KR20090054976A - Microparticles based on an amphiphilic copolymer and on active ingredient(s) with modified release and pharmaceutical formulations containing same - Google Patents

Abstract

The present invention relates to novel microparticles of amphiphilic polyamino acids that transport active ingredient(s)-AI(s)-, in particular protein and peptide active ingredient(s), and also to novel modified-release pharmaceutical formulations containing said microparticles of AI. The objective of the invention is to develop novel microparticles loaded with AI, obtained by aggregation of nanoparticles of amphiphilic polyamino acids, and having improved properties, in particular in dry solid form, with regard to their capacity for dispersion and, as regards the reconstituted suspension, with regard to its stability and its ability to be readily handled and injected. In a first aspect, the invention relates to microparticles of amphiphilic polyamino acid (PO) containing at least one AI (associated noncovalently), spontaneously forming a colloidal suspension of nanoparticles in water, at pH 7.0, under isotonic conditions; wherein said microparticles are characterized a. in that they are obtained by atomization of a colloidal suspension or solution of PO comprising at least one AI, b. by a size of between 0.5 and 100 mum, and c. in that they are dispersible in a colloidal suspension. The invention also relates to the method for preparing these microparticles, to a liquid formulation comprising a suspension of these PO/AI microparticles, to a method and a kit for reconstituting this formulation, and to a dry form of this formulation.

Description

Microparticles based on the active ingredients and amphiphilic copolymers of modified release and pharmaceutical preparations containing the same

The present invention relates to novel transport agents for the active ingredient (AP), in particular protein and peptide active ingredients, and to novel modified release pharmaceutical preparations containing the AP transport agent. This formulation is applicable to several treatments (human and animal).

Code AP, as used throughout this specification, refers to one or more active ingredients.

The term "modified release" means delayed release and / or sustained release and / or pulsed release.

More specifically, the novel AP transporting agents of the present invention are fine particles formed from amphiphilic polymers such as polyamino acids modified with hydrophobic groups. These microparticles comprise one or more APs associated with a polymer and may be provided in a colloidal suspension form or in a dry form.

In the field of delayed release of pharmaceutical APs, in particular therapeutic peptides / proteins, in many cases, the aim is to reproduce the plasma protein or peptide concentration of the patient as closely as possible to the value observed in healthy subjects.

This goal is impaired because of the short lifespan of the protein in plasma, which requires repeated injections of the therapeutic protein. The plasma concentration of the therapeutic protein exhibits a "sawtooth" profile characterized by high peaks and very low peaks. The peak concentration is much higher than the basal concentration in healthy subjects, with a very significant adverse effect attributable to the high toxicity of therapeutic proteins such as interleukin IL-2. Moreover, since the concentration trough is below the concentration required to have a therapeutic effect, the patient's treatment recovery is poor and suffers severe long-term side effects.

Finally, to reproduce the plasma concentration of a patient's therapeutic protein close to the ideal value for treating the patient, the pharmaceutical formulation releases the therapeutic protein during a delayed period so that the plasma concentration changes little over time. It is important to consider.

Moreover, such active agents preferably meet the following points, which are already known to those skilled in the art.

1-delayed release of active and non-denatured therapeutic proteins, such as human or synthetic proteins, such that plasma concentrations are maintained at therapeutic levels,

2-injection viscosity low enough for easy injection,

3-biocompatible and biodegradable form,

4-a form that exhibits neither a toxic or immune response,

5-Form with good local tolerance.

In attempting to achieve these goals, one of the best approaches proposed in the prior art is to develop a form for delayed release of therapeutic proteins consisting of a low viscosity liquid suspension of nanoparticles containing therapeutic proteins. It was. These suspensions facilitated the administration of therapeutic natural proteins.

Accordingly, therapeutic proteins have been associated with nanoparticles consisting of copolyamino acids containing hydrophobic and hydrophilic groups.

Patent US-B-5,904,936 discloses quasi-particulates (NPV) having an average particle size of 0.01 to 0.5 μm of an amphipathic polyamino acid copolymer comprising two or more amino acids, one of which is neutral and hydrophobic and the other which is ionic; Particulates (MPV) with an average particle size of 0.5 to 20 μm are described. Proteins such as insulin are adsorbed spontaneously on the surface of these particles in an aqueous solution. The polyamino acid copolymer is, for example, a block copolymer of poly (L-leucine-bL-sodium glutamate). The patent includes monovalent cationic salts (ammonium sulfate) or polyvalent cationic salts (Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Ca ²⁺ , Al ²⁺ , Al ³⁺ or Cu ²⁺ ), acids (HCl) or Aggregation of NPV into MPV is described by adding a cationic polymer (polylysine) to a colloidal suspension of poly-Leu / Glu.

Patent application WO-A-03 / 104303 discloses polyglutamate grafted with alpha tocopherols derived from these residues (such as synthetic or natural-derived alpha tocopherols) containing aspartic acid residues or glutamic acid residues and having at least one alpha-tocopherol unit. Or amphiphilic polyamino acids comprising at least a portion of polyaspartates). In water, these “hydrophobically modified” homopolyamino acids spontaneously form colloidal suspensions of nanoparticles that can easily associate with one or more active proteins (insulin) in an aqueous suspension at pH 7.4.

Suspensions according to US-B-5,904,936 and WO-A-2003 / 104303 can advantageously increase the in vivo release time of "vectorized" active proteins (eg insulin).

The increase in release time has been partially achieved by the pharmaceutical formulations described in PCT application WO-A-05 / 051416. In this patent application, a hydrophobically modified colloidal suspension of poly (L-sodium glutamate) nanoparticles (0.001 to 0.5 μm) forms a gel in the patient's field upon contact with endogenous serum albumin after subcutaneous injection. Injections are made at the concentrations desired. The protein is then slowly released, usually over a week. However, as in the case of human growth hormone, when the dosage concentration of the therapeutic protein is relatively high, for example, the release time is limited to only a few days.

The disadvantage of the prior art is that

After parenteral (eg subcutaneous) administration, it is possible to obtain in vivo delayed release time of undenatured high concentration (eg several mg / ml) AP (eg therapeutic proteins and peptides and small molecules),

And stable during storage in physiochemical and biological

It is possible to overcome by providing a pharmaceutical formulation for delayed release of AP.

To this end, it is possible to agglomerate into microparticles, using polyatomic ions of opposite polarity to that of the ionic groups (IG) of the amphiphilic polyamino acids in clearly defined proportions with the nanoparticles of the preparations according to WO 05/051416. have. This allows the selection of particulates of a particular population which significantly delay the release time of the AP (eg, protein or peptide) to which these particulates are bound. Some polyvalent ions such as Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Fe ²⁺ , Cu ²⁺ or mixtures thereof and / or Al ³⁺ , Fe ³⁺ or mixtures thereof are liquids for AP delayed release. Imparts good resistance to pharmaceutical preparations. This formulation comprises, for example, a low viscosity colloidal aqueous solution based on microparticles of polyglutamate grafted with synthetic alpha-tocopherol with a particle size of 0.5 to 100 microns, with the polyvalent ions Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Fe ²⁺ , Cu ²⁺ Al ³⁺ or Fe ³⁺ , and the product r is represented by the following formula.

Where

N is the valence of the polyvalent ion,

[ MI ] is the molar concentration of the polyvalent ion,

[ IG ] is the molar concentration of the ionic group IG, and r is from 0.3 to 10.

These selected microparticles consist of agglomerated several nanoparticles of an amphipathic copolymer.

Therefore, particulate suspensions filled with AP (eg, hGH) are required to aggregate, mature and wash with the polyvalent ions Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Fe ²⁺ , Cu ²⁺ Al ³⁺ or Fe ³⁺ . Is manufactured. The suspension is then lyophilized, sprayed and then reconstituted with water to prepare an injectable formulation.

It is clear that these dry powdery fine particles are extremely advantageous in terms of storage. Specifically, they should be able to increase the physical stability of these particulates and the stability of the AP.

However, in the use of these particulate powders for parenteral administration of therapeutic agents, it is easy to disperse these particles at room temperature, for example for several minutes and indeed for at least several ten minutes when used to obtain a reconstituted liquid particulate form, more specifically a stable suspension. The difficulty is to be able to (or suspend) it. Such stability is particularly required in order to be able to easily handle such reconstitution suspensions. It is also important that such reconstituted suspensions be readily injectable by delivery through a syringe or an empty syringe with an injection pen (insulin pen type) attached. Finally, it should be borne in mind that the reconstituted suspension must be suspended and can be handled and injected by the patient or healthcare practitioner.

In this respect, an essential object of the present invention is to provide novel microparticles obtained by agglomerating nanoparticles of biodegradable and water-soluble amphiphilic polymers, such as those according to the formulations described in WO-A-05 / 051416. It contains AP and may exhibit improved properties, particularly in terms of its ability to disperse ("dispersibility") in relation to dry solid forms, and in terms of its stability, handleability and injectability with respect to reconstituted suspensions. Refers to a characteristic that has been acquired.

It is another object of the present invention to provide novel microparticles obtained by agglomeration of nanoparticles of biodegradable and water-soluble amphiphilic polymers, wherein the microparticles contain AP, and whether the microparticles are in aqueous or oily form It is a feature that it shows good dispersibility while maintaining the structure of.

It is another object of the present invention to provide novel microparticles obtained by agglomeration of biodegradable and water-soluble amphiphilic polymer nanoparticles, which are characterized by containing AP and exhibiting excellent stability in solid drying.

It is another object of the present invention to provide a novel method for producing solid dry fine particles,

¡Æ the microparticles are obtained by agglomerating biodegradable and water soluble amphiphilic polymer nanoparticles, such as those according to the formulations described in WO-A-05 / 051416,

¡Æ the particulates contain AP

→ the microparticles further exhibit the characteristic as defined for the purpose.

Another object of the present invention is to provide a method for producing solid dry fine particles which can achieve the above objects in a simple, economical and large-scale industrial.

Another object of the present invention is to provide pharmaceutical formulations comprising biodegradable and water-soluble amphiphilic polymer nanoparticles, for example novel microparticles obtained by agglomeration of those according to the formulations described in WO-A-03 / 104303. Is to provide

¡Æ the particulates contain AP

≧ the particulates are as defined for this purpose

Indicates further features.

Another object of the present invention is to overcome the disadvantages of the prior art and, in particular, after parenteral (eg subcutaneous) injection, undenatured AP (eg protein, therapeutic peptide or small molecule) may be delayed release in vivo. It is to provide a pharmaceutical formulation for delayed release of the AP.

It is another object of the present invention to provide pharmaceutical preparations which, after parenteral (eg subcutaneous) injection, delay the release of highly concentrated, therapeutic proteins or peptides such as several mg / ml in vivo.

It is another object of the present invention to provide a pharmaceutical formulation for delayed release of an in vivo AP which is physiologically and biologically stable upon storage.

Another object of the present invention is to provide a pharmaceutical formulation for delayed release of AP in vivo, which exhibits at least one property of biocompatibility, biodegradability, non-toxicity and good local resistance.

It is still another object of the present invention to provide a pharmaceutical formulation for slowing the delayed release of AP in vivo comprising microparticles of amphipathic polymer PO (PO / AP microparticles) that self-associate with at least one AP. Is a water-soluble biodegradable polymer containing a hydrophobic group (HG) and a hydrophilic group (preferably an ionic group (IG) that is at least partially ionized), which can spontaneously form colloidal nanoparticles in water, the backbone of which polymer PO Such as formed by aspartic acid residues or glutamic acid residues, wherein at least a portion of the unit is modified by grafting one or more hydrophobic groups HG in the chain and / or at the chain end.

Another object of the present invention is to provide a kit for reconstructing a formulation as defined in the above-mentioned object, which means that the kit is easy to use for example for easy use by a patient or medical practitioner.

Another object of the present invention is to provide a method for reconstitution of an agent as defined in the above-mentioned object, which means that it can be easily performed by a patient or medical practitioner, for example.

It is another object of the present invention to provide a solid pharmaceutical formulation for delayed release of AP, in particular a dry powdered pharmaceutical formulation for pulmonary inhalation and administration.

And based on the AP and the associated at least one PO particles as defined in the above-mentioned object, and

And or, it means obtained from the preparation as defined in the above-described purpose.

In order to achieve the objects as described above, the inventors of the present invention, after a steady study in the laboratory, spray the preparation based on amphiphilic PO (such as copolyamino acid) and AP, it is stable, and the aqueous suspension of the particles in a liquid medium It has surprisingly been found that it is possible to reconstruct and to provide dry PO / AP particulates having a particle size of 0.5 to 100 microns.

In addition, the inventors have developed a means for reconstructing these particulate suspensions, which means means for optimizing their dispersion in both aqueous and oily liquid phases.

This is a high quality, stable and easily dispersed dispersion because it is a prerequisite for the use of reconstituted suspensions from dry PO / AP microparticles as injectable preparations.

Spraying is a known industrial technique for producing dry particles from solutions or suspensions of constituent materials that are the particulates. Spraying (or spray drying) consists of very rapidly evaporating in hot air or hot inert gas vapor and spraying a drop of such solution or suspension.

In the pharmaceutical field, spraying techniques can be problematic because they are exposed to heat stress, which has proven to be unacceptable for heat sensitive APs, such as APs based on proteins or other peptide compounds. Therefore, spraying to prepare excipients and AP-based dry particles is self-evident except selecting an excipient that prevents and finally minimizes peptide AP degeneration.

Accordingly, patent application US-A-2005 / 0158392 describes a preparation by spraying solid lipophilic particulates containing peptide AP. This spraying technique involves spraying an aqueous solution comprising hyaluronic acid, AP and a lipophilic surface active agent (eg lecithin), or other surfactants of class Tween ^® 80 as the first step, or hyaluronic acid, AP and optionally Spraying an aqueous solution containing a Tween ^® 80 surfactant to obtain a primary particle and, as a second step, spraying an alcohol solution of a lipophilic surfactant (such as lecithin) in which the primary particles are dispersed. By that. The key to this US application is the protective combination of hyaluronic acid with a lipophilic surfactant (such as lecithin), which is intended to form a coating film for hyaluronic acid and AP based particulates. This document does not mention the use of amphiphilic PO, nor does it describe the formation of particulates together with an amphiphilic copolyamino acid or a transport agent of AP as an excipient or a transport agent.

Examples of other spraying techniques are described in Maa et al., J. Pharm. Sci., 87 (2), 152-159, 1988. According to this document, the AP (human growth hormone) is combined with zinc in the presence of a biodegradable surfactant and then sprayed to form fine particles.

Therefore, spraying proteins is a complex that contains multiple excipients that are particularly important for certain sensitive molecules, such as human growth hormone (hGH), to protect AP during processing and to ensure stability upon storage. It is clear that there are difficulties in requiring the use of the formulation.

Moreover, the inventors further note that there is no teaching in the prior art regarding the problem of stabilizing and dispersing dry particulates (particularly with respect to fine particles of amphiphilic PO) developed for the purpose of delayed release of AP. Considered.

From the above, the first aspect of the present invention relates to fine particles of a polymer (PO) containing at least one active ingredient (AP), wherein the polymer (PO)

And a hydrophobic group (HG) and a water-soluble and hydrophilic group containing biodegradable amphiphilic

(Co) polymer,

And the spontaneous appearance of a colloidal suspension of nanoparticles in water of pH = 7.0 under the condition Castle

Form as enemies,

It is associated with the AP and non-covalent associative,

The fine particles,

a . By spraying a solution or colloidal suspension of PO containing at least one AP

Obtained,

b . Particle size measured by T test, 0.5 to 100 microns, preferably 1 to 70

Microns, preferably 2 to 40 microns,

c . Dispersible in colloidal suspension in "dispersibility" test DP1

It is characterized by that.

In a second aspect, the present invention relates to a process for producing PO particulates associated with one or more active ingredients (AP), wherein such PO / AP particulates are specifically defined according to the first aspect of the present invention. Saying,

i. The polymer (PO) is,

And a hydrophobic group (HG), and preferably a hydrophilic group [this is at least partially ionized

Warm group (IG)], which is a water-soluble and biodegradable amphiphilic (co) polymer,

To cut a colloidal suspension of nanoparticles in water at pH = 7.0 under isotonic conditions.

Can be formed with redness,

And it is associated with the AP and non-covalent associative,

ii. The particle size of the microparticles ranges from 0.5 to 100 microns, preferably from 1 to 70 microns, preferably from 2 to 40 microns, as measured by a T test, and the process essentially consists of a solution or colloidal suspension of PO containing the AP. It is characterized by spraying. According to an interesting variant embodiment, the microparticles obtained by spraying are redispersed in an essentially aqueous liquid medium (preferably containing polyvalent ions as a dispersing means) and then the resulting dispersion is freeze dried.

According to a third aspect, the present invention relates to a liquid pharmaceutical formulation for delayed release of AP, wherein the formulation comprises a "low" viscosity colloidal suspension based on PO particulates containing at least one AP, which particulates It is defined according to the first aspect of the present invention or obtained by the method defined in the second aspect of the present invention.

According to a fourth aspect, the present invention relates to a reconstitution kit, in particular a reconstitution kit for reconstitution of an agent defined according to the third aspect of the invention.

Fine particles containing at least one AP (these fine particles are those described in the first aspect or those obtained by the method described in the second aspect),

And → essentially be adult liquid

    ¡Æ essentially an organic water-miscible liquid

    ¡Æ essentially a reconstituted liquid selected from the group comprising organic water immiscible liquids

Characterized in that it comprises a.

In a fifth aspect, the invention relates to a reconstitution method, in particular to a reconstitution method for reconstitution of a formulation as defined in the third aspect of the invention, which method comprises the following steps:

And ⇒ PO fine particles (fine particle containing at least one AP is based on the point of view of a first

Or those obtained by the method described in the second aspect),

    ⇒ → essentially aqueous liquid

       ¡Æ essentially an organic water-miscible liquid

       ¡Æ essentially an organic water immiscible liquid

 Mixing the reconstitution liquid selected from the group comprising:

And the step of shaking the mixture

Essentially includes.

In a sixth aspect, the invention relates to a solid pharmaceutical formulation for delayed release of an AP, the formulation comprising a dry powder form for inhalation and pulmonary administration,

PO microparticles containing and at least one AP is or is based on (the fine particles are the first ones that are defined in terms of one of the invention, or those obtained by a method as defined in terms of the second of the present invention),

And or, it means obtained from the preparation as defined in the third aspect of the present invention.

Advantages:

The hydrophobic groups of the polymer PO are thought to play an important role in the stabilization process later, because they form a hydrophobic domain upon bonding and play an important role in the process for modified release of AP, but are not intended to be limited to this particular theory.

It can be hypothesized about the physical (non-covalent) interactions that occur between the amphiphilic polymer PO and AP, in particular the hydrophobic domains of the protein. Because of the strong affinity of the protein for such polymers, they can be delayed release after subcutaneous administration. This release mechanism is different from the release mechanism observed for the polylactic acid, the polylactic acid-glycolic acid microparticles and may optionally be used in combination with this, or the release of AP is essentially related to the diffusion of the active ingredient, It is different from the release mechanism observed for sodium hyaluronate associated with decay / corrosion and can optionally be combined with it.

Due to the affinity of the protein for the polymer PO, the damage that can occur without the need to re-add other stabilizers (such as sugars, surfactants, etc.) during the spraying process is limited, and the aggregation phenomenon of the protein is also limited. This protective effect of PO facilitates obtaining a stable liquid formulation upon storage. Finally, when PO is essentially amorphous (particularly when PO is a polyamino acid), it gives the fine properties of physical stability to the microparticles when stored in dry form.

Therefore, it is evident that due to the presence of these amphiphilic polyamino acid PO additional means have been introduced to control the stability to modified release, aggregation or possible chemical degradation of AP.

Furthermore, during the spraying process, due to the amphiphilic nature of the polymer PO, it can be made completely aqueous, or completely organic volatile, or a volatile mixture of the aqueous and oily phase, so It gives you great flexibility.

In addition, because of its chemical properties, the microparticles formed from PO of the (co) polyamino acids are biodegradable, biocompatible and generally have good local resistance properties.

The process for producing solid and dry microparticles, ie spraying, according to the invention can easily be carried out smoothly on an industrial scale.

Finally, the microparticles of the invention and formulations containing them are nontoxic and have good local resistance.

Justice:

Throughout this specification, the conjunction "or" is to be understood as meaning "one or the other or both." Thus, for example, "straight chain alkylene which may contain one or more heteroatoms (O, N, S) or one or more unsaturated bonds" may contain unsaturated bonds with two heteroatoms N and S. Say what it is.

Throughout this specification, unlike the microparticles described herein, "particulate particles" or "nanoparticles" are particles having a particle size (measured by the T test described below) of, for example, 1 nm or more and 500 nm or less, preferably 5 Refers to particles that are from 250 nm.

Within the scope of the present invention and the specification of the present invention, the term "polyamino acid" encompasses both natural and synthetic polyamino acids, as well as oligoamino acids comprising 2 to 20 amino acid residues, as well as 20 or more amino acids. Also included are polyamino acids comprising residues.

The preferred amino acid residues of the main polyamino acid chain are of the L form, and the preferred backbone is the alpha form, ie there is a peptide bond between the alpha amino group and the carboxylic acid group of the amino acid at position 1 of the other alpha-amino acid.

Within the context of the present invention, the term "protein" refers to both proteins and peptides, for example whether or not oligopeptides or polypeptides. This protein or peptide can be modified, for example by grafting one or more polyoxyethylene groups.

Within the context of the present invention, the term "association" or "to associate" means that the relationship of the polymer PO with one or more APs is particularly such that the active component is polymerized by non-covalent bonds, such as electrostatic interactions or hydrogen bonds or steric interferences. Used to describe what is bound to the PO.

First Aspect of the Invention: Particulates

Feature (a):

Prior to spraying, for example, the binding of a protein or other peptide compound with an amphiphilic polymer PO, such as an amphiphilic (co) polyamino acid, is not only relevant to the process itself, but also to the properties of the particulates produced by spraying. Provides the benefits of.

Physical spraying of the resulting dry solid particulates results in a unique structure that is the source of these various advantages. To be precise, by spraying this structure characterizes this manufacturing method and also gives this advantage a function.

Feature (b):

This feature is evident by the T test below.

T test for measuring particle size by laser scattering method

a -T0 test when fine particles are dry

1. Equipment and operating conditions

 Laser particle size analyzer  Malvern Mastersizer 2000  unit  Heathrow 2000SM Wet Route  Volume of carrier fluid for dispersion  150 ml  Wavelength (Blue and Red)  466 and 632 nm  Shaking speed  2400 rev / min  Scope of analysis  0.02 μm to 2000 μm  Optical Model (Mie Theory) Refractive Index Used: Dispersion Fluid (Water) Polystyrene Latex m _fluid = 1.39 + i.0 m _{polystyrene latex} = 1.59 + i.0  Shield value for analysis triggers  5% to 20%  Collection time  10 sec

2. Preparation of Sample

0.1% of a solution of Span 80 dissolved in heptane is prepared (by weighing and introducing 0.01 g of powdered Span 80 into a 20 ml flask and then adding heptane to a final weight of 10 g).

Weigh about 6 mg of powder and place in 5 ml test tube.

0.7 g of heptane containing 0.1% span 80 is added to the test tube.

The test tube is placed in an ultrasonic container for 2 minutes to completely disperse the powder.

3. Analysis of Sample

The circulating fluid stored in the idle wet sample dispersion system is emptied and replaced with heptane. The shaking speed of the Hydro 2000 SM device is set at 2400 rev / min.

Measurement is initiated under the experimental conditions described above.

1-alignment of the laser beam

2-recording of background noise

After these steps, the analytical sample is introduced in the following manner: the diluted sample is added dropwise (by Pasteur pipette) until the shielding value is between 5% and 20% and the collection is started. .

Data is obtained for D50, the diameter at which up to 50% of the subjects are found.

Three D50 measurements for three different formulations are taken and their average value is taken.

b-T1 test when the fine particles are in aqueous dispersion form

1. Equipment and operating conditions

Laser particle size analyzer Malvern Mastersizer 2000 unit Hydro 2000SM Wet Root Volume of carrier fluid for dispersion 150 ml Wavelength (Blue and Red) 466 and 632 nm Shaking speed 2400 rev / min Scope of analysis 0.02 μm to 2000 μm Optical Model (Mie Theory) Refractive Index Values Used: Dispersion Fluid (Water) Polystyrene Latex m _fluid = 1.33 + i.0 m _{polystyrene latex} = 1.59 + i.0 Shield value for analysis triggers 5% to 20% Collection time 10 sec

2. Preparation of Sample

The analytical sample may be introduced into the cell as it is, or in the case of a highly scattering sample, it may be re-diluted with water if necessary.

3. Analysis of Sample

Empty the circulating fluid stored in the wet, wet sample dispersion system and replace with demineralized water. The shaking speed of the Heathrow 2000 SM device is set to 2400 rev / min.

Measurement is initiated under the experimental conditions described above.

1-alignment of the laser beam

2-recording of background noise

After these steps, the analytical sample is introduced in the following manner: the diluted sample is added dropwise (by Pasteur pipette) until the shielding value is between 5% and 20% and the collection is started.

Data is obtained for D50, the diameter at which up to 50% of the subjects are found.

Three D50 measurements for three different formulations are taken and their average value is taken.

c-T2 test when fine particles are dispersed in oily solvent

This test is identical to the T1 test. Nevertheless, in this case it is required to use a solvent which is completely miscible in the dispersion and in which the particles cannot dissolve. In many cases heptane is used.

1. Equipment and operating conditions

 Laser particle size analyzer Malvern Mastersizer 2000  unit Hydro 2000SM Wet Root  Volume of carrier fluid for dispersion 150 ml  Wavelength (Blue and Red) 466 and 632 nm  Shaking speed 2400 rev / min  Scope of analysis 0.02 μm to 2000 μm  Optical Model (Mie Theory) Refractive Index Values Used: Dispersion Fluid (Water) Polystyrene Latex m _heptane = 1.39 + i.0 m _{polystyrene latex} = 1.59 + i.0  Shield value for analysis triggers  5% to 20%  Collection time  10 sec

2. Preparation of Sample

To prepare the sample to be analyzed, 400 μl of the sample to be analyzed is diluted with 600 μl of heptane in a 5 ml test tube and prepared by vortexing for 10 seconds (10 ± 5).

3. Analysis of Sample

Empty the circulating fluid stored in the wet, wet sample dispersion system and replace with demineralized water. The shaking speed of the Heathrow 2000 SM device is set to 2400 rev / min.

Measurement is initiated under the experimental conditions described above.

1-alignment of the laser beam

2-recording of background noise

After these steps, the analytical sample is introduced in the following manner: the diluted sample is added dropwise (by Pasteur pipette) until the shielding value is between 5% and 20% and the collection is started.

Data is obtained for D50, the diameter at which up to 50% of the subjects are found.

Three D50 measurements for three different formulations are taken and their average value is taken.

Feature c:

This feature is evident by the DP1 test below.

"Dispersibility" DP1 test of powder:

30 mg of the particulate powder is placed in a 3 ml flask equipped with a septum. Measure the exact amount of particulate (w ₁ ). Add 1 ml of the reconstituted solution to the powder through the septum using a 1 ml syringe equipped with a 25G x 5/8 (0.5 x 16 mm) needle (e.g. BD Microlance 3 type) and shake by shaking occasionally for 15 minutes by hand. . The flask is weighed so that the exact mass (w ₂ ) of the solution introduced can be determined. At the end, the entire suspension was inhaled using a 1 ml syringe (Braun Injeckt-F Luer 1 ml type, ref. 9166017V) equipped with a 25G needle, poured into a flask before use, and the weight of the recovered solution w ₃ Measure

Quality aspects of the dispersion (dispersion tendency, turbidity, visual solution homogeneity), whether or not it can be injected through a 25 G needle and the percentage of solution recovered, ie

% = w ₃ x 100 / (w ₁ + w ₂ )

And the particle size of the fine particles are measured according to the T1 or T2 test (depending on whether the dispersion is an aqueous solution or an oily liquid).

It is considered to be good dispersibility in liquids if one of the following cases is true:

The resulting suspension contains fine particles having an average diameter of 2 to 40 μm

■ The appearance of the suspension is homogeneous

■ If you can inject with a 25 G needle

■ At least 80% of the suspension is recovered

Apart from the above features (a), (b) and (c), the microparticles according to the invention can be characterized as being stable in the following ST1 test and / or ST2 test.

ST1 test to test the physical stability of dry particulate:

The particle size of the particles that are in the form of dry sprayed powder is measured within one week after spraying the powder on a laser particle size meter according to the T0 test. This powder is placed in an oven at 30 ° C. for a week. The "control" sample is placed at 5 ° C. The size measurement is performed again after dispersion under the same conditions as at time t0. The distribution of the fine particles before and after aging is compared.

ST2 test to test the colloidal stability of the microparticles in suspension:

The particulate powder is dispersed in the dispersion with magnetic shaking at the concentration required to be observed to be stable, which is left with magnetic shaking at medium intensity for at least 2 hours (measured t0).

Particulate particle size is measured by a T1 or T2 test depending on the dispersing medium (aqueous or oily) in a laser particle particle size analyzer.

This suspension is left at 5 ° C. for 7 days. The settled pellets are shaken and dispersed for several minutes (until visual observation yields a homogeneous suspension). Particle size measurements are performed after dispersion under the same conditions as time point t0.

Polymer PO

The polymer PO according to the invention is a water soluble, biodegradable polymer containing a hydrophobic group HG and a hydrophilic group [preferably an ionic group IG which is at least partially ionized]. The hydrophobic group HG may have a small portion of the chain and may be attached to the side of the chain, inserted into the chain, randomly distributed (random copolymer), or distributed in sequential or graft form (block airborne). Coalescing or sequential copolymers).

According to a preferred embodiment of the invention, the polymer PO is an amphipathic (co) polyamino acid.

By selecting such PO, it provides good suitability for use with the protein / peptide class of AP. Accordingly, it is possible to use polymer PO to simplify the composition of the starting solution or suspension of the protein / peptide class of AP. Such starting solutions or suspensions may contain only AP and PO in aqueous and / or oily phase form. Since other components are absent, this starting solution or suspension can be filtered (0.2 μm filter) before spraying, so that spraying can be performed under sterile conditions.

According to a preferred embodiment of the present invention, PO is selected from amphipathic (co) polyamino acids and combinations thereof.

Preferably, the polyamino acids according to the invention are oligomers or homopolymers containing glutamic or aspartic acid repeat residues or copolymers containing mixtures of amino acid residues of this glutamic or aspartic acid class. Residues contemplated as such polymers are amino acids having the form D or L or D / L, which are bonded at the α or γ of the glutamic or glutamic acid residues, or at the α or β position of the aspartic acid or aspartate residues.

According to a further preferred embodiment of the invention, the polymer PO is a polyamino acid formed by aspartic acid residues and / or glutamic acid residues, at least some of which contain grafts containing at least one hydrophobic group HG. These polyamino acids refer in particular to the classes described in PCT patent application WO-A-00 / 30618.

According to a first possibility, PO is defined by the following formula 1 (wherein radical -COOR ³ comprises a form wherein the bond between carboxyl and R ³ is an ionic bond -COO ^{_ +} R ³ ).

In the above formula,

R ¹ is H, straight C ₂ to C ₁₀ or branched C ₃ to C ₁₀ alkyl, benzyl, or -R ^4-

[HG], or

■ NHR ¹ represents a terminal amino acid residue,

R ² represents H, straight C ₂ to C ₁₀ or branched C ₃ to C ₁₀ acyl group, terminal pyroglutamate group or -R ^4- [HG],

R ³ is H, or

■ ⁺ R ³ is

   Advantageous from a subgroup consisting of sodium, potassium, calcium and magnesium

     A metal cation selected from

   -The following cations,

An amine cation,

And oligo amine cations,

And the polyamine-based cation (which is especially preferred polyethyleneimine),

And lysine or arginine-based amino acids advantageously selected from cationic acids

      Organic amount advantageously selected from subgroups of systemic cations

      With ions,

   Advantageously from a subgroup consisting of polylysine and oligolysine

      Cationic Polyamino Acids Selected

It is preferably selected from the group consisting of

R ⁴ is a direct bond or a 1-4 amino acid residue-based “spacer

(spacer) "

■ A is independently a -CH ₂ -radical (aspartic acid unit) or -CH ₂ -CH ₂ -radical

(Glutamic acid unit),

N / (n + m) is defined as the molar grafting rate and its value is measured in water at pH = 7 and 25 ° C.

Colloidal suspension of quasi-particulates of PO because it is low enough for dissolved PO

N / (n + m) is preferably 1 to 25 mol%, particularly preferably 1 to 15

Range in mole percent,

(N + m) is defined as the degree of polymerization and is from 10 to 1,000, preferably from 50 to 300

Range,

HG represents a hydrophobic group having 6 to 30 carbon atoms.

In a preferred embodiment of the invention, the hydrophobic group HG is selected from the group comprising alkoxy radicals of the type -OCH ₂ (CH ₂ -CH ₂ ) _3-8 -CH ₃ , oleyl, tocopheryl or cholesteryl, R ⁴ represents a single bond.

According to a second possibility, PO is one of the following formulas (2), (3) and (4) wherein the radical -COOR ^{3 '} is a form in which the bond between carboxyl and R ^3' is an ionic bond -COO ^{_ +} R ^{3 '} It includes).

In each said formula,

HG represents a hydrophobic group having 6 to 30 carbon atoms,

R ³⁰ is a straight divalent C ₂ to C ₆ alkylene chain,

R ^{3 '} is H, or

■ ⁺ R ^{3 '} is

   Free from a subgroup consisting of sodium, potassium, calcium and magnesium

A metal cation selected to

   The following cations, ie

An amine cation,

And oligo amine cations,

And the polyamine (which is especially preferred polyethyleneimine) based cations and,

And Lee New Territories or arginine-based organic cation is advantageously selected from the subgroup consisting of amino acid cations are selected from the free acids with a cation,

   Advantageously selected from subgroups consisting of polylysine and oligolysine

     Polyamino acid cation

It is preferably selected from the group consisting of

R ⁵⁰ is a straight divalent C ₁ to C ₈ alkylene chain, wherein one or two methyls

Each end of the ethylene unit, preferably R ⁵⁰ , may be independently substituted with —O— or —NH.

There is,

R ⁴ is a direct bond or a “spacer” based on one to four amino acid residues

ego,

■ A is independently a -CH ₂ -radical (aspartic acid residue) or -CH ₂ -CH ₂ -radical

(Glutamic acid residues),

(N '+ m') and n "are defined as degrees of polymerization, from 10 to 1,000, preferably 50

To 300.

According to another advantageous form, the R ⁴ group represents a single bond.

According to a third possibility of the present invention, PO is essentially neutral copolyhydroxyalkylglutamine (preferably alkyl is ethyl) containing several pendant hydrophobic groups (HG) groups identical or different from the others. Copolyhydroxyalkylglutamine also contains hydroxyalkylamine groups. These hydroxyalkylamine groups are bonded to the copolymer, preferably via amide bonds. The hydroxyalkylamine groups that can be used to amidate the carboxyl groups of the glutamate residues and which can be used to prepare this copolyhydroxyalkylglutamine are, for example, 2-hydroxyethylamine, 3-hydroxypropylamine, 2, Same or different from the one selected from 3-dihydroxypropylamine, tris (hydroxymethyl) aminomethane and 6-hydroxyhexylamine.

Advantageously, the at least one hydrophobic group HG is included in a hydrophobic graft comprising at least one spacing joint (or unit) (spacer) that allows connection to the hydrophobic group HG of the copolyglutamate chain. This joint may comprise, for example, one or more direct covalent bonds or one or more amide bonds or one or more ester bonds. For example, the joint may be those belonging to the group comprising in particular amino acid residues, derivatives of aminoalcohols, derivatives of polyamines (eg diamines), derivatives of polyols (eg diols) and derivatives of hydroxy acids. Grafting HG to copolyglutamate or polyalkylglutamine chains involves using HG precursors capable of binding to copolyglutamate or copolyhydroxyalkylglutamine chains. Precursors of HG are actually selected from the group comprising alcohols and amines, but are not limited to such compounds, which can be readily functionalized by those skilled in the art. With respect to such copolyhydroxyalkylglutamines (preferably alkyl is ethyl), reference may be made to FR-A-2,881,140.

According to an advantageous alternative, in particular at least one of the third possibilities mentioned above, some or all of the hydrophobic radicals HG in PO are independently selected from the group of radicals comprising:

■ straight or branched chain alkoxy, which may comprise one or more heteroatoms (preferably O, N, or S) and / or one or more unsaturated bonds, and has 6 to 30 carbon atoms,

■ optionally comprising at least one unsaturated bond or at least one hetero atom (preferably O, N, or S), having from 6 to 30 carbon atoms and at least one annealed cycloalkyl Alkoxy containing,

■ Alkoxyaryl or aryloxyalkyl which may comprise one or more unsaturated bonds or one or more heteroatoms (preferably O, N, or S) and having 7 to 30 carbon atoms.

According to another advantageous alternative, in particular at least one of said third possibilities, the hydrophobic group HG group comprises an alkoxy radical of -OCH ₂ (CH ₂ -CH ₂ ) _3-8 CH ₃ , oleyl, tocopheryl or cholesteryls. And R ⁴ represents a single bond.

According to another advantageous alternative, in particular at least one of said third possibilities, the n HGs of PO each independently represent a monovalent radical of the formula:

Where

R ⁵ represents methyl (alanine), isopropyl (valine), isobutyl (leucine), sec -butyl (isoleucine) or benzyl (phenylalanine),

R ⁶ represents a hydrophobic radical containing 6 to 30 carbon atoms,

-l ranges from 0 to 6.

According to a salient feature of the invention, all or part of the hydrophobic group R ⁶ of PO is independently selected from radicals comprising:

■ straight or branched chain alkoxy, which may comprise one or more heteroatoms (preferably O, N, or S) or one or more unsaturated bonds, and has 6 to 30 carbon atoms,

■ alkoxy, optionally containing one or more unsaturated bonds or one or more heteroatoms (preferably O, N, or S), having from 6 to 30 carbon atoms and comprising at least one annealed cycloalkyl,

■ Alkoxyaryl or aryloxyalkyl which may comprise one or more unsaturated bonds or one or more heteroatoms (preferably O, N, or S) and having 7 to 30 carbon atoms.

In fact, the hydrophobic radical R ⁶ in the graft of PO is selected from the group comprising alkoxy radicals of —OCH ₂ (CH ₂ —CH ₂ ) _{3 to 8} CH ₃ , oleyl, tocopheryl or cholesteryls, and R ⁴ is It represents a single bond, but is not limited to this theory.

Advantageously, the backbone of the polyamino acid

Α- L-glutamate or α- L-glutamic acid homopolymer,

Α- L-aspartate or α- L-aspartic acid homopolymer,

Α- L-aspartate / α- L-glutamate or α- L-aspartic acid / α- L-glutamic acid copolymer.

Surprisingly, the distribution of aspartic or glutamic acid residues of the polyamino acid backbone of PO is made in such a way that the resulting polymer can be formed in a random or block copolymer or multiblock copolymer form.

According to another definition, the molar mass of PO is 2000 to 100,000 g / mol, preferably 5000 to 40,000 g / mol.

According to another form, the PO contains one or more grafts of polyalkylene glycol type bonded to glutamate or aspartate residues.

Advantageously, this polyalkylene glycol type graft is represented by the following general formula (V).

Where

R ′ ⁴ is a direct bond or “spacer” bound to 1 to 4 amino acid residues

Indicate,

X is a heteroatom selected from the group containing oxygen, nitrogen and sulfur,

R ⁷ and R ⁸ independently represent H or straight chain C ₁ to C ₄ alkyl,

n ″ ′ is in the range from 10 to 1000, preferably in the range from 50 to 300.

In practice, polyalkylene glycols are for example polyethylene glycols.

According to the invention, the molar ratio grafted to the polyalkylene glycol is preferably varied in the range of 1 to 30%.

Moreover, polyamino acid PO is very advantageous due to the fact that by adjusting the grafting rate, it is dispersed in water (eg phosphate buffer) at pH = 7.4 to form a colloidal suspension.

In addition, active ingredient APs such as proteins, peptides or small molecules can spontaneously bind to nanoparticles comprising such polyamino acid PO.

It should be understood that include an ionic functional group ^- PO is neutral or (e. G., -COOH), or the ionized form (e.g., -COO), depending on pH and composition. For this reason, the solubility in the aqueous phase is a function of the degree of ionized functional groups, ie pH. In the case of carboxyl functional groups in aqueous solution, the counter ions may be organic cations such as polycions such as sodium, calcium or magnesium, or protons of triethanolamine, tris (hydroxymethyl) aminomethane or polyamine, such as polyethyleneimine.

PO of polyamino acids is obtained, for example, by methods known to those skilled in the art. Random polyamino acids can be obtained by grafting hydrophobic grafts previously functionalized with spacers to the polymer by conventional coupling reactions. Block or multiblock polyamino acid PO can be obtained by sequential polymerization of the corresponding N-carboxyamino acid anhydride (NCA).

Polyamino acids that are homopolyglutamate, homopolyaspartate or block, multiblock or random glutamate / aspartate copolymers are prepared, for example, according to conventional methods.

The most common techniques used to obtain α type polyamino acids are described, for example, by "Biopolymers" 1976, 15, 1869 and by HR Kricheldorf [ "Alpha-amino acid N-carboxy anhydride and related". heterocycles " Springer Verlag (1987), based on the polymerization of amino acid N-carboxy anhydride (NCA). When the catalyst is a carboxylic acid functional group, the NCA derivative is preferably NCA-O-Me, NCA-O-Et or NCA-O-Bz derivatives (Me = methyl, Et = ethyl and Bz = benzyl). The polymer is then hydrolyzed under appropriate conditions to obtain a polymer in acid form. These methods are based on the specification of patent FR-A-2 801 226 to the applicant of the present invention. A number of polymers that can be used according to the invention, such as poly ( α- L-aspartic acid), poly ( α- L-glutamic acid), poly ( α -D-glutamic acid) and poly ( γ- L-glutamic acid) of various molecular weights ) Is commercially available. The α, β type polyaspartic acid polymer of the condensation reaction is that the aspartic acid and then (poly succinimide obtaining the imide) is obtained subjected to a basic hydrolysis (cf. Tomida et al., Polymer, 1997 , 38, 4733-36) .

Coupling of the graft and the acidic functional group of the polymer is carried out in the presence of carbodiimide, a coupling agent, and 4-dimethylaminopyridine, a catalyst, if necessary, dimethylformamide (DMF), N-methylpyrroli It is readily carried out by reacting in a suitable solvent such as ton (NMP) or dimethyl sulfoxide (DMSO). The carbodiimide is, for example, dicyclohexylcarbodiimide or diisopropylcarbodiimide. The grafting rate is chemically controlled by chemical stoichiometry or reaction time of the constituents and reactants. Hydrophobic grafts functionalized with the "spacer" are obtained by general peptide coupling or by direct polymerization under an acid catalyst. These techniques are well known to those skilled in the art.

The NCA derivative previously synthesized with the hydrophobic graft is used to synthesize a block or multiblock copolymer. For example, the hydrophobic NCA derivative is copolymerized with NCA-O-benzyl and then selectively removed the benzyl radical by hydrolysis.

Active ingredient (AP)

As for the active ingredient AP, preferably, proteins, peptides, polysaccharides, proteins, glycoproteins, proteins bound to one or more polyalkylene glycol chains (preferably polyethylene glycol (PEG) chains: "PEGylated proteins"), Liposaccharide, oligonucleotides, polynucleotides and mixtures thereof, particularly erythropoietin, oxytocin, vasopressin, corticosteroids, epidermal growth factor, platelet derived growth factor (PDGF), hematopoietic stimulating factor and Mixtures of these, factors Ⅷ and Ⅸ, hemoglobin, cytochrome, prolactin, albumin, luteinizing hormone secreting hormone (LHRH), LHRH antagonists, LHRH competing bodies, human, pig or bovine growth hormone (GH), growth hormone releasing factor, insulin , somatostatin, glucagon, interleukin or mixtures thereof (IL-2, IL-11 , IL-12), α -, β - or γ - interferon, gastrin, tetra gas Lean, pentagastrin, urogastrin, secretin, calcitonin, enkephalin, endomorphine, angiotensin, thyrotropin-releasing hormone (TRH), tumor necrosis factor (TNF), nerve growth factor (NGF), granulocyte colony stimulating factor ( G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), heparin lyase, bone morphogenic protein (BMP), hANP, glucagon-like peptide (GLP-1), Synthetic analogs and enzymes with VEG-F, hepatitis B surface antigen (rHBsAg), renin, cytokines, bradykinin, bacitracins, polymyxins, colistins, tyrosidines, gramicidines, cyclosporins And cytokines, antibodies, antigens and vaccines, and pharmacologically active variants and fractions.

In one variant, the active ingredient belongs to an anthracycline, taxoid or camptothecin system, such as leuprolide or cyclosporine, or mixtures thereof, or "small" Hydrophobic, hydrophilic or optionally ionic organic molecules.

Within the meaning of the present invention, “small” molecules are especially small nonprotein molecules such as small proteins without amino acids.

In another variation, the active ingredient comprises the following active substances: alcohol abuse drugs, Alzheimer's disease drugs, anesthetics, terminal megalopathy drugs, analgesics, anti-asthma drugs, allergy drugs, anticancer drugs, anti-inflammatory drugs, anticoagulants and antithrombotic agents, Anticonvulsants, antiepileptic drugs, diabetes drugs, antiemetic drugs, antiglaucoma drugs, antihistamines, anti-infective drugs, antibiotics, antifungal drugs, antiviral drugs, Parkinson's disease drugs, anticholinergic agents, antitussive agents, carbonic anhydrase inhibitors, cardiovascular preparations, lipid lowering agents, Antiarrhythmics, vasodilators, antianginals, antihypertensives, vasoprotectors, cholinesterase inhibitors, central nervous system disorders, central nervous system stimulants, contraceptives, fertility stimulants, labor and labor inhibitors, mucoadhesive agents Dopamine receptor agonists, endometriosis drugs, erectile dysfunction drugs, fertility drugs, gastrointestinal disorders drugs, Reverse regulators, immunosuppressants, memory disorders, antimigraine, muscle relaxants, nucleoside analogues, osteoporosis medications, parasympathetic stimulants, prostaglandins, psychotherapeutics, sedatives, sleep and nerve stabilizers, neuroleptics, anti-anxiety agents, mental stimulants, Antidepressants, skin disease therapies, steroids and hormones, amphetamines, appetite suppressants, non-anesthetic analgesics, antiepileptic agents, barbiturates, benzodiazepines, hypnotics, laxatives, psychotropics, and any combination of these agents. .

From a quantitative point of view, the percentage by weight of the AP [unassociated AP] not associated with the particulates is

[Non-associative AP] ≤ 1,

Preferably, [unassociated AP] ≤ 0.5

It is especially worth it.

Second Aspect of the Invention: A method for producing particulates by spraying a colloidal suspension or a solution of PO comprising AP

According to a preferred form of the process of the invention, the PO present in the solution or colloidal suspension to be sprayed is at least partially less than 500 nm, preferably 10 to 300 nm, more preferably 10, as the particle size measured by the T1 test. In the form of PO particulates with a particle size of from 100 nm.

Advantageously, the concentration of PO present in the solution or colloidal suspension to be sprayed is for example in the range of 5 mg / ml to 100 mg / ml, preferably in the range of 10 kmg / ml to 40 mg / ml.

In the context of the present invention, AP / PO is prepared before and / or during the spraying step.

For this association, the PO and / or AP may be in the form of a solid (preferably powder) or liquid suspension.

Techniques for bonding PO with at least one AP before the spraying step are described in particular in patent application WO-A-00 / 30618. This method consists, for example, of mixing a colloidal suspension of PO with a solution or suspension of AP. As another method, the powdered AP is mixed with the PO suspension.

According to a variant of the particular production process, the fine particles of the PO polymer associated with at least one active ingredient (AP) are dispersed in an essentially aqueous liquid medium, which medium preferably contains dispersing means M ₁ , and produced. The prepared dispersion is lyophilized.

The lyophilized product thus produced accelerates the preparation of the liquid formulation based on the fine particles (third aspect of the present invention described later), in which the lyophilisate is used in the reconstitution liquid used to prepare the liquid formulation. This is because it is distributed quickly.

Advantageously, this dispersing means M ₁ is selected from the following group.

(i). Polyvalent ions of opposite polarity and polarity of the ionic groups of the PO polymer, contained in the continuous aqueous phase,

(ii). At least one hydrophilic compound (preferably useful for injectable preparations) contained in the atomized PO / AP particulates by addition in the PO suspension / solution for atomization,

(iii). Particulate coating of at least one film of at least one hydrophilic compound (preferably useful for injectable preparations),

(iv). pH change,

(v). A combination of at least two of the means (i) to (iv).

The said means (i) is favorable.

For a detailed description of the embodiment of the means M ₁ (i) to (iv), reference may be made to the description of the dispersion means M ₂ .

Similarly, the liquid medium of the dispersion may also contain the same additives as described in the description of the reconstitution liquid described below.

Third aspect of the invention: liquid formulations based on fines obtained by spraying a colloidal suspension or a solution of PO comprising an AP

The formulation according to the invention may be a liquid medicament for delayed release of an AP comprising a low viscosity colloidal suspension, based on PO particulates associated with one or more APs, the particulates as defined above or as defined above. Microparticles obtained by a method as described above or a method as described in the Examples below.

This formulation is said to be advantageous for parenteral injection and to be liquid under the injection conditions.

According to the invention, the descriptors "low viscosity" or "ultra low viscosity" advantageously correspond to a kinematic viscosity of up to 1,000 mPa.s at 20 ° C. The kinematic viscosity of the formulation measured at 20 ° C. for a shear slope of 1,000 s ⁻¹ is 500 mPa · s or less, particularly preferably 2 to 200 mPa · s, such as 1.0 to 100 mPa · s or 1.0 Preferably from 50 mPa · s.

Measurement of kinematic viscosity

The reference measurement of the kinematic viscosity can be carried out using, for example, AR instruments equipped with cone-and-plate geometry (4 cm, 2 °) at 20 ° C. have. The viscosity v is measured for a shear stress of 10 s ⁻¹ .

This low viscosity facilitates parenteral administration, particularly by injection into the tumor or by mucosal, subcutaneous, intramuscular, intradermal, intraperitoneal or intracranial routes. The preparations according to the invention can also be administered by the oral, nasal, pulmonary, vaginal, ocular or oral route.

The liquid state or low viscosity of the preparations according to the invention is present both at injection temperatures corresponding to room temperature, for example at temperatures between 4 and 30 ° C. and at both physiological temperatures.

Dry particulates formed by spraying amphiphilic PO (such as polyamino acids) can be readily redispersed to form low viscosity particulate suspensions or solutions.

Dispersion means M ₂

Specifically speaking for dispersing or suspending nebulized PO / AP particulates in a reconstitution liquid to prepare a formulation, the formulations provided according to the invention comprise means M ₂ for suspending PO / AP particulates. do.

This dispersing means M ₂ may vary depending on the characteristics of the continuous phase (ie reconstitution liquid) of the suspension used to prepare the formulation.

Therefore, the fourth possibility according to the present invention is as follows.

1. The continuous phase of the suspension is essentially aqueous.

2. The continuous phase of the suspension is essentially an organic water miscible phase.

3. The continuous phase of the suspension is essentially an organic water immiscible phase.

4. The continuous phase of the suspension is essentially an organic water miscible or nonphase.

The term "essentially an aqueous continuous phase" should be understood to mean a liquid comprising, for example, water by mass of at least 60%.

The term “essentially an oily continuous phase” should be understood to mean a liquid comprising, for example, an oily phase in a mass of at least 60%.

1. The continuous phase of the suspension is essentially aqueous

Various forms of dispersing means M ₂ , optionally mixed with others, can be expected to mean that dispersing means M ₂ are for example selected from the following group.

(i). Polyvalent ions of opposite polarity and polarity of the ionic groups of the PO polymer, contained in the continuous aqueous phase,

(ii). At least one hydrophilic compound (preferably useful for injectable preparations) contained in the atomized PO / AP particulates by addition in the PO suspension / solution for atomization,

(iii). Particulate coating of at least one film of at least one hydrophilic compound (preferably useful for injectable preparations),

(iv). pH change,

(v). A combination of at least two of the means (i) to (iv).

The said means (i) is favorable.

(i) dispersion means based on polyvalent ions

If PO represents an ionic group IG, the dispersing means M ₂ may comprise polyvalent ions, which are opposite in polarity and polarity of the ionic group IG (at least partially ionized) of the polymer PO, forming a suspension. Is introduced into the aqueous continuous phase during the reconstitution of the suspension / solution.

Within the meaning of the present invention, the term "polyvalent ion" means for example divalent ions, trivalent ions, tetravalent ions and mixtures of these ions.

When PO represents an IG group, the polyvalent ion consists of a polyvalent cation, preferably a divalent cation, more preferably Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Fe ²⁺ , Cu ²⁺ and mixtures thereof. Selected from the group or trivalent ions, more preferably those selected from the group consisting of Al ³⁺ , Fe ³⁺ or mixtures thereof.

The preparations according to the invention may comprise monovalent ions (such as cations) in addition to the polyvalent ions, which monovalent ions are active upon aggregation of the nanoparticles to form microparticles.

Such polyvalent ions are preferably in the form of aqueous salt (organic or inorganic) solutions, such as sulfate solutions, hydrochloride solutions, acetate solutions, gluconate solutions or glutamate (or other anionic amino acids) of polyvalent cations. It is introduced in the form of a solution.

This dispersing means M ₂ based on polyvalent ions is even more suitable when the amphiphilic PO (such as (co) polyamino acids) is relatively hydrophilic.

Dispersion means M comprising at least one coating based on (ii) and (iii) a hydrophilic compound or on a hydrophilic compound ₂

Dispersion means M ₂ may comprise essentially the following.

At least one hydrophilic compound (preferably which may be useful for use in injectable preparations) present in the sprayed PO / AP particulates added to the PO suspension / solution to be sprayed,

-Coating the microparticles with one or more films of one or more hydrophilic compounds (preferably those which may be useful for use in injectable preparations).

Such dispersing means M ₂ can be incorporated into the formulations according to various methods.

According to a first method, at least one hydrophilic compound selected from those which can be used in the preparation for injection is added to the PO suspension / solution to be sprayed, which hydrophilic compound is preferably selected from the group comprising:

→ amino acids (eg lysine, arginine, glycine),

→ polyalkylene glycols, preferably polyethylene glycol,

¡Æ copolyalkylene glycols, preferably ethylene glycol / propylene glycol copolymers

(Poloxamer or Pluronic or Lutrol type),

Cellulose polymers and derivatives thereof, preferably carboxyalkylcelluloses (eg

Such as carboxymethylcellulose) or alkylcelluloses (such as methylcellulose),

→ hardened or uncured polysaccharides such as trehalose, sorbitol, mannitol or water

cross,

→ polyols such as propylene glycol or glycerol,

→ gelatin, preferably hydrated gelatin,

→ nitrogen-containing (co) polymers, preferably polyacrylamides, poly-N-vinylamides,

Present in the group comprising polyvinylpyrrolidone (PVP) and poly-N-vinyllactam

those,

→ poly (vinyl alcohol) (PVA),

→ poly (sodium glutamate),

→ and mixtures thereof.

The hydrophilic coating compound preferably comprises at least one hydrophilic polymer.

According to a second method, the microparticles are coated with one or more layers of one or more hydrophilic compounds as defined above.

In this second method, the hydrophilic compound preferably comprises at least one hydrophilic compound selected from hydrophilic compounds as defined above.

Dispersion means M ₂ (ii) based on at least one hydrophilic compound has proven to be particularly suitable for amphiphilic PO, which exhibits a level of at least 10 mol% HG groups relative to PO which is sufficiently high in hydrophobicity, such as at least one polyamino acid. .

Coating of the microparticles with a layer of hydrophilic and biocompatible compounds [means M ₂ (iii)] can be achieved, for example, by two stages of continuous spraying, with the second spraying a suspension of the microparticles in a solvent which is immiscible with the microparticles. , Which contributes to promoting the dispersion of the fine particles.

This dispersing means (iii) by hydrophilic coating of the microparticles is reliable and makes it useful for handling by being stable for at least several days.

Advantageously, mixing the means (i) for dispersion based on divalent ions present in the aqueous phase and the means (ii) or (iii) for dispersing particulates based on hydrophilic compounds during the reconstitution of the formulation accelerate the dispersion. Let it be.

According to another specific embodiment, the aqueous continuous phase or hydrophilic coating comprises one or more injectable surfactants such as Tween ^® 80, lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine or polyoxypropylene / polyoxyethylene copolymers. Poloxamer, Pluronic, Lutrol).

(iv) dispersion means M by pH change ₂

Another dispersing means foreseeable in accordance with the invention preferably consists of dispersing means by changing the pH before spraying. This type of means has proved particularly suitable when the amphiphilic PO (such as polyamino acid) is a compound which has an ionic functional group and is more hydrophilic, in other words, for example when the hydrophobic group HG is less than 10 mol%.

Finally, using this means M ₂ (iv), the pH is changed to a value at which the ionic functional group (such as polyamino acid) of PO is not ionized (e.g., when the PO is a hydrophilic polyamino acid of polyGlu or polyAsp type) Increase the hydrophobicity of the amphipathic PO.

Preferably this dispersing means M ₂ (iv) by changing the pH before the spraying step is applied to the PO suspension / solution just before spraying.

Such dispersing means M ₂ (iv) due to a change in pH before spraying may or may not be combined with the following.

And after spraying, a polyvalent ion distribution means based on the M ₂ (i), or in the reconstruction step

And preferably in, the dispersion means is based on a hydrophilic compound M ₂ (ii) by 1 M ₂ (iii) to coat the particles of the hydrophilic polymer or more species, or (iii).

2. The continuous phase of the suspension is essentially an organic water miscible phase.

The dispersing means consists of an organic water miscible phase.

This phase can be, for example, ethanol, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), isopropyl, glycerol or glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether).

3. The continuous phase of the suspension is essentially an oil immiscible water miscible phase.

According to an advantageous embodiment, the dispersing means comprises a lipophilic liquid whose melting point is preferably below 15 ° C. and is present in the water immiscible oily continuous phase.

Preferably the lipophilic liquid may comprise one or more mixtures of triglycerides of saturated fatty acids or one or more vegetable oils, or one or more lipids or one or more lipid derivatives or one or more fatty acids or one or more fatty acid derivatives. .

More preferably, the lipophilic liquid includes the following.

And derived from a saturated coconut oil C ₈ to C ₁₀ fatty acid triglycerides of one type of

Mixtures of the foregoing, such as those obtained by Sasol under the trade name "Miglyol 812"

Plate,

ㆍ at least one vegetable oil, preferably soybean oil, palm oil, flaxseed oil, cottonseed oil, sesame seed

Oil, sunflower oil or peanut oil,

At least one lipid, preferably liquid lecithin, synthetic or natural vitamin E,

At least one lipid derivative, preferably arachidylylphosphatidylcholine and stearate

Loyle Forcepatidylcholine,

At least one fatty acid, preferably oleic acid, myristic acid, palmitic acid, stearic acid

And salts thereof,

At least one fatty acid derivative, preferably mono-, di- or triglyceride oils

Conductor, ethyl oleate, lauryl lactate, glyceryl stearate, sorbitan palmitate,

Sorbitan stearate, sorbitan monooleate or polysorbate,

And derivatives thereof

Wherein, under conditions where some of the products taken separately are liquids, for example at temperatures below 15 ° C., these products may be mixed with other products to be liquids, for example at temperatures below 15 ° C.

Derivatives of fatty acid triglycerides are particularly preferred, especially those sold under the trade name “Miglyol 812” by triglyceride mixtures of saturated C ₈ -C ₁₀ fatty acids derived from coconut oil, such as Sasol. Other triglycerides, including long chain fatty acids, are especially present in vegetable oils such as soybean oil, palm oil, flaxseed oil, cottonseed oil, sesame oil, sunflower oil or peanut oil.

The sprayed PO / AP powder is easily dispersed in this water immiscible oily phase, resulting in a stable particulate suspension that is maintained for several days, thus facilitating handling. This dispersion process takes place quickly, especially without the need for adding other means of dispersion.

4. Continuous phase of a suspension that is essentially an organic water miscible or water immiscible phase

According to another advantageous method compatible with the second and third possibilities with respect to the nature of the continuous phase of the suspension / solution constituting the formulation, the means for dispersing is preferably at least one film forming coating compound (preferably usable in the preparation for injection). Coating the particulates.

Preferably, the film forming coating compound is polylactide, polyglycolide, poly (lactide-co-glycolide), polyorthoester, polyanhydride, poly (hydroxybutyric acid), polycaprolactone, poly ( Alkyl carbonates), water-insoluble PO polymers, derivatives thereof, and mixtures thereof.

According to another alternative, the film forming coating compound has lipid-like properties, preferably having a melting point of 15 ° C. or higher, at least one lipid or at least one lipid derivative, or at least one vegetable oil, or one At least one fatty acid or at least one fatty acid derivative or at least one mixture of triglycerides of saturated fatty acids or mixtures thereof.

According to another advantageous embodiment, the organic continuous phase, for example hydrophobic, or hydrophobic coating also comprises one or more injectable surfactants such as Tween ^® 80, lecithin, phosphatidylethanolamine, phosphatidylserine or polyoxypropylene / Polyoxyethylene copolymers (trade names: Poloxamer, Pluronic, Lutrol).

Excipients / Stabilizers

In order to promote the dispersibility of the aqueous phase and / or to further improve the stability of the aqueous suspension solution, it is preferable that the injectable preparation contains other additives, ie those listed in the "Excipients / Stabilizers" column, in other words conventional excipients. May be advantageous.

Excipients / stabilizers can be selected from the group consisting of:

≧ nanoparticles of at least one polymer PO, wherein PO is a hydrophobic group (HG) and at least

Water soluble biodegradable amount with partially ionized ionized hydrophilic group (IG)

As a lipophilic copolymer, the call of nanoparticles in water with isotonic conditions pH = 7.0

Spontaneously forming an Lloyd suspension.

→ amino acids,

→ polyalkylene glycols, preferably polyethylene glycol,

¡Æ copolyalkylene glycols, preferably ethylene glycol / propylene glycol copolymers

(Poloxamer, Pluronic or Lutrol type),

→ cellulose polymers and derivatives thereof, preferably carboxyalkylalkylcelluloses (eg

Such as carboxymethylcellulose) or alkylcelluloses (such as methylcellulose),

→ esters of sorbitan and fatty acids, preferably polyoxyalkylenes (eg ethyl

Ethylene) glycols and esters of one or more acids (such as oleic acid), Tween (or poly)

Resorbate) mold,

Phospholipids and polyalkylene glycols, preferably based on polyethylene glycol

Surfactants,

→ hardened or uncured such as trehalose, sorbitol, mannitol or sucrose

Polysaccharide,

→ polyols such as propylene glycol or glycerol,

→ gelatin, preferably hydrated gelatin,

→ nitrogen-containing (co) polymers, preferably polyacrylamides, poly-N-vinylamides,

Selected from the group comprising polyvinylpyrrolidone (PVP) and poly-N-vinyllactam

Hitting,

→ poly (vinyl alcohol) (PVA),

Mixtures thereof.

One of the preferred excipients (stabilizers) according to the invention is formed by nanoparticles of at least one polymer PO, which is the same or different (preferably the same) constituting the microparticles.

The amount of excipient / stabilizer used in the formulation is preferably 0.01 to 10% by weight, preferably 0.1 to 5% by weight.

With regard to stabilizers comprising nanoparticles, the nanoparticles are advantageously used in the formulation at 1.5 to 3.5% by weight, for example at a ratio of 2.0 to 3.0 (about 2.5)% by weight.

Reconstitution liquid

In addition to the aforementioned dispersing means, the reconstitution liquid used in the preparation of the preparations according to the invention may comprise, for example, the following.

-For example, one species of concentration 0.001 M and 0.1 M, preferably 0.005 M to 0.02 M

One or more buffers or one or more injectable salts (phosphate buffer, citrate

Buffer, sodium chloride), this buffer or injectable salt to adjust the pH of the solution

To help.

At least one injectable surfactant, preferably polysorbate type, such as

Tween ^® 20 or Tween ^® 80, or Poloxamer type such as Lutrol ^® F38, Lutrol

^® F68 or Lutrol ^® F127, for example concentrations from 0.01% to 2%, preferably 0.05

To 0.5%.

The reconstitution liquid may further comprise a density enhancer such as sucrose, D-mannitol or trehalose in a concentration of 0.1 to 10%, preferably 0.5 to 5%. Reconstitution liquids are also injections selected from the group consisting of polysaccharides, synthetic polymers (such as sodium carboxymethylcellulose), poly (vinyl alcohol), polyvinylpyrrolidone, polyalkylene glycols (such as polyethylene glycol) and mixtures thereof Possible viscosity enhancing polymers may also be included.

By using dry fine particles of amphiphilic PO (e.g. polyamino acids), and in particular by means of reconstitution means, it is possible to achieve two objectives simultaneously, i.e. stable and easy to be parenterally injectable. It is possible to obtain pharmaceutical formulations in dispersed form.

In addition to the aforementioned excipients / stabilizers for improving dispersibility and stability, the suspensions / solutions to be sprayed or other conventional excipients which may be added to the preparations according to the invention, such as antibiotics, buffers, antioxidants or isotonic agents known to those skilled in the art Agents may be added that allow for adjustment of See Injectable Drug Development , PK Gupta et al., Interpharm Press, Denver, Colorado, 1999.

The addition of conventional excipients can be carried out in aqueous dispersion phase or in solution / suspension prior to spraying.

Application of liquid formulations according to the invention

The preparations according to the invention are preferably pharmaceutical preparations, but may also be in the form of cosmetics, dietary supplements or plant protection preparations if they comprise at least one PO and at least one AP as defined above.

Fourth Aspect of the Invention: Kits for Reconstitution of a Formulation According to the Invention

PO / AP particulates and essentially aqueous reconstituted liquids, essentially organic water miscible reconstituted liquids and essentially oily water immiscible reconstituted liquids are as defined above.

Fifth Aspect of the Invention: Reconstitution Method for Reconstitution of a Formulation According to the Invention

PO / AP particulates and essentially aqueous reconstituted liquids, essentially organic water miscible reconstituted liquids and essentially organic water immiscible reconstituted liquids are as defined above.

According to the invention, it is also possible to sterilize the liquid suspension of the nanoparticles from the microparticles of the preparations according to the results of the invention, for example through a filter having a pore size of 0.2 μm. Agglomeration under sterile conditions according to the manufacturing method as described above makes it possible to inject the formulation directly into the patient.

Sixth Aspect of the Invention: Solid Pharmaceutical Formulations

Such delayed release formulations of AP also include dry powder forms for inhalation or pulmonary administration based on PO / AP particulates as described above.

Other Aspects of the Invention

The invention also relates to a solid product derived from the PO particulates according to the invention.

Indeed, the products thus derived can be prepared in the form of powders, gels, implants or films.

Therefore, the present invention relates to products derived from the preparations according to the invention, regardless of the method of preparation.

The present invention also relates to a method of treatment consisting essentially of administering a formulation as described herein to parenteral, mucosal, subcutaneous, intramuscular, intradermal, intraperitoneal or intracranial or cancer. The preparations according to the invention can be administered by oral, nasal, pulmonary, intravaginal or intraocular route.

According to a particular alternative of the invention, this method of treatment is administered parenterally, i.e. by subcutaneous, intramuscular, intradermal, intraperitoneal or intracranial injection or cancer, by administering the agent as described above, preferably the injection site. Consisting of forming a layer deposited on it.

The present invention describes the synthesis of PO formed by polyamino acids grafted with hydrophobic groups and the conversion to a system for delayed release of AP, i.e. the preparations (powder fine particles) according to the invention and the stability and redispersibility of such systems. It will be better understood from the present embodiment, its advantages and other embodiments will be apparent.

Example 1 Synthesis of Amphiphilic Polymer PO-A

synthesis α Synthesis of Polyglutamate Grafted with Tocopherols

15 g of poly ( α- L-glutamate) (equivalent to about 16,900 Da with respect to polyoxyethylene standards, obtained by hydrolysis after polymerization of NCAGluOMe as described in patent application FR-A-2 801 226) ) Is dissolved in 288 ml of dimethylformamide (DMF) by heating at 80 ° C. until the polymer is dissolved. The solution was cooled to 15 ° C., 2.5 g of D, L- α -tocopherol (> 98%, obtained from Fluka ^® ) pre-dissolved in 8 ml of DMF, and 280 mg of 4-dimethylaminopyridine pre-dissolved in 1 ml of DMF. , 1.6 g of diisopropylcarbodiimide previously dissolved in 6 ml of DMF is added continuously. After shaking for 3 hours, the reaction medium is poured into 1200 ml of water containing 15% sodium chloride and hydrochloric acid (pH = 2). The precipitated polymer is recovered by filtration and washed with 0.1 N hydrochloric acid, water, diisopropyl ether. This polymer is dried in a vacuum oven at 40 ° C. The yield is about 90%. The molar mass measured by size exclusion chromatography is 15,500 relative to the polyoxyethylene standard. The level of grafted tocopherols measured by proton NMR spectroscopy is 5.1 mol%. The nanoparticle suspension of the polymer in water is obtained by dissolving in water, and the pH is adjusted to 7 ± 1 (neutralization of carboxylate).

Example 2: Synthesis of Amphiphilic Polymer PO-B

Example 2 is a modification of Example 1 in which the grafting rate is 20%.

Example 3: IFN- α Method for producing dry fine particles of polymer PO-A containing 2b

Preparation of a solution containing 20 mg / g of polymer and 0.25 mg / g of IFN

200 g of a 30 mg / g solution of polymer PO-A are introduced into a 500 ml flask. 68 g of water are introduced into the flask containing the polymer. A frozen solution of IFN- α- 2b concentrated at 2.8 mg / g is thawed at 25 ° C. for 1 hour and 26.8 g of this thawed solution is introduced into a flask containing polymer solution. This mixture is left at room temperature for 14 hours.

This solution is filtered on a 0.2 μm sterile filter.

Spray of Polymer-IFN Solution

This solution is sprayed in a spray dryer of type Buchi B290. The solution is aspirated at a rate of 5 ml / min and sprayed through a spray nozzle supplied with nitrogen (7 bar, 900 l / h). Suction flow rate (dry air) is 40 m ³ / h. The inlet temperature is maintained at 90 ° C., so that the outlet temperature is 45 ° C. under these conditions.

Characterization of the produced particulate

The particle size (according to T0 test) of the resulting particles is D50 = 5 μm.

After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying and no degraded form is detected.

Example 4: IFN- α Preparation of Dry Fine Particles of Polymer PO-A Containing 2b and Polysorbate 80

A solution consisting of polymer PO and interferon was prepared in the same manner as in Example 3. 0.9 g of polysorbate 80 is added to this solution before spraying.

Spraying of the polymer-IFN solution in the presence of polysorbate

This solution Sprayed under the same conditions as described in Example 3.

Characterization of the produced particulate

The particle size of the resulting fine particles (according to T0 test) is D 50 = μm.

After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying and no degraded form is detected.

Example 5: IFN- α Preparation of Acidified Dry Fine Particles of Polymer PO-A Containing 2b

A solution of polymers PO-A and IFN was prepared in the same manner as in Example 3. Dilute by adding water so that the concentration of polymer PO-A is 15 mg / g (then the IFN concentration is 0.188 mg / g). After filtration using a 0.2 μm filter, the solution is acidified by addition of 1N HCl until the pH is 4. The solution thus obtained gives off an opalescent light.

Spray of Acidified Polymer-IFN Solution

This solution is sprayed under the same conditions as described in Example 3.

Characterization of the produced particulate

The particle size (according to T0 test) of the resulting particles is D50 = 5 μm. After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying, and no degraded form is detected.

Example 6: IFN- α Preparation of Dry Fine Particles of Polymer PO-A Containing 2b and Coated with PVP

Powders made of dry particulates of polymer PO-A and IFN are obtained from a PO-A / IFN mixture according to the protocol used in Example 3.

As a result of the spraying step, 1.5 g of this powder is resuspended in an ethanol solution containing 7 g / l polyvinylpyrrolidone (PVP) of the injection grade PVP K30. This ethanol suspension was shaken for 2 hours and then sprayed a second time in a Buchi B290 type powder drying apparatus equipped with a discharge trap for organic solvent (inactivation loop at -20 ° C) and operated as a short circuit under nitrogen. The solution is aspirated at a flow rate of 5 ml / min and sprayed with a spray nozzle supplied with nitrogen (7 bar-670 l / h). Suction flow rate (dry air) is 40 m ³ / h. The inlet temperature is maintained at 80 ° C., for this reason the outlet temperature is 55 ° C. under these conditions.

Characterization of the produced particulate

The particle size of the resulting particles (according to T0 test) is D50 = 6 μm. After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying, and no degraded form is detected.

Example 7: IFN- α Preparation of Dry Fine Particles of Polymer PO-B Containing 2b

The polymer-IFN solution preparation and spray protocol is the same as in Example 3 except that PO-B was used instead of PO-A.

Characterization of the produced particulate

The particle size of the resulting particles (according to T0 test) is D50 = 4 μm. After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying, and no degraded form was detected.

Example 8: Preparation of Dry Fine Particles of Polymer PO-A Containing hGH

Preparation of hGH Concentrated Solution

60 g of recombinant human growth hormone solution (concentration 3.9 mg / g) were thawed at 25 ° C. for 1 hour and 30 minutes, followed by six frontal ultrafiltrations through a 10 kD membrane until the concentration was 24 mg / g. Concentrate by filtration.

Mixture with polymer solution

52 g of 30 mg / g concentrated solution of polymer PO-A is diluted to 19.5 mg / g by adding 28 g of water. Pour 8 g of 24 mg / g hGH solution slowly into the polymer solution. The mixture is left at room temperature overnight and then filtered through a 0.2 μm sterile filter.

Spray of polymer / hGH solution

This solution is sprayed under the same conditions as described in Example 3.

Characterization of the produced particulate

The particle size (according to T0 test) of the resulting particles is D50 = 5 μm. After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying, and no degraded form is detected.

Example 9: Preparation of Dry Fine Particles of Polymer PO-A Containing IL-2

Preparation of IL-2 Concentrated Solution

100 g of the IL-2 frozen solution stabilized at 2 g / l with SDS is thawed at room temperature, and then the solution is cooled to 0-2 ° C. 100 g of pure ethanol is added slowly to this solution to precipitate the protein. The precipitate is recovered by filtering through a 0.65 / 0.45 μm membrane in a frontal diafiltration cell and washing with 200 g of water. Nitrogen is applied to the precipitate and dried. This precipitate is dissolved in 10 g of precooled (<5 ° C.) 0.02N sodium hydroxide to give a pH = 12 clear solution of 20 mg / g without SDS. Measured by HPLC method for accurate analysis of this solution.

Mixture with polymer solution

Example 1 (30 mg / g) was diluted by adding 39 g of water to 96 g of a concentrated solution of polymer PO. 9 g of the above-described 20 mg / g IL-2 concentrate is slowly poured into the polymer solution.

The mixture containing 20 mg / g of polymer PO and 1.25 mg / g of IL-2 is left overnight at room temperature and then filtered through a 0.2 μm sterile filter.

Spray of Polymer-IL-2 Solution

This solution is sprayed under the same conditions as described in Example 3.

Characterization of the produced particulate

The particle size (according to T0 test) of the resulting particles is D50 = 5 μm. After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying, and no degraded form is detected.

Example 10 Preparation of Dry Fine Particles of Polymer PO-A Containing Insulin

Preparation of 40 g of 20.3 mg / g insulin concentrate

0.83 g of recombinant human insulin (powder) with an activity of 28.7 UI / g and a water content of 2.5% are introduced into the glass flask. 23.62 g of water are added and the insulin is dispersed by slow magnetic shaking. 6.22 g of 0.1N HCl is added until a clear acidic insulin solution is obtained. 9.32 g of 1N sodium hydroxide is added to obtain a final clear solution with a pH of 7-8. Insulin solution is filtered through a 0.2 μm sterile filter.

Mixture with polymer solution

37.66 g of the above-described insulin concentrate is slowly poured into 220 g of 30 mg / g polymer PO solution (which is magnetically shaken). Add 72.34 g of water to the solution. The mixture is left at room temperature for 4 hours and then filtered through a 0.2 μm filter.

Spray of Polymer-Insulin Solution

This solution is sprayed in a spray drying apparatus of type Buchi B290. The liquid solution is aspirated at a flow rate of 5 ml / min and sprayed at a spray nozzle supplied with nitrogen (7 mbar-900 dl / h). Suction rate (dry air) is 40 m 3 / h. The inlet temperature is maintained at 120 ° C., so the outlet temperature is 70 ° C. under these conditions.

Spray of Polymer-Insulin Solution

The solution is sprayed under the same conditions as described in Example 3.

Characterization of the produced particulate

The particle size (according to T0 test) of the resulting particles is D50 = 5 μm. After dispersing the powder in water at the spray concentration, the result of IFN analysis by HPLC is the same as the solution before spraying, and no degraded form is detected.

Example 11 Characterization of Particles Produced According to the Invention: Particle Size and Stability

Particulate particle size is measured by T0 test and stability is measured by S1 test. Protein degradation is assessed by HPLC by comparing chromatograms of the preparation before and after spraying.

These results demonstrate that spraying a protein in the presence of an amphipathic polyamino acid allows to maintain the stability of the protein. In solid form, the powder is stable as described herein. This is a condition that may be inactivated and it is expected that the stability of the powder will be maintained at 5 ° C. for at least 2 years.

Spraying is a way to break down proteins. HPLC analysis showed that no degradation form was observed when comparing the chromatograms before and after spraying.

Spraying proteins in the presence of amphipathic polyamino acids has been shown to produce stable particulates over time and not to cause degradation of the proteins.

These powders may be used in solid form (eg inhalation, injection without needle into the gun under pressure) or in the form of injection suspensions after reconstitution in a suitable medium.

Example 12: Reconstitution of the Suspension Starting from the Powders of Examples 3-10

This powder is reconstituted in the following three different media according to the protocol described for conducting the DP1 test.

A. Phosphate Buffered Saline at pH 7.4

B. 0.1 M magnesium chloride solution

C. Capric Triglyceride Solution (Miglyol ^® 812)

These samples were evaluated by performing the following steps.

Dispersing in one of the above-mentioned media,

Injecting through a 25G needle into a syringe.

This suspension is considered to have good performance when at least 80% is recovered by inhalation / injection through a 25G needle.

result:

During the stability test, reconstitution in media A, B and C was followed to see if the protein was degraded.

These results show that only the composition of Example 7 is stable and capable of injectable suspension of microparticles in water buffered with PBS at pH 7.4. For most of the examples (except 5), reconstitution media containing divalent salts make it possible to improve these properties. In Miglyol medium, all formulations are readily dispersed, injectable and stable.

Combinations of these properties are not readily obtainable depending on the polymer used. The inventors have found that, after a number of efforts, certain excipients and reconstitution media can achieve these properties.

Example 13: Viscosity Measurement of Reconstituted Formulations

The 30 mg / ml dispersion of the powder in Medium B and C (Medium B for Example 5) of the above-described examples made a stable suspension of low viscosity, and for Medium B, the measured viscosity was all less than 10 μmPa · s In the case of medium C, all the viscosity is about 30-40 mPa.s.

In comparison, the composition of the same polymer in the form of nanoparticles used in the prior art did not yield acceptable viscosity values (<100 μmPa · s) at this concentration (30 mg / ml).

Example 14 IFN- obtained in the form of lyophilized powder α Preparation of Dry Fine Particles of Polymer PO-A Containing 2b

Initially, the microparticles are prepared according to the manner described in Example 3. The spraying step is performed under sterile conditions and recovered in a sterile container.

Mg ²⁺ Redispersion of Particles in the Presence of Ions

Immediately after the spraying step, the fines are recovered and redispersed in sterile conditions in 0.1 M MgCl ₂ aqueous solution. The amount of MgCl ₂ solution added is adjusted so that the concentration of the PO-A polymer solution in the suspension is 40 mg / ml. The pH is adjusted to 6.5 by addition of 1N sodium hydroxide.

Freeze drying

The suspension is divided into Lyoguard ^® -type containers that keep the suspension sterile during freeze drying. The vessel is then lyophilized in a lyophilizer (Laboratory Table Christ, Osterode, Germany) for 72 hours in a sterile manner. The powder is dispensed into the flask in a sterile manner before use.

Example 15 Comparison of the Reconstruction of the resulting particulate powder starting from Example 3 and the particulate powder of Example 14

For this comparison, the volume of the reconstituted suspension is the same in each case (about 1 ml) and the flask used for reconstitution is also the same (3 ml glass flask). The amount of powder has a suspension Adjust to contain 40 mg / ml final polymer and 0.5 mg / ml IFN- α 2b. Example 3 The powder is reconstituted with 0.1 M MgCl ₂ aqueous solution, and the Example 14 powder (which already contains Mg ²⁺ ions) is reconstituted with pure water.

First shake the flask by hand. If at least 15 minutes are required for the powder of Example 3 to be redispersed, the powder of Example 14 is dispersed more quickly, ie within 5 minutes, to obtain a homogeneous suspension. Place the magnetic shake bar into the flask and shake the two flasks in the same manner for 1 hour.

At the end of shaking, the characteristics of the two suspensions are compared. The particle size and viscosity of both suspensions are similar.

Example 16: Pharmacokinetics to IFN After Subcutaneous Injection of Amphiphilic Polyamino Acids in Particulate Form into Dogs

Eight beagle naive dogs (weight: 9 ± 0.6 kg) are treated with the following formulations.

IFN IR (IR for immediate release) corresponds to recombinant human interferon solution (PCGen, IB05.0516 batch) adjusted for concentration, pH and osmotic pressure ([IFN] = 0.5 mg / ml, pH = 6.5 and 354 mOsm) do.

Particles of Formulation 1 are prepared according to Example 14 starting from the same interferon PCGen batch. The lyophilized powder is resuspended in water in a sterile manner (a hood with a thin air stream) according to the same method as described in Example 15.

Pharmacokinetic results are shown in the following table.

Where

C _max is the maximum IFN plasma concentration,

T> 50 pg / ml is the time when the IFN plasma concentration is higher than 50 pg / ml,

AUC represents the area under the curve corresponding to IFN plasma concentration as a function of time,

RBA represents a bioavailability with respect to preparations for immediate release,

-T _{50% auc} represents the time required for 50% of the total IFN amount precipitated as a salt.

IFN IR shows a rapid release profile with a maximum plasma concentration of 25.2 ± 0.4 ng / ml, obtained after a median time of 5 h (range: 3 to 5 hours). Blood IFN is no longer quantified after 24 hours.

Formulation 1 has sustained release properties and IFN pharmacokinetics with a maximum plasma concentration of 0.5 ± 0.2 ng / ml (50 times lower than the maximum plasma concentration in IR form), obtained after an intermediate time of 60 hours (range: 48-96 hours). Provide major changes in the profile. A common feature of pharmacokinetics is the flat nature with pseudo-plateau. IFN concentrations in blood are concentrations that are not quantifiable between 192 and 240 hours (between 8 and 10 days). This formulation has a low AUC, relative biological availability (RBA = 34%) T 66 % loss of _50% is more _auc about 20 times higher than the T _50% of the IFN IR _auc.

Example 17 Pharmacokinetics of Insulin Following Subcutaneous Injection in Dogs with Formulations Based on Amphiphilic Polyamino Acids in Particulate Form

For this example Lantus ^® is a modified insulin analogue (glargin insulin -Sanofi Aventis). Modification of two amino acids on the primary structure of human insulin is induced to exhibit delayed release properties over a 24-hour period through in situ precipitation.

Groups of 6 healthy beagle dogs (11.4 ± 1 kg body weight) were treated with Lantus ^® preparations during the 2nd crossover trial, and 4 groups of 8 healthy beagle dogs (11.8 ± 1 kg body weight). It is processed twice during the crossover attempt period. A table for each treatment is presented below.

The microparticles of Formulation 2 are prepared according to Example 10 at a ratio of 30 mg of polymer PO to 3.5 mg of insulin (100 IU). This formulation is prepared by dispersing the sprayed powder in 0.1 M MgCl ₂ and then magnetically shaking the suspension for 1 hour. The pH of the formulation is 6.2 and the osmotic pressure is 348 mOsm. The particle size of the particles measured by T1 test is D50 = 12 μm, and the viscosity of the formulation is about 5 mPa · s at 20 ° C.

Hyperglycemia is confirmed by enzymatic methods (hexokinase) with a biochemical blood analyzer (Advia 1650, Bayer Diagnostics).

The pharmacokinetic analysis results are expressed in% of baseline blood glucose over time.

Pharmacokinetic data is presented in the following table.

In the table above,

C _min is the minimum percentage of the reference blood glucose observed,

Area Percent Glycemia Curve (APGC _0-36h ) represents the interface between baseline blood glucose (100%) and a curve representing blood glucose change (expressed in% of baseline blood glucose) between 0 and 36 hours after administration,

T _{50% APGC} represents the time required to obtain 50% of APGC _0-36h .

Because of the administration of the baseline Lantus ^® , blood glucose levels dropped rapidly during the first hour. The blood glucose of glargin insulin is maintained for 18 to 36 hours (hyperglycemia returns to baseline after an average of 30 hours).

In comparison, blood glucose is rapidly reduced during the first hour due to administration of Formulation 2. The percentage of baseline blood glucose remains on peak for an average of 36 hours. C _min obtained in preparation 1, since on average higher than the C _min of the reference Lantus ^® agent, which may mitigate the severe hyperglycemia in diabetic patients.

The duration of action of Formulation 2 is obviously longer than the long duration of action of the reference Lantus ^® agent. This is also illustrated by the higher mean T50% _APGC value when using Formulation 2.

Criteria using Formulation 2 with respect to the Lantus ^® were observed with a APGC _0-36h lost only 24%.

Claims (42)

As fine particles of a polymer (PO) containing at least one active ingredient (AP), the polymer (PO) And a hydrophobic group (HG) and the hydrophilic amphipathic (兩water-soluble and biodegradable containing 공중합 性 copolymer, And isotonic (等張性) a colloidal suspension of nanoparticles in water of pH = 7.0 under the conditions Spontaneously forming fluids, And the AP and non-covalent associative and the associated (會合), The fine particles, a . By spraying a solution or colloidal suspension of PO containing at least one AP Obtained, b . Particle size measured by T test, 0.5 to 100 microns, preferably 1 to 70 Microns, preferably 2 to 40 microns, c . Dispersible in colloidal suspension in "dispersibility" test DP1  Particles characterized in that. The particulate as claimed in claim 1, which is stable in the ST1 test or the ST2 test. The particulate of claim 1, wherein the PO is a block or a random copolymer. The microparticle according to any one of the preceding claims, wherein the hydrophilic group of PO is an ionic group (IG) which is at least partially ionized. The microparticle according to any one of the preceding claims, wherein the polymer PO is an amphipathic (co) polyamino acid or a blend of amphipathic (co) polyamino acids. The method according to any one of the preceding claims, wherein the PO is a polyamino acid whose main chain is formed by aspartic acid units and / or glutamic acid units, at least some of these units being at least one in the chain or at the chain end. Particles modified by grafting hydrophobic groups (HG). The method according to any one of the preceding claims, wherein PO is defined by the following Formula 1 (radical -COOR ³ comprises a form wherein the bond between carboxyl and R ³ is an ionic bond -COO ^{_ +} R ³ ) Fine particles which become. [Formula 1]

In the above formula, R ¹ is H, straight C ₂ to C ₁₀ or branched C ₃ to C ₁₀ alkyl, benzyl, or -R ^4- [HG], or ■ NHR ¹ represents a terminal amino acid residue, R ² is H, straight C ₂ to C ₁₀ or branched C ₃ to C ₁₀ acyl group, or -R ^4- [HG] Indicates, R ² represents a terminal terminal pyroglutamate group, R ³ is H, or ■ ⁺ R ³ is    Advantageous from a subgroup consisting of sodium, potassium, calcium and magnesium      A metal cation selected from    -The following cations, An amine cation, And oligo amine cations, And the polyamine-based cation (which is especially preferred polyethyleneimine), And lysine or arginine-based amino acids advantageously selected from cationic acids       Organic amount advantageously selected from subgroups of systemic cations       With ions,   Advantageously selected from a subgroup consisting of polylysine and oligolysine     Silver cationic polyamino acids It is preferably selected from the group consisting of R ⁴ is a direct bond or a 1-4 amino acid residue-based “spacer (spacer) ", ■ A is independently —CH ₂ -radical (aspartic acid unit) or —CH ₂ —CH ₂ —radical (Glutamic acid unit), N / (n + m) is defined as the molar grafting rate and its value is measured in water at pH = 7 and 25 ° C. Colloidal suspension of quasi-particulates of PO because it is low enough for dissolved PO N / (n + m) is preferably 1 to 25 mol%, particularly preferably 1 to 15 Range in mole percent, (N + m) is defined as the degree of polymerization and is from 10 to 1,000, preferably from 50 to 300 Range, HG represents a hydrophobic group having 6 to 30 carbon atoms. A method according to any of the preceding one of the preceding claims, PO is to correspond to any one of the following general formula (2), (3) and (4) of (a radical -COOR ^{3 'is} carboxyl, and R ^3' is an ionic bond between the bonding -COO ^{_ + In the} form of R ^{3 '} ). [Formula 2]

[Formula 3]

[Formula 4]

In each said formula, HG represents a hydrophobic group having 6 to 30 carbon atoms, R ³⁰ is a straight-chain divalent C ₂ to C ₆ alkylene chain, R ^{3 '} is H, or ■ ⁺ R ^{3 '} is    Free from a subgroup consisting of sodium, potassium, calcium and magnesium A metal cation selected to    The following cations, ie An amine cation, And oligo amine cations, And the polyamine (which is especially preferred polyethyleneimine) based cations and, And Lee New Territories or amino acid cations advantageously selected from the arginine-based cation acids An organic cation advantageously selected from the subgroup consisting of:    Advantageously selected from subgroups consisting of polylysine and oligolysine Polyamino acid cation It is preferably selected from the group consisting of R ⁵⁰ is a divalent straight chain alkylene C ₁ to C ₈ chain, wherein one or two methyls Each end of the ethylene unit, preferably R ⁵⁰ , may be independently substituted with —O— or —NH. There is, R ⁴ is a direct bond or a “spacer” based on one to four amino acid residues Indicates ■ A is independently —CH ₂ -radical (aspartic acid unit) or —CH ₂ —CH ₂ —radical (Glutamic acid unit), (N '+ m') and n "are defined as degrees of polymerization, from 10 to 1,000, preferably 50 To 300. The microparticle according to claim 7 or 8, wherein the R ⁴ group represents a single bond. The "intrinsic neutral" copolyhydroxyalkylglutamine (preferably alkyl is ethyl) according to any one of claims 1 to 6, wherein said PO contains several of the same or different pendant hydrophobic groups (HG). Microparticles containing one or more). The method according to any one of claims 6 to 10, wherein all or part of the hydrophobic radical HG of PO is ■ at least one hetero atom (preferably O, N, or S) or at least one fire Straight or branched chain, which may contain saturated bonds and having 6 to 30 carbon atoms Alkoxy, ■ at least one unsaturated bond or at least one hetero atom (preferably O, N, Or S), optionally 6 to 30 carbon atoms, at least one annealing Alkoxy, including annealed cycloalkyl, ■ at least one unsaturated bond or at least one hetero atom (preferably O, N, Or a) alkoxyaryl having 7 to 30 carbon atoms or Ryloxyalkyl Particles that are independently selected from the group of radicals comprising a. The hydrophobic group HG according to any one of claims 6 to 10 is an alkoxy radical of the type -OCH ₂ (CH ₂ -CH ₂ ) _{3 to 8} -CH ₃ , oleyl, tocopheryl or cholesteryl. And R ⁴ represents a single bond. The fine particle according to any one of claims 6 to 10, wherein each of the n HG groups in the PO independently represents a monovalent radical of the following formula.

In the above formula, R ⁵ represents methyl, isopropyl, isobutyl, sec -butyl or benzyl, R ⁶ represents a hydrophobic radical containing 6 to 30 carbon atoms, -l ranges from 0 to 6. The method according to claim 13, wherein all or part of the hydrophobic group R ⁶ of the PO, ■ at least one hetero atom (preferably O, N, or S) or at least one fluoride Straight or branched chain eggs containing 6 to 30 carbon atoms, which may include a chemical bond Coxy, ■ at least one unsaturated bond or at least one hetero atom (preferably O, N, Or S), optionally 6 to 30 carbon atoms, at least one annealing Alkoxy, including ring-treated cycloalkyl, ■ at least one unsaturated bond or at least one hetero atom (preferably O, N, Or a) alkoxyaryl having 7 to 30 carbon atoms or Ryloxyalkyl Particles that are independently selected from the group of radicals comprising a. 15. The alkoxy radical of claim 13 or 14, wherein the hydrophobic radical R ⁶ in the graft of PO is -OCH ₂ (CH ₂ -CH ₂ ) _3-8 -CH ₃ , oleyl, tocopheryl or cholesteryl type. Particles, characterized in that selected from the group comprising. The microparticle according to any one of claims 6 to 9 and 11 to 15, wherein the main chain of the polyamino acid is α- L-glutamate or α- L-glutamic acid homopolymer. The particulate according to any one of claims 6 to 9 and 11 to 15, wherein the backbone of the polyamino acid is α- L-aspartate or α- L-aspartic acid homopolymer. The method according to any one of claims 6 to 9 and 11 to 15, wherein the main chain of the polyamino acid is α- L-aspartate / α- L-glutamate or α- L-aspartic acid. / α- L-glutamic acid copolymer. 19. The method according to any one of claims 6 to 18, wherein the PO comprises at least 10 mol%, preferably at least 15 mol%, of HG in (n / n + m) in formula (I). Particulates. The particulate of claim 6, wherein the molar mass of PO is in the range of 2,000 to 100,000 g / mol, preferably in the range of 5,000 to 40,000 g / mol. As a manufacturing method of the polymer (PO) microparticles | fine-particles containing one or more active component (AP) as described in any one of Claims 1-20, i. The polymer (PO) is, And a hydrophobic group (HG), and preferably a hydrophilic group [this is at least partially ionized Warm group (IG)], which is a water-soluble and biodegradable amphiphilic copolymer, To cut a colloidal suspension of nanoparticles in water at pH = 7.0 under isotonic conditions. Can be formed with redness, And it is associated with the AP and non-covalent associative, ii . The particle size of the fine particles is 0.5 to 100 microns, Groups range from 1 to 70 microns, preferably from 2 to 40 microns, The method is characterized by spraying essentially a solution or colloidal suspension of PO containing said AP. The PO of claim 21, wherein the PO present in the solution or colloidal suspension is at least partially determined by T1 test and has a particle size of less than 500 nm, preferably 10 to 300 nm, more preferably 10 to 100 nm. The preparation method is in the form of nanoparticles. The particulate of claim 21 or 22, wherein the particulates of the PO polymer associated with at least one active ingredient (AP) are dispersed in an essentially aqueous liquid medium, which medium preferably contains dispersing means M ₁ , The resulting dispersion is freeze dried. The method of claim 23, wherein the dispersing means M ₁ , (i). Polyvalent ions of opposite polarity and polarity of the ionic groups of the PO polymer, contained in the continuous aqueous phase, (ii). At least one hydrophilic compound (preferably useful for injectable preparations) contained in the atomized PO / AP particulates by addition in the PO suspension / solution for atomization, (iii). Particulate coating of at least one film of at least one hydrophilic compound (preferably useful for injectable preparations), (iv). pH change, (v). A combination of at least two of the means (i) to (iv) And the means (i) is good. A PO particulate-based low viscosity colloidal suspension containing at least one AP, wherein the particulates are those according to any one of claims 1 to 20, or any one of claims 21 to 24. A liquid pharmaceutical preparation for delayed release of AP, which is obtained by the method described in. The pharmaceutical formulation of claim 25 comprising means M ₂ for dispersing PO particulates associated with one or more APs. 27. The pharmaceutical formulation of claim 25 or 26, wherein the continuous phase of the suspension is essentially aqueous. The pharmaceutical formulation of claim 25 or 26, wherein the continuous phase of the suspension is essentially an organic water miscible phase. 27. The pharmaceutical formulation of claim 25 or 26, wherein the continuous phase of the suspension is essentially an organic water immiscible phase. The dispersion means M ₂ according to any one of claims 25 to 27,  (i). Reverse polarity and polarity of the ionic groups of the PO polymer, in the continuous aqueous phase Fused polyvalent ions, (ii). The spray by adding in the PO suspension / solution to spray At least one hydrophilic compound contained in the purified PO / AP fine particles (preferably Useful for the formulation used), (iii). Particulate nose with at least one film of one or more hydrophilic compounds Ting (preferably useful for preparations for injection), (iv). pH change, (v). A combination of at least two of the means (i) to (iv) A pharmaceutical formulation selected from the group consisting of wherein said means (i) is good. The method of claim 30 wherein the hydrophilic coating compound → amino acids, → polyalkylene glycols, preferably polyethylene glycol, ¡Æ copolyalkylene glycols, preferably ethylene glycol / propylene glycol copolymers (Poloxamer or Pluronic or Lutrol type), Cellulose polymers and derivatives thereof, preferably carboxyalkylcelluloses (eg Such as carboxymethylcellulose) or alkylcelluloses (such as methylcellulose), → hardened or uncured polysaccharides such as trehalose, sorbitol, mannitol or water cross, → polyols such as propylene glycol or glycerol, → gelatin, preferably hydrated gelatin, → nitrogen-containing (co) polymers, preferably polyacrylamides, poly-N-vinylamides, Present in the group comprising polyvinylpyrrolidone (PVP) and poly-N-vinyllactam those, → poly (vinyl alcohol) (PVA), → poly (sodium glutamate), → mixtures thereof A pharmaceutical formulation selected from the group consisting of, wherein said hydrophilic coating compound preferably contains at least one hydrophilic polymer. 29. The lipophilic liquid of any one of claims 25, 27 or 28, wherein the dispersing means comprises a lipophilic liquid, preferably having a melting point of less than 15 ° C, present in a water miscible or water immiscible continuous phase. Phosphorus pharmaceutical preparation. 33. The lipophilic liquid of claim 32, wherein the lipophilic liquid comprises at least one mixture of triglycerides of saturated fatty acids or at least one vegetable oil or at least one lipid or at least one lipid derivative or at least one fatty acid or at least one fatty acid derivative. Pharmaceutical formulation. The lipophilic liquid of claim 33, wherein the lipophilic liquid is And derived from a saturated coconut oil C ₈ to C ₁₀ fatty acid triglycerides of one type of Mixture of more than ㆍ at least one vegetable oil, preferably soybean oil, palm oil, flaxseed oil, cottonseed oil, sesame seed Oil, sunflower oil or peanut oil, At least one lipid, preferably liquid lecithin, synthetic or natural vitamin E, At least one lipid derivative, preferably arachidylylphosphatidylcholine and stearate Loyle Forcepatidylcholine, At least one fatty acid, preferably oleic acid, myristic acid, palmitic acid, stearic acid And salts thereof, At least one fatty acid derivative, preferably mono-, di- or triglyceride oils Conductor, ethyl oleate, lauryl lactate, glyceryl stearate, sorbitan palmitate, Sorbitan stearate, sorbitan monooleate or polysorbate, And derivatives thereof Wherein only a portion of said products, taken separately, are liquids, for example at temperatures below 15 ° C., such products are mixed with other products such that they are liquid at temperatures below 15 ° C., for example. (No content) 35. The method of any one of claims 25, 27, 28, 32, 33, or 34, wherein the dispersing means comprises at least one film forming coating compound (preferably an injectable formulation). Pharmaceutical coating), which comprises coating the microparticles with which can be used. 37. The method of claim 36, wherein the film forming coating compound is polylactide, polyglycolide, poly (lactide-co-glycolide), polyorthoester, polyanhydride, poly (hydroxybutyric acid), polycaprolactone And at least one hydrophobic polymer selected from the group consisting of poly (alkyl carbonates), water-insoluble PO polymers, derivatives thereof, and mixtures thereof. 37. The saturated fatty acid triglyceride of any one of claims 25, 27, 29 and 31-36 wherein the film forming coating compound is lipidic, preferably has a melting point of at least 15 ° C. A pharmaceutical formulation comprising one or more mixtures or one or more vegetable oils or one or more lipids or one or more lipid derivatives or one or more fatty acids or one or more fatty acid derivatives or mixtures thereof. 39. A reconstitution kit for reconstructing the pharmaceutical formulation of any one of claims 24 to 38, wherein the kit is PO microparticles containing and at least one AP (of the fine particles of claim 1 to claim 20, wherein The method according to any one of claims, or the method according to claim 21 or 22. Obtained by And → essentially be adult liquid   ¡Æ essentially an organic water-miscible liquid   ¡Æ essentially an organic water immiscible liquid Reconstituted liquid selected from the group comprising Kit comprising a. A method for reconstitution of a formulation according to any one of claims 24 to 38, And ⇒ PO fine particles containing at least one AP (the fine particles of claim 1 to claim 20 22. Those described in any one of the claims, or described in any one of claims 21 or 22. Obtained by the method),     ⇒ → essentially aqueous liquid        ¡Æ essentially an organic water-miscible liquid        ¡Æ essentially an organic water immiscible liquid   Mixing the reconstitution liquid selected from the group comprising: And the step of shaking the mixture Reconstruction method comprising essentially. And based on the first PO fine particles containing at least one AP (the fine particles are those obtained by the process described in things or claim 21 or 22 as described in any one of claims 1 to 20, wherein) or , And or, delayed release solid pharmaceutical preparations for the AP comprising the claim 24 to claim 38, obtained from the dry powdered preparation according to any one of claims. The solid pharmaceutical formulation of claim 41, which is for inhalation or for pulmonary administration.