MXPA97004503A - Steril powder of interlocked materials and its - Google Patents

Abstract

The present invention relates to a sterile powder comprising microparticles of 0.1 to 50 um in diameter obtainable by means of spray drying and bonding with a water soluble material having free fusional groups, characterized in that the microparticles are hydrophilic, can reconstitute in water to give a monodisperse suspension and have said retained groups available for derivation. The particles bind to drugs or other functional molecules and are used as vehicles in the therapeutic

Description

STERILE POWDER OF INTERLOCKED MATERIALS AND THEIR USE SUCCESS OF LR INVENTION This invention relates to interlaced microparticles and their use as therapeutic vehicles.

BACKGROUND OF THE INVENTION The particle transport systems are attracting increasing attention for use in the parental release of therapeutic and diagnostic agents. A large number of systems and chemistries have proliferated in the area of microparticle technology as vehicles for releasing agents subcutaneously, intravenously and interarterially. There are many key aspects to the "ideal vehicle". These include size, size of distribution, payload, index of biodegradation, ease of use, released kinetics and reproducible production at scale. The individual aspects of this "ideal vehicle" have been successfully managed by others, mainly drug payload, biodegradation index and, in part, size and size distribution. The known vehicles have been manufactured by means of several techniques, very solvent and based on emulsion. A disadvantage of these procedures is that control of the key elements of the vehicle was attempted within one or two key elements of the vehicle was attempted within one or two steps of production. In this way, the size, the size distribution, the payload and the biodegradation index were imparted to the product in a unique and dynamic environment, typified by simple and double emulsion systems or solvent evaporation techniques. Typically, for the emulsion processes, in the drug solution, the polymers and surface modification agents are mixed with an insoluble, emulsified, heated or stabilized solvent to fix the particles, and then washed to remove oils or solvents that do not They are compatible with parental use. The reaction vessel of the emulsion or solvent evaporation system is a major feature of the prior art. Within this vessel, the control of the morphology of the microparticles is achieved by balancing the interfacial forces of the oil and the components of the water, the interaction of the solute in the interface, the balance between the agitation, the heating and the formation of the capsule and, of course, the incorporation of the asset into the polymer matrix. However, such technology is largely incompatible with the large-scale pharmaceutical manufacturing required by a parent agent. Almost without exception, the control of the size and the size distribution of the known microparticles was much lower than the size control achieved by means of the spray drying techniques described in the PCT publications UO-P-9112823, UO-R- 9218164 and UO-R-9408627, in the production of microparticles for use in contrast images by echo and other potential parental uses, The acute toxicity of intravenous microparticles is extensively associated with capillary block in the pulmonary circulation, concurrent decrease in pulmonary venous pressure and loss of elasticity. The relationship between particle size and LD toxicity is well recorded. The information shows the precipitated elevation in the toxicity of the iv particles, with an average size greater than 6 m, the notional capillary size in the lung tissue for non-deformable nycrocapsules. Typically, the larger the average size of the capsules, the more important the size distribution will be significantly larger, and the scale of the sizes of the microparticles can increase by two orders of magnitude. For therapeutic use, such as quirnioerbolization, the prospect of injecting a microparticle preparation containing particles having an average size of 5-100 is much more consistent with the concept of highly regionalized release. At the upper end, exempt the prospect of bolizar main vessels up to a diameter of 100 and more, with the existing risk of suffering necrosis in large areas of perfusion; at the smallest end of the scale, it becomes possible what essentially arrives at the systematic distribution.

The mechanism by which previously sustained release in microparticle systems has been most frequently achieved has been the control of the erosion matrix and the release of the surrounding media from embedded or absorbed assets. The asset has been incorporated either in the time of production of the particle or absorbed in the matrix that follows the fixation or stabilization. The incorporation of drugs in the matrix of the known micro-capsules that require heating in the presence of water and, inevitably, oxygen. This would lead the vehicle almost certainly to the adulteration of the drug by oxidation damage or uncontrolled cross-linking. In these cases, where chemical stabilization is used, the potential loss of assets would be even worse. Another mechanism for decreasing the rate of drug release rates or modifying them from soluble polymeric transporters has been to bind the active ingredients by means of covalent bonds to the soluble polymer. In general, it has not been applied to microparticle systems in which drugs, ligands or antibodies are bound to particle transporters. The main problem for binding active ingredients to microparticles of the prior art is the relative hydrophobicity of the latter. Because many of the chemical reactions required to achieve the binding are carried out in an aqueous medium, said hydrophobic particles are almost impossible to derive. When previous workers have produced hydrophilic microcapsules, they require the formation of a complex in the presence of a hydrophilic polymer, in an emulsification process. The rate of biodegradation of the microcapsules is determined to a large extent by the extent of the entanglement. In prior art systems, changes in entanglement have detrimental effects on drug loading and have the subsequent ability to formulate the microparticles. A small effort was made to try to manipulate this parameter to control the rate of biodegradation and drug release. The microparticles of the prior art have required significant amounts of surfactants or are intended to achieve an onodisperse suspension in the aqueous medium. Even when they have been reconstituted, the microparticles have a tendency to agglomerate and therefore are difficult to administer through hypodermic syringes.

BRIEF DESCRIPTION OF THE INVENTION This invention is based on the discovery that the microparticles of the type described in the publications of PCT UO-fi-9112823, UO-fi-9218164 and UO-O-9408627, which have good particle characteristics, can retain, even after thermal entanglement, their hydrophilic properties and the ability to be reconstituted in water to give a suspension rnonodiepersa. In addition, the functional groups co or the carboxyl, amino, hydroxyl or sulfhydryl groups are retained in the starting material and can be derived. According to the present invention, a sterile powder comprises soft, spherical microparticles with a diameter of 0.1 to 50 μ, of interlaced materials, the microparticles being hydrophilic and capable of being reconstituted in water to give an onodisperse suspension, and which also comprises a component physiologically or diagnostically active linked directly or indirectly to microparticles by means of a free functional, ie, amine, hydroxyl, carboxyl or sulfhydryl groups thereof.

DETAILED DESCRIPTION OF THE INVENTION This invention preferably uses a microparticle production technique of the type described in the PCT publications, supra (of which the contents are incorporated herein by reference), in which there is strict control over the size, size distribution , payload, biodegradation index and kinetic release, which provides ease of use and production at scale. The microparticles of this invention may have the size that is desired to suit the application while retaining, in all cases, the ability to produce the loaded vehicle in scale at high levels of pharmaceutical practice and always with the same level of control, In addition, to control the size, independent control is possible in the individual stages for the size distribution; the index of fixation or, reciprocally, the index of biodegradation; the drug loading, and the formulation and finished. As described above in the PCT publications, supra, applicants have a fully classified process that can produce microparticles of the specified nature. The procedure can be operated for pharmaceutical standards are the entry of foreign particles that certainly should be avoided for the parental use of microparticles produced by means of many of the prior art procedures. The present invention relates to the production of microparticle preparations for intravenous, interarterial and ex vivo use. Intravenous suspensions of particles, in the reconstitution diluent, preferably contain particles of 5% by volume of particles greater than 6 μm. In addition, the size distribution resembles Gaussian in shape, with approximately 50% of particles within the scale of 5", preferably 3 μm, preferably 2 μ and most preferably 1.2 μ. A desirable distribution has 80% of particles on a scale of 3 μm. (All distributions cited in a volume or base mass). A preferred embodiment of the invention are powders in which 95% of the particles are smaller than 6 μ, and 80% of the particles are in the range of 1 to 6 μm, especially for iv administration. Another preferred embodiment is powders in which 90% of the particles are less than 20 μm, and less than 5% by volume are less than 6 μm, especially for interarterial administration. For larger particle systems, using a combination of highly controlled spray drying and a subsequent fractionation stage, it is possible to produce microparticles of sufficient size and strict size distribution so that, following interarterial administration, the systemic release is eliminated. and only vessels less than 20 μrn are ernbolized. In one embodiment of the present invention, the active has been incorporated into the supply for spray drying and subsequently the particle has been stabilized. The advantage obtained is far superior to the morphology and payload control of the previous methodologies. The method used in this invention may involve the deposition of the material and the drug in a dry powder state, with negligible water content, capable of being reconstituted to the original soluble components. In this invention, the links can be used to control the release rates of assets. For example, in one modality, the extension of the link, and therefore the biodegradation index, is "established" before the union of the assets. Then the ligand of the active can be bound, and it shows a controlled release profile determined by the extent to which the matrix is bound. This has the advantage that a stable rate of release is observed without the well-known "burst" effect. Another aspect of this invention is link manipulation. The potential levels of derivable groups and the temperature / time required to bind can be controlled by incorporating additives that alter these parameters. The inclusion of lysine or polylysine, glutamate or poly lutamate, phenylalanine and tyrosine in the supply may have the effect of the final preparation of microparticles. Adernáe. the incorporation of these additives can significantly increase the number of potential groups to which the assets, or ligands, can join. Furthermore, the incorporation of these additives can reduce the time / temperature at which the stabilization of the microparticle occurs during the heating of the soluble particles formed after spray drying. Therefore, because of this, while the present invention provides microparticles that are especially suitable for delivery in defined places, which belongs to its narrow particle size distribution, has been found a combination of the desired characteristics that makes the icroparticles especially useful. It has been found that microcapsules manufactured using the techniques described have many superior properties over prior art microparticles. including the ability to handle, derive and formulate the microparticles. Thus, for example, applicants' technology allows the production of microparticles that apparently retain a substantial degree of secondary structure, and thus are hydrophilic instead of denatured, insolubility subdued, with hidden functional groups, as in the art. previous. In particular, the microparticles: (i) possess significant levels of amine, hydroxyl or carboxyl groups, or combinations thereof, for derivatization, (ii) are highly hydrophilic with full access through an aqueous medium for the derivation of the active groups, (iii) can be handled in the dry or wet state to achieve dry final formulations that do not require surfactants or sonication to achieve monodisperse particle suspensions, (iv) diodegradabl.es principles and release of active ingredients at a given index by means of the The composition and binding of the capsule, (v) can have sizes ranging from 0.01 μrn to 100 μm, and (vi) are retained in circulation in vivo for periods of 80 minutes or longer, considerably longer than for known hydrophobic microparticles. , and longer than for liposomes; opsonization is reduced, and the potential trinogeneic is minimized. The size of the particles is preferably less than 4 μm for intravenous administration, and between 8 to 30 μm for interarterial administration. Especially for larger particles, fractionation is an optional additional step. This particle size scale can be expressed in such a way that the ratio of the interquartile scale to the average diameter ee of 0.2 to 0.5. The microparticles of this invention can be derived by means of the conjugation of drugs, ligands, peptides or proteins directly with the transported using the carboxyl or amino groups of the basic capsules or additives made for the supply for spray drying. For example, conjugation can be accomplished using glutaraldicide, EDCI, terephthaloyl chloride, cyanogen bromide or reductive ination. Alternatively, the ligand, drug, protein or peptide can be linked by means of a biodegradable hydroxy acid linker of the content type of which it is incorporated by reference. A further advantage of this invention is based on the ability to formulate and present the product as a dry sterile powder.

The icrocapsules of this invention in powder form do not have an absolute requirement for surfactants to ensure a monodisperse suspension in reconstitution.

Once reconstituted, they can not agglomerate and can be administered by means of a syringe. One aspect of the present invention is a water compatible system made of biocompatible materials. It could not be anticipated that the microparticles of the present invention could be insolubilized by heating and retain sufficient secondary structure to remain highly hydrophilic. The evidence that secondary structure retention is obtained by examining the particle isoelectric point (PI) which, at a pH of 4.5 to 5, is very similar to native albumin. Normally, complete denaturation of the albumin leads to a significant increase in PI, to a value of 5.5 to 7.0. In addition, the digestion of protein particles with protease produces the peptides which, when compared to digestion of the starting soluble protein, show almost identical profiles, by means of analogous HPLC. In addition, acid hydrolysis of protein microparticles and the protein starting material show odor content similar to that of amino acid. This two analyzes support the observation that the protein in the microparticles is very native. The novel particles are hydrophilic and have the potential to circulate for periods exceeding one hour, offering for the first time a transportable biopsy that shows a prolonged circulatory life with highly specific affinity for the ligands. The specificity of the microparticulate is "established" during manufacture and imparts a high-affinity ligand-binding capacity normally associated with chromatography matrices or enzymes. The particles also offer potential for use in contact with biological fluids, bioassays in serum or blood and the separation of blood components for reintroduction into the body. The following examples illustrate the invention.

EXAMPLE 1 This example illustrates the fixation of soluble icrocapsules, to form insoluble microcapsules or soluble nenos, binding the protective material. The microcapsules can be linked by various methods, including the use of heating or chemical means. Adjustment of the degree of fixation results in the subsequent degree in which the microcapeules will be dissolved in an appropriate medium. Moreover, any binding of active or encapsulated compounds within the nicrocapeulae will be released at this point of dissolution. In addition, the degree to which the microcapsules are fixed is also reflected in the degree to which they can be digested enzymatically. The greater the degree of fixation, the greater the resistance of the rnicrocapsulae to enzymatic digethion. Lae microcapeulae HSA are produced from an asperion drying supply containing 150 rnl of 25% ethanol containing 10.0 g / ml of HSA. The conditions of spray drying used to form the microcapsules are detailed in Table 1, below.

TABLE 1 Spray Drying Conditions Ambient Inlet temperature 220 ° C Inlet temperature 85.2 ° C Exit temperature- end 84.0O C Atomization pressure 7.5 bar Regulatory environment- 0. 5 feed rate 3.88 g / min Stored solution 25% ethanol 100 rng / rnl HSA The spray drying procedure yielded 17.21 g of microcapsules. The microcapsules of this single production charge were divided into aliquots of the same size and heated to a fixed temperature of 175 ° C for 45, 55 and 75 minutes respectively. The process of heat-based fixation produces the soluble microcapsules of the insoluble spray-drying process, linking some of the amino acids with the albumin structure. The three different heat-set microcapsules were given the size in a system I run a Multis zer II (Coulter Electronics). The microcapeulae have an average size of 3.28 + _ 0.6 μm and with 90% of the mass within 2-5 μm. Before joining any active component to the microcapsules, those fixed on a heat basis for 55 minutes were analyzed in several types of forms to suit their drug transformers.

FREE THYOL ANALYSIS The free thiol group present in the albumin molecule is very susceptible to modification and therefore can be used as a measure of the state and condition of albumin. Similarly, it must be present within the structure of the microcapsule which provides that the albumin molecule does not decompose during formation. The analysis of the free thiol group was carried out by reacting the albumin microcapsules with DTNB, ie, 5,5'-dithiobis (2-nitrobenzoic acid). If the free thiol is present, it reacts with the DTNB to produce a nitrobenzoic acid derivative that absorbs at 412 nm. The absorbance of a 12 mg / ml suspension of microcapeules was measured at 412 nm. 50 microns of a 20% solution of DTNB was added to the suspension in a TRIS pH regulator incubated for 10 minutes at room temperature and measured the absorbance. The difference between the doped builders was calculated and the concentration of the free thiol present in the microcapsule was calculated from the molecular extinction coefficient of the reaction product. The molecular ratio of the thiol groups measured in the microcapsules was 0.4785. It is compared with a value of 0.5045 for native albumin. This was not an important difference and it was concluded that the free thiol group did not change during the manufacture of the rnicrocapsule. The microcapsulae (both soluble and insoluble) and the native albumin are decomposed to their amino acid constituents by means of hydrolysis of the vapor phase using concentrated HCl at 120 ° C for 24 hours. The ureaters were derived by the addition of triethylamine in 50% ethanol and followed by triethylamine and PITC in ethanol. Lae derived samples were analyzed by HPLC, and the amino acids were detected at a wavelength of 245nrn. Table 4 (at the end of the description) shows the resulting amino acid compositions. Unexpectedly, there is no important difference between the different molecules, with only small losses of amino acids containing carboxyl, hydroxyl and amino groups after the insolitization of the microcapsules.

PEPTIDE ANALYSIS The pepsin digestion of the microcapsules and the albumin were carried out using an acidified solution of microcapsulae or albumin to which 20 μm of a 1% pepsin solution was added. Digestion was carried out at 37 ° C for 24 hours followed by a second addition of pepein and a further incubation at 37 ° C until the samples were completely digested. HPLC analysis of the lysate was performed using an acetonitrile gradient in 0.1% TFA, measuring the absorbance at 214 nm. The trypsin digestion of the microcapsules was carried out in the samples initially treated with guadinine-HCl, DDT and iodoacetamide, to open the protein structure. 0.2% trypsin was added to these samples and incubated at 37 > C until completely digested (if needed, additional trypsin can be added). The HPLC analysis of the lieate was performed as detailed above. The HPLC analysis showed no significant differences between the structures of the icrocapsule and the albumin. This confirms that there is significant retention of a secondary or tertiary protein structure after insolubilization of the microcapeula. The FITC (fluoroescein isothiocyanate) is covalently bound to amino groups in the microcapsules and exemplifies the principle of deriving charge groups, essentially reefed from lieine, with drugs that are suberequently liberated by degradation of the microcapsule matrix. The FITC was covalently linked to three charges of microcapsules. A ratio of microcapsules for FITC of 15: 1 was used. 12.5 mg of FITC was added to the suspended microcapsules and the mixture was incubated at 30 C for 30 minutes. Excess fluoroescein was removed by washing the microcapsule until no fluoroescein remained in the wash, that is, no leaching of the marker was observed. The microcapsulee were digested with Proteinae K in a concentration of 0.4 EU / l. Fluoroescein was released from the microcapsules as they were digested, and was measured by sampling the suspension of icrocapeulae at various intervals. The released FITC was separated from the microcapsules by centrifugation and quantified by measuring the absorbance at 493 n. The results showed that, between hot-set microcapsules, the initial release of fluoroescein is faster. However, after 25 minutes, all the samples had released more than 90% of the fluoroescein. The amount of FITC linked to the different microcapsules fixed based on heat was similar, charging approximately 10 + 0.5% mole / mole for the three loads. The release rates for linked assets can thus be adjusted by "establishing" the degradation rate of the microcapsules prior to the binding of the assets.

The microcapsules of Example 1 were incubated with whole human blood for 30 minutes at 37 ° C to determine if the microcapsules were capable of stimulating platelet activation. The concentration of microparticles was equivalent to the dose of 2000 x 10 * particles / kg. After 30 minutes of incubation, the serum was tested for effect on platelet aggregation stimulated with ADP collagen and arachidonic acid. In general, the effects of the hernestatic rnechanisnoe were determined by measuring the procoagulation activity, the partial thromboplastin time; the prothrombin time due to the appearance of 1 + 2 fragments and fibropeptide A; and fibrolytic activity by means of erythrobulin lieie time examination. In this concentration, there was no evidence of any adverse effect in the tested trials. In this way, the results suggest that the microparticles are inert and hydrophobic, with different particles made by an emulsion process. In a further test, the microparticles made by the method of Example 1 were sterilized by means of gamma radiation by exposure to a Ce * source and received a dose of 25-35 Kgras'. The microcapsules were reconstituted in an aqueous diluent at a concentration of 1.5 x 109 icroparticles / ml and administered to healthy male volunteers in dosie of the 25-300 x 106 mic oparticulae / kg scale under the approval of the ethical committee. The microparticles were emptied and contained air that allowed for their pause and persistence in the bloodstream to be followed by the use of ultrasound images. To monitor the circulatory life time that follows IV dosing, Acuson-128 is used, with double-dimension images of the gray area of the right and left ventricle. For the opacity of the left and right ventricle to occur, important levels of microparticles must be present in the chambers. In the doses of 25 x 106 upwards, the opacity in the right and left ventricles persisted for a period of 1 hour or more, showing that the important quantities of particles remained in the circulation. This information shows that the Bacic microcapsule vehicle is inert to the coagulation machinery in the blood, and that is why it is ideally suited for performing therapies. This is completed in a manner contrary to that of the microparticles made by emulsion procedures that exhibit rapid increases RES and circulating half-life of 10 minutes or rnenoe. In addition, the icroparticles do not need derivation with co-block polymers to improve the circulatory half-life.

EXAMPLE 2 This example shows that the additives can be included in the supply of the spray drying of the material forming the wall of the microcapsule, so that the microcapsules can be fixed based on heat at a low temperature. The additives that allow entanglement at lower temperatures (insolitization) of the rnicrocapsule polymer have utility when the active drugs are dried by coasperation and thereby incorporated into the matrix. Using these additives, the microcapsules with heat-sensitive active agents can be solubilized at advantageously lower temperatures. 5 rag / ml of tyrosine were added to the spray drying feed and the microcapsulee were formed using the method detailed in example 1. No change was required in the spray drying conditions to obtain microcapsules. The collected microcapsules were fixed on a heat basis or above, but at a temperature of 100 ° C for 55 minutes, to achieve the same or link. The microcapsules produced had an average size of 3.28 + _ 0.6 μn with 90% of the mass within 2-5 μm.

EXAMPLE 3 Example 1 details the production of microcapsules of 3 μm. This example shows that, by adjusting the spray drying conditions and the use of a step of the secondary stage classification process, larger microcapsules can be produced with excellent control over size and size distribution. 20% HSA was spray-dried under the conditions shown in Table 2. The collected microcapsules were fixed at 175 ° C for 55 minutes, desalinated and then classified using a jet classifier elbow (see Table 3).

Table 2 Input temperature 220 ° C Initial output temperature 89.1 ° C Final output temperature 89.2 »C Spray pressure 2.0 bar Regulating environment 0.5 feeding index 20.1 g / rnin Stored Solution 20% HSA Table 3 Conditions of Classification Environment Primary air 0.6 bg. Secondary air 2.0 bg. Venturi air 8.0 bg.

The average classified fraction was collected and reformulated, as the classification procedure removes much of the excipient. The dry powder free of re-emerging fluid was characterized as above. The microcapsules had an average size of 12 μm, virtually no microcapeulae lower than 6 μm, and 85% of the mass between 9-18 μm. Removing particles smaller than 6 μrn prevents systemic circulation, followed by interarterial administration due to the capillary network. This has the advantage of locating the drug store to achieve the therapeutic activity in the desired place. This is desirable, particularly in the case of cytokines since the toxicity if thermal is the main cause of harmful side effects. The antibody was then bound to the wall surface of the nicrocapsule. FITC IgG was used to aid in the detection of bound antibody. 35 mg of sodium periodate was added to 5 mg of FITC ~ IgG. The mixture was incubated at room temperature for 1 hour, after which 20 g of icrocapsules were added. The suspension was stirred for 10 minutes and then the activated antibody was bound to the microcapsule by the addition of 30 mg of borohydride. The reaction was allowed to continue for 2 hours at room temperature, after which the microcapeulae were collected and washed. A cadherin of rnicrocapsulae was reduced, releasing the light chains of the bound antibodies. The microcapsules were removed and the resulting filtrate was collected. The presence of the light antibody labeled FITC in the filtrate was measured by the use of a fluorometer.

The binding of antibodies to the microcapsules can also be achieved by means of tri and tetrapeptide spacers. The peptides are covalently linked to the activated sugar ring in the antibodies using the periodate and borohydride reaction discussed above. The antibodies are linked to the microcapsules by means of this peptide spacer using EDCI, as described in detail in Example 6.

Example 4 This example shows that the incorporation of additives to the supply of spray drying, for example HSA, will alter the chemical properties of the nicrocapeulae produced in Example 1, so that the number of chemical bonding sites can be greatly increased. The polylysine was incorporated into the spray drying agent at a concentration of 5 mg / ml. The spray-drying process was carried out as described in detail in Example 1. The microprocessors of this modified warehouse had an average size of 3.5 + _ 0.3 μrn with physical characteristics similar to those of the rnicrocaps. HSAs such as re-suspension properties. The FITC was attached to the polylysine microcapsules as described in detail in Example 1. The icrocapeula was washed until no FITC residue was observed. The FITC remained attached to the microcapsulee HSA, and showed no release of aqueous suspension. The microcapsulee were digested as detailed in Example 1 and the total amount of fluoroescei was measured to be attached to the microcapsules. The rate of release of fluoroescein in the digethion showed a faster release than that of the common microcapsules. However, a total of 20 molar% of fluoroescein bound to the microcapsules was measured, a 50% increase over the common icrocapsule composition.

EXAMPLE 5 In this example, the assets can be linked to the envelope and their subsequent release rate can be governed by degradation of the microcapsule itself. 25 mg carbodiinide was added to a solution of 10 mg / ml methotrexate. The solution was stirred for 4 hours to initiate and ensure complete activation of methotrexate. 50 mg of HSA microcapsules were added, produced as in Example 1, to the activated drug and stirred for 3 hours at room temperature. Methotrexate was chemically bound to the microcapeulae by amino groups in albumin. The microcapsules were collected and washed to remove any freely bound methotrexate. The bound methotrexate microcapeulae were characterized and also analyzed for drug content. The microcapsules had an average size of 3.2 + _ 0.6 μm with 90% mass between 2-5 μm. The analysis of the drug content showed that the microcapsules do not release the drug when res? Spendidas are found in an aqueous medium. In addition, a three-month stability trial showed that there was no drug release after this time. Digestion of proteinase K from the albumin microcapsules released the bound drug that was shown to bind only a limited number of amino acids and small peptides.

EXAMPLE 6 Doxorubicin is conjugated to microcapsules produced using the general method described in Example 1, with carbodii idaa. 3 mg of doxorubicin, 6 mg EDCI and 100 mg of microcapsules were discharged in distilled water at pH 6.6 and 37 ° C for 20 hours with continuous shaking and resorption and formulating. Lae microcapsules were analyzed to see the drug content and characterized. The results showed no change in the characteristics of the microcapsulae during the conjugation of the drug. Drug loading was achieved in a sample of enzyme digested from microcapsules by HPLC analysis. A load of 2% ee was measured and the drug was bound only to a limited number of amino acids or small peptides. He has previously shown that the activity of doxorubicin linked to polypeptide transporters has benefits in tumors that show a drug-resistant phenotype.

EXAMPLE 7 The icrocapeulae produced by the procedure of Example 1 were derived to have 2'-deoxy-5-fluorouridine (FUdR). The drug was activated with succinic anhydride. 40 mg of FUdR was added to 0.2M of phosphate pH regulator at pH 8.3. To this solution was added 200 mg of succinic anhydride and the reaction mixture was stirred at 30 ° C for 2 hours, while the pH was maintained at 8.3 with IM NaOH. The pH of the reaction mixture was adjusted to 6.5, and 0.5 g of microcapeulae was added. After stirring the euepension for 10 minutes, EDCI and N-hydroeuccinarnide were added in a ratio of 15: 1, and the coupling reaction was allowed to continue at room temperature. After 24 hours, the microcapsules were collected and washed, and the presence of FUdR in the capsules was confirmed by the HPLC analysis. Acid hydrolysis of the drug-bound microcapsules was carried out in an ASTED system using 1% TFA, and the subsequent release was monitored at 269 nm. It was found that 15% w / w FUdR is bound to the microcapsules by the hydrolysable bond of the acid. Cytotoxic activity The cytotoxic activity of the drug microcapeula conjugates the ploe axis 5 to 7 was measured in vitro. The HSN cell line was used. This is a chemically induced colonic sarcoma in the rat, which produces hepatic tumors in a model animal with a vascular pattern similar to that seen in a human colorectal hepatic metastasis. The malignant cell was measured indirectly with MTT. This ee reduces to form in an active nitocondrio, a rnetabolito with color. The HSN cells were incubated in multiwell plates to which the drug control doeie or the drug strand was added in the microcapeula. After a scale of exposure times, MTT was added to the cavities and the formazan concentration was measured as an indication of cell death. The assay was calibrated for this cell line against a known cell number scale. The three drugs (methotrexate, 5-FUdR and doxorubicin) have similar dose response curves for the native drugs and the drug microcapsule lysate, which indicate that the cytotoxic activity was similar. The maximum reduction in cellular activity of the three drugs and Used was approximately 80%. For 5-FUdR and methotrexate, the steepest part of the dose response curve was 1 μ / ml up to 0.01 μ / ml and for doxorubicin 0.1 μm / ml, within the serum scales seen in vitro.

The lees of control of non-derived icrocaps did not show any cyto + ototoxic activity.

EXAMPLE 8 This example illustrates the binding of active compounds, not directly to the shell wall of the musculocapsule by means of a degradable spacer or linker. This allows greater control of the binding and release of the active compound. Using the technology described in detail in IO-A-9317713 for the linking of drugs with soluble carriers, naproxen binds to the microcapsules using a lactic acid spacer. 20 mmol of triethylamine and 10 mmol of pentane-l-benzyl chloride (PMB) were added to a 10-mmol euepene of L-lactic acid in dinitic form. The mixture was heated to a solution and then kept at room temperature. Excess sodium carbonate was added after incubation of the solution overnight, and the precipitated ether, L-lactic acid-PMB, was collected, washed and dried. 11 mmol of dicyclohexylcarbodiirnide solution was added to a solution containing 10 mg of naproxen, L-lactic acid-PMB and 4-dirnethylaminopyridine. The reaction mixture was stirred at 25 ° C and e monitored the formation of the naproxen linker. To complete the reaction, the naproxen-L-lactic acid linker was collected, washed and dried.

The protecting group PMB was removed by means of the linker-reaction of naproxen with anisol and trifluoroacetic acid at room temperature for 2 minutes. The excess reagent was removed under vacuum and the residue was collected and washed. The acidification of the washed residue produced naproxen-L-lactic acid, which was extracted, washed and dried or emptied at 50 ° C. The naproxen-L-lactic acid was activated by means of its 1: 1 reaction with carbodumide, followed of the addition of 1 H-hydroxye-ccinirnide mnn.Naproxen-L-lactic acid-NHS was added to the HSA microcapsules at a ratio of 5: 1 in a borate pH regulator.The resulting product was collected and Dry lapa ricrocapeulae was formulated, resulting in a fluid-free powder, with an average microcapella size of 3.5 + 0.6, 90% of the mass was between 2 and 5 μm. performed the capillary zone electrophoresis (Bec man, UK), showing the preemption of the drug using eeterasee and subsequent analyzes of the released naproxen carried out using an ASTED system together with a Gileon HPLC (Anachen UK). The drug is intact and in its native form. EXAMPLE 9 The production of spray drying of the microcapsules allows the control over many facets of the procedure and the characteristic of the microcapeulae. The characteristics of the surface of the micro-culinae can be altered in such a way that the ligands for enzymes or receptors can multiply in the envelope of the microcapsule. In this example, the number of refunds of a gi a is increased, and this increase is used to link TPA. Poly-arginine was added to the aerated aeration under the method of Example 1 and 5. The microcapoulae are produced using the same conditions described in Example 1. The micro-escaulae have an average size of 3.31 ± 0.6 μm and 90% the mass is between 2-5 μrn. To an eolion containing 250 μg TPA, 100 mg of microcapeulae are added. The suspension is stirred for 2 hours after which the microcapsules are removed and washed a little. The concentration of TPA remaining in the reaction eol ee measured by means of RP-HPLC having the peptide or reduced by incubation in 20 mM DTT at 37 ° C for 30 minutes in the presence of 8 M Urea. The analysis of the fragments ee carried out with a gradient of 10-40% acetomtpla-water and 0.1% of TFA during 60 minutes. Lae inicrocapeulae-TPA are analyzed for the presence of IPA using the clot lysis assay. A fibrin clot is produced by combining fibrinogen, trinbin and lae microcapeulae of TPA. Then plasminogen is added to the clot and a glass bead is added to the surface to allow the endpoint of the assay to be determined, that is to say, the coagulum. The fall of the glass bead through the coagulum lieado mueetra that the TPA is linked to the microcapeulae and that it is still active. In addition, the amount of TPA bound to the microcapeulae is determined by a modified fibrin assay. To a microtiter plate cavity, a small layer of agarose gel containing fibrinogen and troin is added. To the gel ee they add 20 μl of euepeneion of microcapeulae of TPA and plaeminogeno. After 30 minutes, the plate is washed and the reduction in the turbidity of the gel is determined by using a reader-plate reader at 340 nm. The concentration of TPA present in the micro-pipeline is determined using the appropriate TPA standards. The results show that between 15 and 20% of TPA is linked to lae icrocape? Lae. The microcapeulae of TPA may have utility as a thrombolytic deposition agent for ad imetration when angiography, similar to the proposal in lO-A-9408627 as a deposit contrast echo agent, the advantage being the maintenance of a localized TPA reagent. in the myocardium TABLE 4 COMPOSITION OF NATIVE ALBUMINO AMINO ACID AND MICROCAPSULES

Claims (16)

NOVELTY OF THE INVENTION CLAIMS

1. - A sterile powder of interlaced materials comprising soft and spherical microparticles of 0.1 to 50 μ in diameter, the microparticles being hydrophilic and capable of being reconstituted in water to give an onodieperea euspeneion, and which also comprises a physiologically or diagnostically active component directly linked or indirectly to microparticles through free functional groups of the same.

2. A powder according to claim 1, further characterized in that the material is an amino acid, a polyarnino acid or another polypeptide.

3. A powder according to claim 1 or 2, further characterized in that it incorporates an additional water soluble material that facilitates enzymatic biodegradation.

4. A powder according to any preceding claim, further characterized in that the active component is a drug, a chemical separator, a ligand for an enzyme or receptor, or? N antibody.

5. A powder according to any of the preceding claims, obtainable from said material, which is soluble in water and has said groups, (i) drying by aepereion and interlacing, such that said groups are retained in free form , and (ii) joining the active component to the interlaced material by means of said groups.

6. A powder according to claim 5, further characterized in that step (i) comprises spray drying a solution of water-soluble material and bonded with heat-dried material in the presence of less than 4% moisture.

7. - A powder according to any of the preceding claims, further characterized in that the functional groups are amino, hydroxyl, carboxyl or sulfhydryl.

8. A powder according to claim 7, further characterized in that the functional groups are amino, hydroxyl or carboxyl.

9. A powder according to any of the preceding claims, further characterized in that the particles have an interquartile scale ratio to average a diameter of 0.2 to 0.5.

10. A powder according to claim 9, further characterized in that 95% of the particles are less than 6μm, and 80% of the particles are on a scale of 1 to 6μm.

11. A powder according to claim 9, further characterized in that 90% of the particles are less than 20 μm and less than 5% by volume and less than 6 μm.

12. A microparticle euepension as defined in any preceding claim, suitable for parental adiection.

13. A euepeneion according to claim 12, characterized in that the microparticulate eethan according to claim 10, for intravenous administration.

14. A suspension according to claim 12, further characterized in that the microparticles are in accordance with claim 11, for intra-arterial administration.

15. A powder according to any one of claims 11 to 11, characterized in that the active component is a cytotoxic agent.

16. The use of doxorubicin for the manufacture of a medicament comprising a powder according to claim 15, for the treatment of tumors that show the phenotype resistant to the drug.