Patent Application
20070248686
The field of the present invention is that of Vector Particles (PV), which are useful for the administration of active principles (PA). The latter are preferably medicaments or nutrients for administration to an animal or human organism by the oral, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intracerebral or parenteral route, or the like. However, this may also involve cosmetic products or plant-production products, such as herbicides, pesticides, insecticides or fungicides, or the like. In terms of chemical nature, the PAs most particularly, but without limitation, involved in the invention are, for example, proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, oligonucleotides, polynuclides and organic molecules.
The present invention relates, more precisely, to colloidal suspensions of Vector Particles, advantageously of the submicronic type, based on polyamino acids (PAA). The present invention relates to both uncoated particles as such, and vector systems for PAs, consisting of particles loaded with the PA(s) considered. The present invention also relates to pulverulent solids comprising these PVs. The invention also relates to methods for preparing said colloidal suspensions of particles, with or without PAs.
The encapsulation of PA into the PVs is intended in particular to modify their duration of action and/or to convey them to the site of treatment and/or to increase the bioavailability of said PAs. Numerous encapsulation techniques have already been proposed. Such techniques are intended, on the one hand, to allow the transport of the PA to its site of therapeutic action, while protecting it against attacks by the body (hydrolysis, enzymatic digestion and the like) and, on the other hand, to control the release of the PA at its site of action, in order to maintain the quantity available for the body at the desired level. The PAs involved in these vicissitudes of transport and of existence in the body are, for example, proteins, but may also be products which are completely different, organic molecules of synthetic or natural origin.
The review by M. J. HUMPHREY (Delivery system for peptide Drugs, published by S. DAVIS and L. ILLUM, Plenum Press, N.Y. 1986) reports the problem relating to the enhancement of the bioavailability of the PAs and the advantage of vector and controlled release systems.
Among all the materials which can be envisaged for forming PVs, polymers are increasingly used because of their intrinsic properties. As regards the specifications which it is desired to obtain for the PVs, they are particularly demanding and comprise, in particular, the following specifications.
The earlier technical proposals, which are described below, have tried to satisfy all these specifications.
By way of illustration, there may be mentioned the earlier proposals (a) to (h):
It is therefore evident from the above that the earlier technical proposals described above, and in particular proposal (i), incompletely satisfy the new specifications indicated above, and in particular a capacity for sterilizing by filtration, a high rate of degradation, adaptability to constraints for administering medicaments by injection, low cost and high PA load factor.
As regards the sterilizing filtration capacity, it is important that the PV particles are sufficiently small to pass, in suspension in a liquid, across filters whose cut-off is less than or equal to 0.2 μm, without clogging. Such ease and efficiency of filtration sterilization are particularly appreciated for injectable medicaments.
As regards the capacity for injection of the PVs, it is appropriate, for a given dose of PA, to be able to inject small volumes of liquid suspension, and that this suspension is not very viscous. This involves being able to reduce the quantities of excipient (PV) compared with the targeted therapeutic dose of PA and to provide PVs having a size which is as small as possible, while increasing the loading capacity of PA.
As regards the specification relating to biodegradability of the PVs, the smaller the size of the PVs, the better it is and it allows their rapid elimination.
In addition, it is appreciable to be able to reduce the quantities of excipient (PV) for economic reasons and so as to enhance the tolerance of the injectable medicament.
Under these circumstances, an essential objective is to be able to provide novel PVs which spontaneously form, and without the aid of surfactants or of organic solvents, stable aqueous suspensions of PV. Another essential objective of the present invention is to provide novel PVs in stable aqueous colloidal suspension or in pulverulent form and based on poly(amino acids) (PAA), these novel PVs having to satisfy as much as possible specifications 1 to 8 of the abovementioned specifications.
Another essential objective of the invention is to improve the particles disclosed in PCT application WO 96/29991.
Another essential objective of the invention is to provide a novel suspension of PV whose characteristics are perfectly controlled, in particular in terms of PA load factor and in terms of control of kinetics of release of PA.
Another essential objective of the invention is to provide injectable medicinal suspensions. The specifications, which are required for such suspensions, are a low volume for injection and a low viscosity. It is important for the mass of colloidal particles per injection dose to be as low as possible, without limiting the quantity of active principle PA transported by these particles, so as not to damage the therapeutic efficacy.
Another essential objective of the invention is to provide an aqueous colloidal suspension or a pulverulent solid comprising particles for carrying active principles satisfying the specifications targeted above and which constitutes an appropriate and suitable galenic form for administration, for example oral administration, to humans or animals.
Another essential objective of the invention is to provide a colloidal suspension comprising particles for carrying active principles which can be filtered on 0.2 μm filters for sterilization purposes.
Another essential objective of the invention is to propose a method for preparing PAA particles (dry or in suspension in a liquid) which are useful in particular as vectors for active principles, it being necessary for said method to be simpler to use, nondenaturing for the active principles and, in addition, to always allow fine control of the mean particle size of the particles obtained.
Another essential objective of the invention is the use of the abovementioned particles in aqueous suspension or in solid form for the preparation:
Another essential objective of the present invention is to provide submicronic PV suspensions based on PAA and capable of serving as vector for a PA, in particular one which is medicinal, for administration of said PA to a human or animal organism, or alternatively for a nutritional, plant-protection or cosmetic PA.
Another objective of the present invention is to provide a medicament, such as the system for prolonged release of active principles, which is easy and economical to produce and which is, in addition, biocompatible and capable of providing a very high level of bioavailability of the PA.
Another essential objective of the invention is to provide a system for carrying a vaccine, which is intrinsically nonimmunogenic and in combination with one or more antigens.
The objectives relating to the products (inter alia) are achieved by the present invention which relates, first of all, to a stable colloidal suspension of submicronic structured particles which can be used, in particular for carrying active principle(s) PA(s), these particles being individualized (discrete) supramolecular arrangements:
The procedures Md and Ma for the Dh and Ta measurements are detailed below.
Procedure Md:
One of the inventive bases of these novel vector particles PV, in stable aqueous colloidal suspension or in the form of pulverulent solid, is due to the innovative selection of a group of polymers and of an innovative methodology which make it possible to obtain particles of submicronic size, which form a stable aqueous colloidal suspension in the absence of surfactants or solvents.
Another inventive basis of these novel vector particles PV, in stable aqueous colloidal suspension or in the pulverulent solid state, is due to the innovative selection of a group of particular submicronic structured particles by their load factor Ta≧7% and their size Da≦150 nm. This selection is the result of major and long research studies on the method for producing the particles. Indeed, the reduction in size and the increase in the loading capacity of the particles of polyamino acids was a priori not obvious. Thus, using the method for producing nanoparticles of polyamino acid taught in PCT application WO 96/29991, persons skilled in the art were not able to obtain, “to measure”, particles which correspond to the new specifications, as defined above.
Finally, it is by modifying the compositions of the polymers and the operating conditions that the inventors were able to isolate these structured particles of very small size which are based on PAA and which have, quite surprisingly and unexpectedly, a load capacity Ta for insulin which may be up to three times higher than that characteristic of the particles according to WO 96/29991.
Advantageously, the suspension according to the invention is characterized in that the submicronic particles do not acquire their cohesion from the presence of the following three compounds:
The structure of the PAA polymers and the nature of the amino acids are chosen such that:
Thus, by modifying the particular structure of the PAA, it is possible to control the phenomena for combining and releasing the PA from the kinetic and quantitative point of view.
It is to the applicant's credit to have chosen, as constituent material of the PVs, a particular composition of polyamino acids which are amphiphilic and which therefore possess properties of the PVs in PAA, namely:
It was possible to obtain these new properties by virtue of the primary technical functions of the PVs which are the small nanometric size and the high load factor.
To define these PAAs a little further, it is possible to indicate that they can be of the alternating sequential, ordered (block) type or of the random sequential, disordered type.
Thus, according to a first embodiment of the PVs according to the invention, the constituent PAAs are of the “block” type and are characterized by an AAO/(AAI+AAO) molar ratio such that:
Advantageously, the absolute length of each AAO block, expressed as number of AAO is such that:
According to a second embodiment of the PVs according to the invention, the constituent PAAs are of the “random” type, that is to say are prepared by simultaneous copolymerization of AAI and AAO monomers, and the AAO/(AAO+AAI) molar ratio is such that:
Advantageously, the molar mass Mw of these random PAAs is such that:
According to a preferred characteristic of the invention, the constituent block or random PAAs of the particles have degrees of polymerization DP of between 30 and 600, preferably between 50 and 200, and still more preferably between 60 and 150.
Advantageously, the constituent PAAs of the PV particles are “diblock” PAAs.
The present invention relates, not only to suspensions of uncoated particles, as defined above, but also to particles comprising at least one active principle PA. Preferably, the suspension according to the invention is aqueous and stable. These particles, loaded or not with PA, are advantageously in dispersed form in a liquid (suspension), preferably an aqueous liquid, but may also be in a pulverulent solid state, obtained from the suspension of PV as defined above.
Accordingly, the invention relates, apart from to a colloidal suspension (preferably aqueous suspension) of PV, to a pulverulent solid comprising PVs and obtained from the suspension according to the invention.
Another essential subject of the invention relates to the preparation of the selected particles (as described above), both in the form of a colloidal suspension and in the form of a pulverulent solid. The method of preparation considered essentially consists in synthesizing precursor PAAs and in converting them to structured particles.
More precisely, this includes, first of all, a method for preparing submicronic structured particles capable of being used, in particular for carrying active principle(s), these particles being discrete supramolecular arrangements:
This method is characterized in that:
The first step of the method is based on known techniques of polymerization of anhydrides of N-carboxy-(-amino acids (NCA), described, for example, in the article “Biopolymers, 15, 1869 (1976)” and in the book by H. R. KRICHELDORF “(-Aminoacid-N-carboxy Anhydride and Related Heterocycles” Springer Verlag (1987). The use of judiciously chosen polar nonaromatic aprotic copolymerization solvents, while avoiding any precipitation and the use of acid hydrolysis in the presence of water and of nonaromatic polar organic solvent, constitute novel and inventive modalities which lead to structured, discrete and submicronic particles with a high PA load capacity, and which form a stable colloidal suspension in aqueous medium. These particles are not at all comparable to a macroscopic agglomerated precipitate of the type mentioned above in relation to the earlier proposal (d).
According to one variant, at the end of step 1, the copolymer poly(AOO) (pAAI) obtained is precipitated—preferably in water—and this precipitate is recovered. This variant corresponds to a batch mode for preparing particles, in which the copolymer poly(AAO) (pAAI) is isolated in the form of a precipitate forming a stable intermediate product. This precipitate may be, for example, filtered, washed and dried.
Still more preferably, the NCAs-pAAI are NCAs of O-alkylated glutamic or aspartic acid, for example NCA-Glu-O-Me, NCA-Glu-O-Et or NCA-Glu-O-Bz (Me=methyl-Et=ethyl-Bz=benzyl).
In a known manner, the copolymerization takes place at a temperature between 20 and 120° C., at atmospheric pressure and in the presence of an amine-containing initiator, e.g.: NH3. Other experimental parameters, such as the concentration of NCA and/or polymer in the nonaromatic polar solvent (preferably NMP), and/or the concentration or the nature of the protic cosolvent, during the synthesis, will be adjusted according to the desired effects known to persons skilled in the art.
The acid hydrolysis (step 2) is carried out using water and at least one inorganic acid such as phosphoric or hydrochloric aid—the latter being preferred—and/or an organic acid, such as TriFluoroAcetic acid (TFA), acetic acid, dichloroacetic acid or organosulfonic acids.
The water/acid ratios—expressed in parts by weight—in an acidic aqueous phase for hydrolysis are advantageously:
The acidic aqueous phase for hydrolysis/NMP ratios—expressed in parts by weight—are advantageously:
Other parameters, such as the polymer concentration, the temperature of the reaction mixture, the mode of adding the acidic aqueous phase for hydrolysis, the use of reduced pressure, the duration of the reaction, and the like, are adjusted according to the desired effects and are well known to persons skilled in the art.
The neutralization (step 3) is carried out in practice, for example, using sodium hydroxide.
The salt formed after neutralization as well as the solvent are then removed by any appropriate physical separation treatment, for example by diafiltration (dialysis) (step 4), filtration, pH modification, chromatography and the like.
This gives an aqueous suspension of structured particles which may be concentrated, for example, by distillation or any other suitable physical means: ultrafiltration, centrifugation.
To separate, in step 6, the particles from their liquid suspension medium, the aqueous phase is optionally removed, for example, by drying (e.g. in an oven) by freeze-drying or any other suitable physical means: ultrafiltration, centrifugation. A white pulverulent solid is recovered at the end of this step 6.
According to one variant, the concentration step may be carried out by a chemical treatment, such as a reduction in the pH, which converts to an acid the hydrophilic part of the glutamate monomers, making them insoluble in water. These acidic PAA intermediates may be filtered, washed and dried. Said acidic intermediates may be neutralized with a chemical base in a subsequent step in order to obtain a suspension of particles.
It should be noted that the use of steps 1, 2, 3, 4 and optionally 5 of the above method corresponding to a preparation of a colloidal suspension of submicronic particles and to a high load factor with the PAs.
During this preparation of colloidal suspension, the amphiphilic PAAs poly(AAO)(AAI) of step 2 are placed in an aqueous medium in which at least part of the AAIs is soluble and at least part of the AAOs is insoluble. The PAAs exist in the form of nanoparticles in this aqueous medium.
An alternative for preparing the PV suspension according to the invention consists in bringing the pulverulent solid, as described above and as product and by its method of production, into contact with a nonsolvent aqueous medium for the AAOs.
To carry out the combination of one or more PAs with the particles, it is possible to use several methods in accordance with the invention. Nonlimiting examples of these methods are listed below.
According to a first method, the combination of PA with the particles is carried out by bringing a liquid phase (aqueous or otherwise) containing the PA into contact with the colloidal suspension of particles.
According to a second method, the combination of the PA with the particles is carried out by bringing a PA in the solid state into contact with the colloidal suspension of particles. The solid PA may be, for example, in freeze-dried, precipitate or powdered form or the like.
According to a third method, the pulverulent solid (PAA), as described above as product and by its production characteristics, is brought into contact with a liquid phase (aqueous or otherwise) containing the PA.
According to a fourth method, the pulverulent solid, as described above as product and by its production characteristics, is brought into contact with the PA in solid form. This mixture of solids is then dispersed in a liquid phase, preferably an aqueous solution.
In all these methods, the PA used may be in pure or preformulated form.
Given the nanometric size of the particles, the suspension may be filtered on sterilizing filters, which makes it possible to obtain, easily and at a lower cost, sterile injectable medicinal liquids. The fact that it is possible, by virtue of the invention, to control the size of the particles and reach Dh values of between 25 and 100 nm, is a major advantage.
The present invention also relates to novel intermediate products of the method described above, characterized in that they consist of PAA copolymers which are precursors of particles.
According to another of its aspects, the invention relates to a suspension and/or a pulverulent solid, as defined above and/or as obtained by the method presented above, this suspension and this solid comprising at least one active principle preferably chosen from:
The invention also relates to a suspension and/or the pulverulent solid loaded with nutritional, plant-protection or cosmetic PA.
Finally, the invention relates to a pharmaceutical, nutritional, plant-protection or cosmetic proprietary product, characterized in that it comprises a suspension and/or the pulverulent solid loaded with PA and as defined above.
According to another of its subjects, the invention also relates to the use of these PVs (in suspension or in solid form) loaded with PA, for the manufacture of medicaments such as systems with controlled release of PA.
In the case of medicaments, they may be, for example, those which can be administered, preferably by the oral, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intracerebral or parenteral route.
The cosmetic applications which may be envisaged are, for example, compositions comprising a PA combined with the PVs according to the invention and which can be applied by the transdermal route.
The relevant plant-protection products may be, for example, herbicides, pesticides, insecticides, fungicides and the like.
The following examples will make it possible to better understand the invention in its various product/method/application aspects. These examples illustrate the preparation of particles of polyamino acids loaded or otherwise with active principles, and they likewise present the structural characteristics and the properties of these particles.
112.4 g of NCA-GluOMe (0.60 mol) and 449 g of N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into a 1 liter reactor thermostated at 20° C. After dissolution, 21.38 g of a 0.34 M solution of ammonia in 1,4-dioxane (1.25 mol %/NCA) are added. The polymerization is monitored by measuring the carbon dioxide emitted into a gas bell jar and verified by disappearance of vibration bands characteristic of the NCAs at 1860 and 1790 cm−1. After 30 min, a solution of 47.17 g of NCA Leucine (0.30 mol) in 631 g of NMP is introduced. After 10 min of reaction, the temperature is increased to 60° C. The polymerization is monitored as above and is complete after 2 hours. The temperature of the reaction mixture obtained is increased to 80° C. 31.5 g of aqueous concentrated hydrochloric acid (35%, 12 M) are added, with mechanical stirring over 30 min to 350 g of the reaction mixture obtained at the end of step 1. The reactor is then placed under reduced pressure regulated at 600 mbar for 6 hours. A mixture of 31.5 g of 35% hydrochloric acid and of 126 g of water is then added over 60 min, followed by a second phase of vacuum at 250 mBar for 18 hours. In this example, the overall water/pure hydrochloric acid ratio is 7.6/1 by mass and the acidic aqueous phase/NMP ratio is 60/100 by mass.
The reaction mixture is then cooled to 50° C. and then neutralized with aqueous sodium hydroxide (35% by mass). The NMP and the sodium chloride formed during the neutralization are removed by diafiltration against 20 volumes of Milli Q water, on a membrane with an MWCO of 1 000 Daltons (Pellicon II system, Millipore). A stable aqueous colloidal suspension of vector nanoparticles is thus obtained. The suspension of nanoparticles is finally freeze-dried.
The contents of leucine motifs are determined by proton nuclear magnetic resonance (signals at 2.10, 2.22 and 2.58 ppm for 4H of Glu and at 0.85 ppm for 6H of Leu). The mean hydrodynamic diameter (Dh) is 70 nm (according to Md).
The procedure Ma is used. The concentration of free insulin, assayed by HPLC chromatography is equal to 0.59 mg/ml and the combined insulin concentration equal to 1.51 mg/ml is deduced therefrom. The load capacity for a colloidal solution of 10 mg/ml reaches 1.51 mg/ml of insulin. Thus, the ratio of the mass of combined insulin to the bLE (Ta) mass is 15.1%.
146.4 g of NCA GluOMe are dissolved in 586 g of NMP to which 18.43 g of a 0.48 M solution of ammonia in methanol are added. When the polymerization of the NCA GluOMe is complete, a solution of 43.9 g of NCA Leu in 708 g of NMP is introduced and the polymerization of the NCAs Leu is continued until disappearance of the monomers is obtained. The medium is then heated to 80° C. and 129.4 g of 35% HCl are added dropwise thereto over 30 min to 1 hour. A 600 mBar vacuum is applied for 6 hours, and then an additional 129.4 g of 35% HCl are added as a mixture with 517.5 g of water. A 250 mBar vacuum is then applied for 18 hours. After this step, the temperature is reduced to 50° C., 1 liter of water is introduced, followed by 280 ml of 35% NaOH in order to bring the pH to 7.4. The suspension is then filtered (5 μm), dialyzed (cut-off 1 000 Da) in water, in order to remove the solvent and the salts, and finally filtered (0.22 μm). This suspension may be directly used or may be subjected to subsequent treatments, such as distillation of the water (step 5) or freeze-drying (step 6).
The mean hydrodynamic diameter Dh (according to Md) is 14.8%. The insulin load factor Ta, determined according to the procedure Ma, is 35 nm.
38.9 g of NCA-GluOMe (0.208 mol) and 156 g of N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into a 0.5 liter reactor thermostated at 30° C. After dissolution, 5.79 g of a 0.407 M solution of ammonia in methanol (1.25 mol %/NCA) are added. The polymerization is monitored by measuring the carbon dioxide emitted into a gas bell jar and verified by disappearance of vibration bands characteristic of the NCAs at 1860 and 1790 cm−1. After 30 min, a solution of 23.3 g of NCA Leucine (0.148 mol) in 263 g of NMP is introduced. After 10 min of reaction, the temperature is increased to 60° C. The polymerization is monitored as above and is complete after 1-2 hours. The temperature of the reaction mixture obtained previously is increased to 80° C. 41.9 g of aqueous hydrochloric acid (35% of the mass) are added, with mechanical stirring over 30 min, to the reaction mixture. The reactor is then placed under reduced pressure regulated at 600 mbar for 6 hours. A mixture of 41.9 g of 35% hydrochloric acid and of 167.5 g of water is then added over 60 min, followed by a second phase of vacuum at 250 mbar for 18 hours. The reaction mixture is then cooled to 50° C. and then neutralized with aqueous sodium hydroxide (35% by mass). The NMP and the sodium chloride formed during the neutralization are removed by diafiltration against 20 volumes of Milli Q water, on a membrane with an MWCO of 1 000 Daltons (Pellicon II system, Millipore). A stable aqueous colloidal suspension of vector nanoparticles is thus obtained. The suspension of nanoparticles is finally freeze-dried.
The mean hydrodynamic diameter Dh is measured according to Md on aqueous suspensions of the freeze-dried products. The insulin load factor Ta is determined according to the procedure Ma.
38.9 g of NCA-GluOMe (0.208 mol) and 156 g of N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into a 0.5 liter reactor thermostated at 30° C. After dissolution, 5.78 g of a 0.452 M solution of ammonia in methanol (1.25 mol %/NCA) are added. The polymerization is monitored by measuring the carbon dioxide emitted into a gas bell jar and verified by disappearance of vibration bands characteristic of the NCAs at 1860 and 1790 cm−1. After 30 min, a solution of 23.3 g of NCA Leucine (0.149 mol) in 5 219 g of NMP is introduced. After 10 min of reaction, the temperature is increased to 60° C. The polymerization is monitored as above and is complete after 1-2 hours. The temperature of the reaction mixture obtained previously is increased to 80° C. 42.0 g of aqueous hydrochloric acid (35% of the mass) are added, with mechanical stirring over 30 min, to the reaction mixture. The reactor is then placed under reduced pressure regulated at 600 mBar for 6 hours. A mixture of 42.0 g of 35% hydrochloric acid and of 167.9 g of water is then added over 60 min, followed by a second phase of vacuum at 250 mBar for 18 hours. The reaction mixture is then cooled to 50° C. and then neutralized with aqueous sodium hydroxide (35% by mass). The NMP and the sodium chloride formed during the neutralization are removed by diafiltration against 20 volumes of Milli Q water, on a membrane with an MWCO of 1 000 Daltons (Pellicon II system, Millipore). A stable aqueous colloidal suspension of vector nanoparticles is thus obtained. The suspension of nanoparticles is finally freeze-dried.
The contents of leucine motifs are determined by proton nuclear magnetic resonance (signals at 2.10, 2.22 and 2.58 ppm for 4H of Glu and at 0.85 ppm for 6H of Leu). The mean hydrodynamic diameter Dh is measured according to Md on aqueous suspensions of the freeze-dried products. The insulin load factor is determined according to Ma.
46.4 g of NCA-GluOMe (0.248 mol) and 186 g of N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into a 0.5 liter reactor thermostated at 30° C. After dissolution, 6.90 g of a 0.19 M solution of ammonia in methanol (1.25 mol %/NCA) are added. The polymerization is monitored by measuring the carbon dioxide emitted into a gas bell jar and verified by disappearance of vibration bands characteristic of the NCAs at 1860 and 1790 cm−1. After 30 min, a solution of 12.97 g of NCA Leucine (0.083 mol) in 218 g of NMP is introduced. After 10 min of reaction, the temperature is increased to 60° C. The polymerization is monitored as above and is complete after 1-2 hours. The temperature of the reaction mixture obtained previously is increased to 80° C. 40.3 g of aqueous hydrochloric acid (35% of the mass) are added, with mechanical stirring over 30 min, to the reaction mixture. The reactor is then placed under reduced pressure regulated at 600 mBar for 6 hours. A mixture of 40.3 g of 35% hydrochloric acid and of 161.3 g of water is then added over 60 min, followed by a second phase of vacuum at 250 mBar for 18 hours. The reaction mixture is then cooled to 50° C. and then neutralized with aqueous sodium hydroxide (35% by mass).
The NMP and the sodium chloride formed during the neutralization are removed by diafiltration against 20 volumes of Milli Q water, on a membrane with an NWCO of 1 000 Daltons (Pellicon II system, Millipore). A stable aqueous colloidal suspension of vector nanoparticles is thus obtained. The suspension of nanophases is finally freeze-dried.
The contents of leucine motifs are determined by proton nuclear magnetic resonance (signals at 2.10, 2.22 and 2.58 ppm for 4H of Glu and at 0.85 ppm for 6H of Leu). The mean hydrodynamic diameter Dh is measured according to Md. The insulin load factor is determined according to Ma.
The particles obtained by the teaching of patent WO 96/29991 are those which appear in
The differences in morphology and size appear blatantly on comparing
The pulverulent powder of Example 2 is dissolved in an amount of 60 mg/ml of powder in a phosphate buffer. The pH was adjusted to 7.3 and the osmolality of the suspension was adjusted to 300 mOsm/kg using a 5 M NaCl solution. The solution was filtered (0.22 μm) before being distributed at the rate of 5 ml into sterile 10 ml bottles. The stability of the samples was evaluated over a period of 4 months. Half of the samples were kept at 4° C. (±2° C.) while the other samples were maintained at laboratory temperature: 25° C. (±5° C.). At given times, the samples are collected from the site of storage and equilibrated for 1 hour at room temperature before the analysis. The analytical methods are detailed, the results being presented in the form of two tables.
1) Verification of the homogeneity of the colloidal solution: Without stirring the suspension, 100 μl samples are collected three times in order to represent the state of the solution at the top, in the middle and at the bottom of the bottle. The refractive index of each sample is measured at 25° C. on an Abbe refractometer calibrated relative to pure water. Three readings are made for each sample and the three mean values are compared. Any variation in the concentration of the solution results in a difference in refractive index.
2) Measurement of the hydrodynamic diameter: A 100 μl sample of the solution to be analyzed is diluted 120-fold with a 0.15 M NaCl solution and the Dh of the colloidal particles is measured according to the protocol Md.
3) Measurement of the viscosity: The measurements are carried out on 0.75 ml samples using an AR1000 rheometer (TA instruments) equipped with a Cone/Plane geometry (cone 4 cm/2° C.) at a temperature of 20.0° C.+/−0.1° C. (regulation by Pelletier effect). The viscosity curve as a function of the shear gradient is recorded for gradients varying from 1 to 100 s−1. At these concentrations, the solutions are slightly rheofluidizing and the viscosity value selected is taken for a gradient of 10 s−1.
The results obtained after aging at 4° C. and 25° C. are assembled in Tables I and II.
A formulation is prepared from PV (of Example 3) and insulin, the quantity of each being determined according to measurements of combination rate (Ma).
A group of 4 beagle dogs (males and females) weighing between 10 and 12 kg are fasted for 18 hours. A preparation is formulated and is composed of 80 IU of insulin t 56 mg of PV in 1 ml of PBS buffer. The dogs then receive a subcutaneous administration of this insulin preparation at the rate of 2 IU/kg of weight. Blood sample collected for glucose and insulin assay before (−2 h, −1 h and 0 h) and after (1 h, 2 h, 4 h, 6 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h) the injection. The glucose concentrations are measured in the samples by the glucose oxidase method and serum insulin is assayed using a radioimmunological method.
This example shows, through the biological activity, the nondenaturation of the protein as well as the possibility of prolonging the release by >24 h, two advantageous aspects of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR 99 14751 | Nov 1999 | FR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10130783 | Aug 2002 | US |
| Child | 11783801 | Apr 2007 | US |
