Advantageously, the mass of paracetamol is between 120 mg and 170 mg, typically between 125 mg and 165 mg, and is in particular equal to 125 mg or 165 mg.

(12) Preferably, the volume of the hydroalcoholic solution is between 1.2 mL and 1.7 mL, typically between 1.25 mL and 1.65 mL, and is in particular equal to 1.25 mL or 1.65 m L.

(13) Advantageously, the concentration of paracetamol in said hydroalcoholic solution is between 90 mg/mL and 105 mg/mL, preferably between 95 mg/mL and 105 mg/mL, and is in particular equal to 100 mg/mL.

(14) Preferably, the hydroalcoholic solution consists of a water/ethanol mixture, in which the volume of ethanol represents between 45% and 60% of the total volume of said hydroalcoholic solution, advantageously between 48.5% and 52.5%, typically 50%.

(15) In the context of the present invention, “ethanol” refers to a commercial binary solution consisting of water and ethanol, and containing 96% to 99.8% (by volume) ethanol, advantageously 96% (by volume) ethanol.

(16) Advantageously: the mass of paracetamol is between 120 mg and 170 mg, typically between 125 mg and 165 mg, the volume of the hydroalcoholic solution is between 1.2 mL and 1.7 mL, typically between 1.25 mL and 1.65 mL, and the hydroalcoholic solution consists of a water/ethanol mixture, wherein the volume of ethanol represents between 50% of the total volume of said hydroalcoholic solution.

(17) In a first particular embodiment: the mass of paracetamol is equal to 125 mg, the volume of the hydroalcoholic solution is equal to 1.25 mL, and the hydroalcoholic solution consists of a water/ethanol mixture, wherein the volume of ethanol represents 50% of the total volume of said hydroalcoholic solution.

(18) In a second particular embodiment: the mass of paracetamol is 165 mg, the volume of the hydroalcoholic solution is 1.65 mL, and the hydroalcoholic solution consists of a water/ethanol mixture, wherein the volume of ethanol represents 50% of the total volume of said hydroalcoholic solution.

(19) Preferably, the viscosity of the hydroalcoholic solution is greater than 1.5.Math.10.sup.−3 Pa.Math.s.

(20) Advantageously, the hydrodynamic radius at 40° C. of the paracetamol dissolved in the hydroalcoholic solution as defined above is less than 2.1 Å.

(21) For the purposes of the present invention, “hydrodynamic radius”, or R.sub.H, means the radius of a solute in solution, said solute being assumed to be spherical, as defined by the following Stokes-Einstein relation [Edward, J. T. (1970) J. Chem. Educ. 47, 261; Einstein, A. (1905) Annalen Der Physik 17, 549-560; Einstein, A. (1906) Annalen Der Physik 19, 371-381]:

(22) $D=\frac{k_{B}T}{6\mathrm{\pi \rho }{R}_H}\mathrm{where}\text{:}$ D is the diffusion coefficient of the solute in the solvent (expressed in m.sup.2.Math.s.sup.−1), k.sub.B is the Boltzmann constant, and is k.sub.B=1.3806.Math.10.sup.−23 J.Math.K.sup.−1, T is the temperature of the medium (expressed in K), ρ is the viscosity of the medium (expressed in Pa.Math.s, or equivalent in kg.Math.s.sup.−1.Math.m.sup.−1), and R.sub.H is expressed in m.

(23) The measurement of the diffusion coefficient in solution of a molecule considered spherical thus makes it possible to determine the size of the molecule, more precisely its hydrodynamic radius.

(24) In a particular embodiment, the hydroalcoholic solution includes a flavoring and/or a sweetener.

(25) For the purposes of the present invention, a “flavoring and/or a sweetener” is defined as any pharmaceutically acceptable substance that makes more pleasant the taste perceived by the patient to whom the pharmaceutical formulation is administered. “Pharmaceutically acceptable” means what is generally safe, non-toxic and neither biologically nor otherwise undesirable, and which is acceptable for both veterinary and human pharmaceutical use.

(26) In a particular embodiment, the hydroalcoholic solution contains at least one active ingredient other than paracetamol.

(27) In particular, any lipophilic substance capable of providing an adjuvant that is analgesic, decongestant, sedative for the airways, sinuses, rhino and oropharyngeal pathways, which is compatible with dissolution in a hydroalcoholic solution, may be combined with paracetamol. In particular, paracetamol may be combined with pseudoephedrine, triprolidine, promethazine, pheniramine, meclizine, diphenhydramine, dimenhydrinate, cyproheptadine, dextopropoxyphene, and/or codeine.

(28) In addition, the present invention also relates to a pharmaceutical formulation as defined above for use to accelerate the crossing of the blood-brain barrier by paracetamol.

(29) Finally, the present invention also relates to a pharmaceutical formulation as defined above, for use as a drug, in particular for the treatment of pain, typically mild to moderate, and/or fever.

(30) The pharmaceutical formulation according to the invention may be packaged in a single-dose package of 0.5 to 3 mL, which package must limit the adsorption of paracetamol on its surface, be impermeable to the hydroalcoholic solution, and guarantee the stability of the solution, while allowing the complete delivery of the pharmaceutical formulation to the mandibular vestibule.

Examples

(31) The following abbreviations have been used:

(32) C: concentration

(33) EBM2: Endothelial Basal Medium 2

(34) HEPES: 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid

(35) HPLC: High-performance liquid chromatography

(36) PTFE: Polytetrafluoroethene

(37) QS: As much as suffices

(38) NMR: Nuclear Magnetic Resonance

(39) 2D .sup.1H-NMR: Two-dimensional proton NMR

(40) v/v: volume ratio

(41) x.sub.E: mole fraction of ethanol in water

(42) 1. Effect of Dilution of the Hydroalcoholic Solution on the Hydrodynamic Radius of Paracetamol

(43) Materials and Methods

(44) Materials

(45) A. Paracetamol Solutions U 95: 95 mg/ml paracetamol solution in a water/ethanol mixture (50/50 v/v) (C=628 mM), and PERFALGAN: 10 mg/mL commercial paracetamol solution for infants and children+excipients (C=66 mM).

(46) B. References Ethanol (lot MPA1003583): 10 mL vial, Paracetamol powder (lot MPA10031465): 1 g (M=151.16 g/mol), Heavy water (D.sub.2O): purchased from Eurisotop (Groupe CEA, Saclay), Saline (Gilbert Laboratoires): aqueous solution containing 0.9 g/100 mL (C=154 mM) sodium chloride in 5 mL dose units, and Milli-Q water, produced with a filtration and reverse osmosis system from Millipore (Molsheim, France).

(47) Preparation of Samples

(48) For NMR analyses of the liquid, the following samples were prepared and placed in 5 mm diameter glass NMR tubes or 4 mm diameter zirconia rotors (Cortecnet, France). A small amount of heavy water (D.sub.2O) is added to ensure spatial and temporal homogeneity during acquisitions. EU-95: 450 μL U 95+50 μL D.sub.2O (C=565 mM, x.sub.E=0.236), EU-95-30: 500 μL U 95+215 μL saline+70 μL D.sub.2O (C=400 mM, x.sub.E=0.141), EU-95-50: 250 μL U 95+250 μL saline+50 μL D.sub.2O (C=285 mM, x.sub.E=0.092), EU-95-80: 250 μL U 95+1250 μL saline+150 μL D.sub.2O (C=95 mM, x.sub.E=0.033), REF-Saline: 450 μL saline+50 μL D.sub.2O (C.sub.NaCl=138 mM, x.sub.E=0.0), REF: 250 μL EtOH+250 μL Milli-Q water+50 μL D.sub.2O (C=6.9 M, x.sub.E=0.236), REF-30: 250 μL EtOH+250 μL Milli-Q water+215 μL saline+70 μL D.sub.2O (C=4.8 M, x.sub.E=0.141), REF-50: 125 μL EtOH+125 μL Milli-Q water+215 μL saline+50 μL D.sub.2O (C=3.7 M, x.sub.E=0.092), REF-80: 125 μl EtOH+125 μL Milli-Q water+1250 μL saline+150 μL D.sub.2O (C=1.14 M, x.sub.E=0.033), REF-EtOH: 450 μL EtOH (C=13.6 M, x.sub.E=0.999), PERF: 450 μL Perfalgan+50 μL D.sub.2O (C=59 mM, x.sub.E=0.0), and PARA-125: 56 mg paracetamol powder+500 μL EtOH+50 μL D.sub.2O (C=673 mM, x.sub.E=0.735).

(49) Methods

(50) The Bruker SB 400 MHz NMR spectrometer (Wissembourg, France) equipped with a QNP SB .sup.1H/.sup.19F—.sup.13C—.sup.31P probe operating in static mode was used to perform liquid proton NMR analyses.

(51) In 2D .sup.1H-NMR, the sequence used is a DOSY pulse sequence [Morris, K. F., and Johnson, C. S. (1993) J. Am. Chem. Soc. 115, 4291-4299] (spin-echo sequence with Z gradient), with regulation of the temperature at +0.5° C. The 90° pulse is 15 μs, the recycling time is 5 s, the number of acquisitions is 48 with 16 increments for the gradient, for a total acquisition time of 1 hour. The signals are filtered with a decreasing exponential function of constant 2 Hz, before Fourrier transformation in the F2 dimension (.sup.1H chemical shift). An inverse Laplace transform is performed in the F1 dimension and gives the diffusion coefficients directly.

(52) The software used for NMR spectrum processing is Topspin 2.0 developed by Bruker.

(53) Results

(54) Introductory Remark

(55) The sizes of molecules in solution can be estimated by liquid NMR using so-called diffusion experiments to measure the diffusion coefficients of species in solution. The experiment is performed by applying a magnetic field gradient in a given direction, the direction in which the diffusion of particles is measured.

(56) Two treatments are possible following this type of experiment. Peak by peak analysis can be performed by very precisely setting the variation in intensity of NMR peaks as a function of the field gradient (FIG. 1A). The only adjustment variable being the diffusion coefficient, it is then easy to obtain the size of the species in solution using the Stokes-Einstein equation already described. A Laplace transform can also be applied to obtain on a two-dimensional map (2D-NMR), chemical diffusion/shift, the diffusion coefficient being directly measurable along the y axis (FIG. 1B).

(57) Measurement of Diffusion Coefficients of Reference Samples

(58) The DOSY experiments were conducted on water, ethanol, Perfalgan and paracetamol solubilized in ethanol.

(59) For the measurement of diffusion coefficients, an NMR resonance is chosen, usually CH.sub.3 which is isolated, because it provides more accuracy. In this case the accuracy is estimated at ±0.1.Math.10.sup.−9 m.sup.2.Math.s.sup.−1.

(60) The values of the diffusion coefficients of the different compounds that have been determined are shown in Table 1 below. This table also includes viscosity values obtained from the literature [Khattab, I. S., Bandarkar, F., Fakhree, M. A. A., and Jouyban, A. (2012) Korean J. Chem. Eng. 29, 812-817], the mole fraction of ethanol and the hydrodynamic radius calculated from the Stokes-Einstein equation. The accuracy of the value thus calculated is estimated at ±0.1 Å.

(61) TABLE-US-00001 TABLE 1 Measurement of diffusion coefficients by DOSY .sup.1H-NMR, at 25° C., on reference samples. Mole fraction Viscosity Observed D R.sub.H SAM- Ethanol, 25° C. NMR 25° C. 25° C. PLE x.sub.E (10.sup.−3 Pa .Math. s) resonance (10.sup.−9 m.sup.2 .Math. s.sup.−1) (Å) REF- 0 0.8914 H.sub.2O 2.4 1.0 Saline PERF 0 0.8914 CH.sub.3 0.9 2.8 Paracetamol H.sub.2O 2.6 0.9 Mannitol 0.8 3.2 REF- 0.999 1.0995 CH.sub.3 1.2 1.7 EtOH Ethanol H.sub.2O 1.3 1.5 (traces) PARA- 0.735 1.6594 CH.sub.3 0.7 1.8 125 Ethanol CH.sub.3 0.3 4.2 Paracetamol H.sub.2O 0.8 1.5

(62) Measurement of the Diffusion Coefficients of Water-EtOH Solutions During Dilution with Water (Saline Solution)

(63) From the experiments on the reference samples, it appears that the size of the molecules varies from one sample to another, depending very strongly on the solvent: for example, paracetamol has an R.sub.H of 2.8 Å in the Perfalgan sample, and an R.sub.H of 4.2 Å in ethanol.

(64) The viscosity of the sample and complex hydration phenomena are involved here. It is important to monitor the molecule sizes in the different solvents in the study and in particular during dilution studies.

(65) The reference solutions (REF-Saline, REF, REF-30, REF-50, REF-80 and REF-EtOH) were therefore subjected to DOSY experiments to evaluate the effect of dilution with water.

(66) The results obtained at 25° C. and 40° C. are shown in FIGS. 2A and 2B, respectively.

(67) Several conclusions can be drawn from such results.

(68) The viscosity has a maximum for an EtOH/Water mole fraction close to 0.3 (60/40 v/v) at 25° C. and close to 0.2 (50/50 v/v) at 40° C. This is remarkable and indicates that the maximum viscosity is 2 to 3 times higher than that of pure water or of pure ethanol.

(69) The hydrodynamic radii calculated for water and ethanol also have a particular behavior. While ethanol has a radius of 1.5 Å at 25° C., in both pure and highly diluted form, it has a minimum (0.8 Å) to x.sub.E of between 0.05 and 0.1. Water behaves in the same way. This phenomenon has already been encountered in the literature [Price, W. S., Ide, H., and Arata, Y. (2003) The Journal of Physical Chemistry A 107, 4784-4789; Codling, D. J., Zheng, G., Stait-Gardner, T., Yang, S., Nilsson, M., and Price, W. S. (2013) The Journal of Physical Chemistry B 117, 2734-2741] and is explained by the breaking, in the mixture, of water-water or ethanol-ethanol associations found for pure solutions of water or ethanol.

(70) At 40° C., viscosity and R.sub.H are lower than at 25° C., which reflects the effect of temperature: Brownian motion tends to break hydrogen bonds, which has the effect of destroying all the dipole-dipole associations that water and ethanol can form together.

(71) Interestingly, sample EU-95 has a mole fraction of ethanol of 0.236, i.e. in the area where the viscosity has a maximum.

(72) Measurement of the Diffusion Coefficients of Water-ETOH-Paracetamol Solutions when Diluted with Water (Saline Solution) or when the Concentration of Paracetamol in the Same Hydroalcoholic Medium Decreases

(73) Solutions containing paracetamol under different dilution conditions (EU-95, EU-95-30, EU-95-50 and EU-95-80) were also subjected to DOSY experiments. These solutions correspond to x.sub.E values of 0.236, 0.141, 0.092 and 0.033, respectively, which define the shaded area in FIGS. 2A and 2B. The results obtained at 25° C. and 40° C. are compiled in Table 2 below and reported in FIGS. 3A and 3B, respectively.

(74) At 40° C., the temperature closest to that of the human body, the lowest hydrodynamic radius of paracetamol (1.9 Å) is obtained for the undiluted sample of U 95 (EU-95). It differs from the value of paracetamol in Perfalgan (2.5 Å) which is little affected by temperature rise.

(75) TABLE-US-00002 TABLE 2 Measurement of diffusion coefficients by DOSY .sup.1H-NMR, at 25° C. and 40° C., on Perfalgan and U-95 (95 mg/mL) samples during dilutions. Mole Viscosity D R.sub.H fraction (10.sup.−3 Pa .Math. s) Observed (10.sup.−9 m.sup.2 .Math. s.sup.−1) (Å) SAM- Ethanol, 25° C. NMR 25° C. 25° C. PLE x.sub.E 40° C. resonance 40° C. 40° C. PERF 0 0.8914 CH.sub.3 0.9 2.8 0.8914 Paracetamol 1.4 2.5 H.sub.2O 2.6 1.0 4.4 0.8 EU-95 0.236 2.3869 CH.sub.3 0.7 1.3 1.5924 Ethanol 1.5 1.0 CH.sub.3 0.3 3.0 Paracetamol 0.8 1.9 H.sub.2O 1.1 0.8 2.2 0.7 EU- 0.141 2.1337 CH.sub.3 0.8 1.2 95-30 1.4299 Ethanol 1.5 1.1 CH.sub.3 0.4 2.7 Paracetamol 0.7 2.2 H.sub.2O 1.5 0.7 2.6 0.6 EU- 0.092 1.7759 CH.sub.3 0.9 1.4 95-50 1.1862 Ethanol 1.7 1.1 CH.sub.3 0.4 2.8 Paracetamol 0.9 2.1 H.sub.2O 1.6 0.8 2.8 0.7 EU- 0.033 0.9855 CH.sub.3 1.2 1.8 95-80 1.7975 Ethanol 0.2 1.4 CH.sub.3 0.7 3.2 Paracetamol 1.1 2.5 H.sub.2O 2.3 1.0 3.6 0.8

(76) Conclusion

(77) Hydroalcoholic solutions are distinguished by particular macroscopic and molecular aspects, and the paracetamol molecules dissolved in such solutions have remarkable properties.

(78) Water forms a network of pure hydrogen bonds and ethanol forms pure alcohol-alcohol associations. Very low alcohol contents in water lead to water/ethanol associations. For higher ethanol contents, there is a break in the associations and the molecules behave individually, isolated from each other in the mixture.

(79) At the same time, the viscosity of the solution increases, to reach a maximum for an EtOH/Water mole fraction close to 0.3 (60/40 v/v) at 25° C. and close to 0.2 (50/50 v/v) at 40° C., which corresponds to a viscosity 2 to 3 times higher than that of pure water or of pure ethanol.

(80) Sub-nanometer-scale size measurement (DOSY NMR scattering experiments) reveals variations for the paracetamol molecule.

(81) The particular composition of water-ethanol mixtures in the 50/50 (v/v) range leads to the smallest size for paracetamol, particularly at 40° C., the temperature closest to that of the human body. This can be interpreted by a low wetting of the molecule which would make it stealthier and give it a more hydrophobic character, helping its passage through membrane barriers, also hydrophobic.

(82) The contraction of the paracetamol molecule, its stealth, combined with a high viscosity of the preparation and an effect of fluidification of biological membranes by ethanol, a known but poorly characterized phenomenon, contribute to the efficacy of the pharmaceutical formulations according to the invention in buccal/gingival administration.

(83) 2. Transport of Paracetamol Across the Blood-Brain Barrier

(84) Materials and Methods

(85) Materials

(86) A. Paracetamol Solutions U 95: 95 mg/ml paracetamol solution in a water/ethanol mixture (50/50 v/v) (C=628 mM), and PERFALGAN: 10 mg/mL commercial paracetamol solution for infants and children+excipients (C=66 mM).

(87) B. Cellular Model of the Blood-Brain Barrier

(88) It has previously been established in the literature that the hCMEC/D3 cell line may constitute a relevant in vitro model of the blood-brain barrier, which mimics the components of the latter [Weksler et al., FASEB J. 2005 November; 19(13):1872′4. Epub 2005 Sep. 1; Weksler et al., Fluids Barriers CNS. 2013 Mar. 26; 10(1):16. doi: 10.1186/2045′8118′10′1].

(89) The cells were first cultured in cell culture vials (three passages of the culture after thawing frozen cells from the stock culture) and then seeded into the transwell system described in FIG. 4.

(90) Once they reach confluence, which is obtained after 14 days of culture in the transwell system, the cells express the proteins characteristic of blood-brain barrier formation, namely ZO1, occludin, phalloidin and CD31.

(91) The permeability of the barrier thus mimicked is then determined using Lucifer yellow. In this case, the Lucifer yellow does not manage to cross it.

(92) C. Culture Medium

(93) The composition of the culture medium is indicated in Table 3 below:

(94) TABLE-US-00003 TABLE 3 Composition of the culture medium Solution in EBM2 Volume (mL) Fetal calf serum (5%)* 25 Antibiotics (penicillin and streptomycin) 5 Ascorbic acid (5 μg/mL) 2.5 Lipids (1/100) 5 HEPES (10 mM) 5 bFGF (1 ng/mL) 2.5 hydrocortisone (1.4 μM) 0.25 QS EBM2 500 *Some experiments, detailed below, were performed in the absence of serum, or with higher serum concentrations (20% and 50%).

(95) D. HPLC

(96) A specific HPLC method was developed to allow the quantification of paracetamol crossing the in vitro blood-brain barrier model described above, taking into account the culture medium and the sampling protocol.

(97) The equipment used consists of a P4000 pump, a 6000LP UV detector with a 50 mm optical path, an AS 3000 autosampler with a 100 μL loop and an SN 4000 System Controller from Thermo Fisher (Courtaboeuf, France). The acquisition software used is ChromQuest 5.0.

(98) The method developed is based on the following characteristics: Column: XTerra RP18 20*4.6 mm*3.5 μm, UV detector: 242 nm, Flow rate: 1 mL/min, Column temperature: 35° C., Injected volume: 10 μL, and Mobile phase: ammonium formate buffer at 0.02 M and pH 4.

(99) Methods

(100) The following sampling protocol was established, which involves the different steps:

(101) a) Deposition of 104 μL of U 95 in the upper compartment of the transwell system described in FIG. 4, b) Removal of 1000 μL from the lower compartment, c) Protein precipitation by adding 200 μL of a 10% trichloroacetic acid solution, d) Centrifugation at 3000 rpm for 10 min, e) 1/10 dilution using the mobile phase, f) Filtration using a 0.22 μm PTFE filter, g) Injection of 10 μL into the HPLC, and h) Quantification of paracetamol.
Results

(102) Comparison of Blood-Brain Barrier Crossing by Paracetamol “U 95” Vs. Paracetamol “Perfalgan”

(103) The flow of paracetamol through the in vitro model of the blood-brain barrier membrane was determined for both the paracetamol contained in the “U 95” solution and the paracetamol contained in the Perfalgan.

(104) In order to have identical concentrations of paracetamol, in the first case, 10 μL of U 95 solution and 854 of culture medium were added to the luminal compartment of the transwell system, and in the second case, 954 of Perfalgan was used.

(105) The crossing of each of the compounds was studied at 37° C. in the presence of saline in the culture medium (5%), and in the absence of saline.

(106) The cells were cultured at 37° C. for 20-22 h before the measurements were taken.

(107) The flows thus determined have been reported in FIG. 5.

(108) It can be seen that the flow of paracetamol “U 95” in the presence or absence of saline (curves US and U, respectively) is almost constant over time, and significantly higher than that of paracetamol “Perfalgan”, which decreases very rapidly.

(109) It is also noted in FIG. 5 that the transport of paracetamol “U 95” across the in vitro model of the blood-brain barrier membrane is higher in the presence of saline during the first 30 minutes.

(110) Additional experiments were therefore carried out to determine the influence of saline on the transport of the transport of paracetamol “U 95”.

(111) Transport of Paracetamol “U 95” in the Presence of Saline

(112) The change in the abluminal concentration of paracetamol “U95” over time was determined for saline contents in the cell medium of 0%, 5%, 20% and 50%.

(113) The results obtained have been reported in FIG. 6.

(114) It should be noted that the values of abluminal concentrations of paracetamol shown in FIG. 6 correspond to the raw values obtained by HPLC quantification. Due to the dilution performed for HPLC, they should be multiplied by a factor of 12 to correspond to the actual values.

(115) It can be seen that the transport of paracetamol “U 95” across the in vitro model of the blood-brain barrier membrane is enhanced by the presence of saline in the culture medium. It would even seem that there is a proportionality relationship between the amount of saline present and the amount of paracetamol that crosses the barrier.

(116) On the other hand, such an influence of the presence or absence of saline is not observed for paracetamol “Perfalgan”.

(117) Indeed, the change in the abluminal concentration of paracetamol “Perfalgan” over time was determined for saline contents in the cell medium of 0%, 5%, 20% and 50%.

(118) The results obtained have been reported in FIG. 7.

(119) It can be seen that the transport of paracetamol “Perfalgan” across the in vitro model of the blood-brain barrier membrane is less favorable in the presence of saline from 30 min.

CONCLUSION

(120) The results described above show an active transport of the paracetamol contained in the pharmaceutical formulations according to the invention, in which at least one component of the saline solution is involved.