EP1819363A2

EP1819363A2 - Method for the production of albumen conjugates with non-steroidal antirheumatic drugs (nsar)

Info

Publication number: EP1819363A2
Application number: EP05813969A
Authority: EP
Inventors: Hannsjörg SINN; Marcel MÜLBAIER
Original assignee: AlbuPharm Heidelberg GmbH and Co KG
Current assignee: SCHMIEDEBERG-SINN, CHRISTA
Priority date: 2004-11-26
Filing date: 2005-11-25
Publication date: 2007-08-22
Also published as: CA2589455A1; DE102004057196A1; WO2006056463A2; WO2006056463A3; US20080306245A1

Abstract

The invention relates to non-steroidal antirheumatic drug (NSAR) protein conjugates and especially NSAR albumen conjugates, methods for the production thereof, and the use thereof as a medicament, particularly for the treatment of inflammations.

Description

Method for producing albumin conjugates with non-steroidal anti-inflammatory drugs (NSAIDs)

description

The present invention relates to NSAIDs (non-steroidal anti-inflammatory drugs) - protein conjugates, and more particularly to NSAID-albumin conjugates, to processes for their preparation and to their use as medicaments, in particular for the treatment of inflammation.

Non-steroidal anti-inflammatory drugs (NSAIDs), also called NSAIDs (non-steroidal anti-inflammatory drugs), have been used in their low-molecular forms for the treatment of inflammatory processes. A disadvantage of the previously used therapies using NSAIDs is, in particular, the short residence time of the substances used in the circulation, so that they have only a very narrow time window for the development of their effectiveness and, moreover, only a small proportion of the administered medicament reaches the destination. This results in unwanted side effects, which lead to problems especially with longer applications. Thus, by far the largest part of the active ingredients is taken up by healthy organs and causes there partial or severe damage. Observed side effects are, for example, complaints in the gastrointestinal tract and an inhibition of blood clotting. Furthermore, concentration disorders or headaches were also observed. Finally, in some cases even allergic reactions, favoring of asthma attacks (especially in acetylsalicylic acid) as well as the reduction of white and / or red blood cells on.

EP 0 630 263 describes conjugates consisting of a carboxyl group-containing active substance, a spacer and a carrier. The active substance, which may be a non-steroidal anti-inflammatory drug (NSAID), such as naproxen, is via an ester bond to the Spacer coupled, which in turn is linked via a covalent bond to the carrier. Suitable spacers for the controlled release of the active substance to the target tissue are (poly) -α-hydroxy acids and, as carriers, proteins such as isozyme, cytochrome C and aprotein. A disadvantage of these conjugates, however, is that while the active substance at the site of action is cleaved enzymatically from the spacer in the body, the carrier protein initially remains in the body, without as an energy supplier for associated with inflammatory processes cells that have a high turnover of plasma proteins to serve. In addition, the additional use of a spacer to the drug and carrier is associated with a significant amount of work and thus increased costs without being of additional pharmacological benefit.

WO 98/00172 discloses pharmaceutical compositions intended to effect specific delivery of drugs to the target site in the body. They include microbubbles that are encased in a protein shell and that are the actual transporters for the drugs. The protein shell preferably consists of human serum albumin and, as an active ingredient, naproxen may be present as an example. The microbubbles are formed by means of an ultrasound. However, WO 98/00172 does not disclose that there is a direct covalent bond between the albumin and the drug. Although it is the high specificity of the composition, but a higher residence time of the substance in the body, which is based on a direct covalent bond, not disclosed.

Kostiainen et al. (Journal of Chromatography (1993), 647 (2), 361-365) disclose the study of naproxen-lysozyme conjugates by capillary electrophoresis and mass spectroscopy wherein the naproxen is covalently coupled via an amide bond to the lysine group in the lysozyme. However, there is no information on the pharmacological effect of the conjugate, the loading level of the lysozyme with the drug Naproxen, the manufacturing process of the conjugate, etc. made. Thus, the document Kostiainen et al. only physical examinations of the disclosed conjugate.

Manoharan et al. (ChemBioChem (2002), 3 (12), 1257-1260) discuss the binding affinity of antisense oligonucleotides to serum albumin and note that the binding of ibuprofen to the oligonucleotides has an enhancing effect on the affinity of the oligonucleotides to human serum albumin. Thus, this is neither a covalent binding of the drug ^' ibuprofen to the human serum albumin, nor are pharmacological and native properties of an NSAID-HSA conjugate mentioned.

Lister et al. (Am J. Med. 1993 Aug 9; 95 (2A): 2S-9S) merely disclose a clinical comparison of anti-inflammatory non-steroidal

Antirheumatics such as diclofenac, naproxen or ibuprofen and their action in the treatment of osteoarthritis or rheumatoid

Arthritis. Conjugation of these drugs to albumin and associated increased bioavailability of NSAIDs in the body are not disclosed.

Zia-Amirhosseini et al. (Biochem J. (1995), 311, 431-435) describe the synthesis and mass spectrometric. Characterization of human serum albumin modified by covalent binding to tolmetin. However, pharmacological properties of the conjugate are not indicated.

Melgert et al. (Cells of the Hepatic Sinusoid (1997), Vol. 1996, 6, 389-390) describe a naproxen-HSA conjugate wherein naproxen and HSA are covalently linked together. It was the goal of the inventors to more efficiently infiltrate naproxen in copper or epithelial cells of the liver than is possible with the free low molecular weight drugs. The conjugates are used to treat acute / chronic liver inflammation used. A major disadvantage of the in Melgert et al. and Zia-Amirhosseini et al. However, the experiments disclosed in the listed high ratio of NSAID to albumin, since at such a high loading no more can be expected from an active protein. Thus, there is a denatured, non-application-capable product. Thus, it is no longer guaranteed that the active ingredient is released directly at the site of action and thus largely free of side effects.

An object of the present invention was therefore to provide NSAIDs in a form with which the difficulties arising in the prior art can be overcome and with which, in particular, targeted uptake into inflamed tissue can be achieved with simultaneously long half-life in the organism.

This object is achieved according to the invention by an NSAID-protein conjugate comprising an NSAID and a protein. More preferably, the NSAID-protein conjugate is an NSAID-albumin conjugate comprising a NSAID and albumin characterized in that the NSAID is directly covalently linked to the albumin and that the molar ratio of NSAID: protein is 2: 1 to 0 , 1: 1.

By directly coupling NSAIDs to proteins, particularly to carrier proteins, the low molecular weight drugs that are rapidly removed from the body are hidden from the body's excretion or trapping mechanisms, and a long half-life is achieved in the body. This makes it possible to administer small amounts of active ingredient and thus practically eliminate any possible side effects virtually completely.

Toxic effects on healthy tissue or organs are virtually unobservable, since normal, healthy cells have no cause for protein intake. In contrast, proteins and in particular albumin from cells associated with inflammatory processes recorded and thus lead to an active ingredient accumulation in these cells.

Direct covalent coupling of the NSAID to the carrier means that the NSAID is linked to the transport protein through a linker or spacer-free linkage. Preferably, the NSAID is covalently linked to the protein via an acid amide linkage formed from a carboxyl group of the NSAID and an amino group, preferably a lysine group of the protein.

Albumin, in particular serum albumin, and most preferably human albumin or human serum albumin (HSA) are preferably used as protein in the conjugates according to the invention. Basically, a protein is preferably used which is native to the patient for whom the conjugate is intended. By native protein is meant a protein derived from the same species as the species to which the protein is administered. This means, for example, that when administered to humans, human proteins, when administered to mice according to mouse proteins, etc. are used.

Human albumin is an endogenous, ubiquitously distributed and non-immunogenic protein. It has a molecular weight of about 68 kDa and is therefore not excreted via the kidney. Albumin accounts for approximately 60% of the total plasma protein level. In the healthy organism it fulfills transport functions for many substances and serves as a reserve energy source in acute emergencies, which is available everywhere in the body and at any time. Under normal conditions, it is not absorbed by healthy tissue. In contrast, cells associated with inflammatory processes have a high turnover of proteins, especially plasma proteins, predominantly albumin. As a result, the albumin in the inflamed target cells, for which the protein serves as an energy source, degraded and released the drug. This means that the coupling according to the invention of NSAIDs to proteins, especially albumin, a specific uptake and thus an accumulation of the active ingredient at the destination can be achieved. On the one hand, this enrichment makes it possible to use lower doses overall, and on the other hand, little or no side effects are observed since the conjugate is not absorbed by the remaining, healthy body tissue.

The biokinetic behavior of the conjugates according to the invention is determined solely by the macromolecule albumin, but not by the low-molecular weight NSAID. The coupling of the NSAID to the carrier protein, for example albumin, is preferably carried out without limiting its biologically active character. Particularly preferred is a covalent coupling of the active ingredient to the carrier protein. Furthermore, the covalent coupling is preferably selected so that it can be cleaved again in pathologically altered tissues, so that the biological effectiveness of the original pharmaceutical is retained and can be utilized.

Thus, according to the invention, formation of NSAID-protein conjugates, in particular NSAID-albumin conjugates, occurs without altering the biological activity of the active substance and without loss of the biologically active character of the protein used as a carrier, in particular of albumin.

A biologically active protein is understood to mean, in particular, an undenatured protein whose biological function is retained. Preferably, the protein has a biological activity of> 50%, more preferably of> 70%, more preferably of> 80%, and most preferably of> 90%, based on the activity of natural albumin. In the conjugates, the molar ratio of active ingredient to carrier protein is 2: 1 to 0.1: 1, preferably 1.5: 1 to 0.2: 1, and in particular 1, 1: 1 to 0.5: 1. Thus, for example Albumin is still biologically active at 1: 1 loading with an NSAID. Furthermore, it has been found that according to the invention conjugates can be obtained which have no or only a very small proportion of cross-linking during the loading. This further avoids rapid elimination from the circulation and unwanted side effects. The proportion of dimeric albumin in the conjugates according to the invention is advantageously ≦ 5 wt .-%, more preferably ≦ 3 wt .-%.

According to the invention, NSAIDs which are selected in particular from acetylsalicylic acid, diclofenac, indomethacin, naproxen, flufenamic acid, mefenamic acid, tolmetin, ibuprofen, fenoprofen, ketoprofen, acemetacin or niflumic acid are used according to the invention.

In particular, the following advantages can be obtained with the conjugates according to the invention and the preferred embodiments described herein. The active substances are released only in the area of the inflammatory process, in particular by enzymatic cleavage of the protein. The usual side effects that occur when using low-molecular weight NSAIDs, no longer appear because in vivo healthy cells do not absorb albumin or its conjugates. The biological half-life of the conjugates is determined solely by the macromolecule protein, in particular albumin. Thus, the initially available drug concentration falls after about 20 days to about 50% of the initial value. The protein used according to the invention for the formation of the conjugates preferably has a molecular weight of ≥ 18,000 Da, more preferably ≥ 30,000 Da and even more preferably ≥ 50,000 Da.

Due to the long biological half-life, the hitherto very narrow time window of drug availability has been significantly widened without any further side effects occurring. In a preferred embodiment, the conjugate according to the invention is active ingredient in a drug. Such a drug has in particular low side effects and can be administered, for example, on an outpatient basis. The administration is preferably intravenous.

A dosage unit preferably contains 0.1 to 10 mg of active ingredient NSAID per kilogram of body weight per day and in particular 0.5 to 5 mg of active ingredient per kilogram of body weight per day. In particular, the dose can be chosen lower than in the conventional therapy with NSAIDs.

Due to the long half-life of the conjugates in the body, it is possible to provide longer time intervals between administration, such that a dosage unit is administered, for example, at most every seven days.

The conjugates according to the invention are particularly suitable for the treatment of inflammatory processes. All the inflammatory processes that are also treated with the NSAIDs alone can be treated with the conjugates according to the invention. Inflammatory processes which can be treated according to the invention are, for example, rheumatoid arthritis.

Furthermore, it is possible to use the conjugates according to the invention in the combination therapy, for example together with other anti-inflammatory agents. In such a combination therapy, the doses of the respective components continue to reduce.

Another object of the present invention is a method for producing an NSAID-protein conjugate. Particularly preferred within the scope of the present invention is a method of producing an NSAID-albumin conjugate comprising reacting an NSAID with albumin by direct covalent coupling, wherein the molar ratio of NSAID: albumin is 2: 1 to 0.1: 1. In this case, the low molecular weight drug NSAR and the high molecular weight carrier protein are reacted with each other. Preferably, there is a direct covalent coupling of the active ingredient to the carrier molecule, for example albumin, without limiting its biologically active character. Coupling has proven to be particularly advantageous in which a succinimidyl ester is first formed from the low molecular weight NSAID and this is subsequently reacted with the protein. For example, a succinimidyl ester of NSAIDs can be made by reacting low molecular weight NSAIDs with a carbodiimide.

Efficient covalent coupling of the active ingredient to the carrier molecule (ie the protein) is important for the preparation of the conjugates used according to the invention. In particular, the coupling must not lead to an undesired change in the carrier protein and / or the active ingredient. Conventional activation of carboxyl-containing organic compounds with dicyclohexylcarbodiimide (DCC) requires at room temperature or at +4 ⁰ C more than 12 hours (DP 41 22 210 A1; EP 0879604 A1; EP 0820308). In this process, moreover, during activation, insoluble substances are formed, some of which already precipitate during activation and partly when the activated active substance is introduced into an aqueous protein solution and, in order for the conjugate to be administered medically, by time-consuming and expensive filtration steps in addition to the have to be separated from the actual product purification and are never 100% (because of the lipophilic domains in albumin).

There was therefore a need to provide a process for the preparation of drug-protein conjugates in which these problems do not occur and in particular no water-insoluble by-products are formed.

This object is achieved according to the invention by a process for the preparation of a conjugate in which NSAIDs and albumin in the presence of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) as carbodiimide and N-hydroxysuccinimide.

It has surprisingly been found that activation of the carboxyl-containing organic compound and reaction with a carrier protein can be achieved by using EDC 5, in particular in the form of the hydrochloride, without formation of water-insoluble by-products which would be time-consuming and costly to separate off , This method eliminates the need for intermediate cleaning steps and substantially reduces the preparation time and thus also the production costs. Furthermore, problems caused by injecting the conjuate into a human or animal body by insoluble substances or by-products are avoided. 5

The activation is preferably carried out at a temperature of 10 to 100 ⁰ C, more preferably from 20 to 90 ⁰ C and even more preferably from 50 to 75 ⁰ C for a reaction time of 1 minute to 10 hours, more preferably from 20 to 50 minutes. The reaction of the activated active substance with the carrier protein is preferably carried out at a temperature between 10 and 50 ^° C., in particular between 20 and 40 ^° C.

Preferably, the activation of the carboxyl-containing compound, in particular of methotrexate with EDC and N-hydroxysuccinimide in a 5 organic solvent, preferably in dimethyl sulfoxide (DMSO) is performed. Other suitable organic solvents include dimethylacetamide or dioxane. The activation is preferably carried out in the absence of water, in particular in the presence of ≦ 5% by weight of water, more preferably ≦ 1% by weight of water and most preferably completely anhydrous. By activation of the carboxyl group-containing compound with EDC and N-hydroxysucchinimide substances used in an organic, anhydrous solvent They do not react with protein, eg albumin, and do not change their structure.

An essential advantage of the preparation process according to the invention is that the activating reagents used, ie EDC and N-hydroxysuccinimide, have a high water solubility. As a result, unused coupling reagents can be removed in a simple manner from the product obtained, for example by washing with water during the reaction. In contrast, in the coupling reagents used in the prior art, for example when using di-cyclohexyl-carbodiimide (DCC), a non-resolvable residue of coupling reagent remains in the conjugate. Thus, with an NSAID-albumin conjugate using DCC, an undetectable residue of about 13 to 15% by weight of DCC is observed in the conjugate, which is likely bound to a lipophilic domain in albumin. This residue can only be detected with the aid of HPLC and can be separated off preparatively only at considerable expense.

Another preferred aspect of the invention relates to an optimized method of producing a conjugate according to the invention comprising reacting an NSAID with albumin by direct covalent coupling, characterized in that an NSAID and albumin in the presence of N- (3-dimethylaminopropyl) -N'-ethyl -c.arbodiimid reacts as the sole activating reagent.

In a preferred embodiment, the NSAID is preferably a cytostatic or immunosuppressant, more preferably indomethacin, acetylsalicylic acid, diclofenac, ibuprofen and / or naproxen, most preferably indomethacin. The protein is preferably albumin.

Surprisingly, it has been found that the optimized process, which without N-hydroxysuccinimide or other additional Activating reagents works in the cleaning procedure has a positive effect on the simplification of the production. By using EDC for activation without the addition of N-hydroxysuccinimide (HSI), the activation time of indomethacin is only significantly less than the 30 minutes required when using HSI. A further advantage of the optimized method is that after addition of the activated active ingredient to the protein presented, in particular albumin, without N-hydroxysuccinimide, a direct control of the coupling efficiency can take place. When N-hydroxysuccinimide is used, the latter also has a high UV absorption on HPLC when the UV measuring cell is set to 280 nm and disturbs or impedes by its retention time of about 11.5 minutes, during which other low molecular weight compounds appear direct determination of the coupling yield. This means that in many cases a yield determination is possible only at the end of the purification of the conjugate. Due to the optimized process without the use of N-hydroxysuccinimide this factor can now be excluded. This is also a great advantage for product safety. Another advantage of the optimized process is that the coupling yield is surprisingly on average 98-99%.

Thus, the overall cost of each conjugate is significantly reduced by this simplification of the production.

Due to their high purity, the conjugates prepared by the processes according to the invention can be used for numerous uses and, in particular, for intravenous administration. Thus, such conjugates, for example when using an anti-inflammatory NSAID, can be advantageously used for the preparation of medicaments for the treatment of inflammatory processes, in particular for the manufacture of a medicament for the treatment of rheumatoid arthritis. The invention will be further elucidated by the following example and the attached figures.

FIG. 1 shows an HPLC chromatogram of indomethacin alone, and FIG. 2 shows the chromatogram of the indomethacin-HSA conjugate prepared according to Example 1.

example 1

Indomethacin-HSA conjugate

Starting materials:

Indomethacin (IMC ₁ SIGMA-ALDRICH, Taufkirchen), N- (3-dimethylaminopropyl) -N'ethylcarbodiimide hydrochloride (EDC, SIGMA-ALDRICH, Taufkirchen), N-hydroxysuccinimide (Sigma-Aldrich, Taufkirchen) and albumin (Göricke, Dessau) , Instead of indomethacin, the other NSAIDs: acetylsalicylic acid.Diclofenac, ibuprofen, naproxen, etc. can be used for conjugation with albumin.

Standard batch on a laboratory scale:

About 10.5 mg of indomethacin (IMC, MW 357.8) is added together with about 11.3 mg of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC MW 191, 71, molar ratio 1: 2) and about 34 mg N-hydroxysuccinimide (NHS, MW 115.9, molar ratio 1:10) in a test tube with NS 14.5 ground and stopper. After the addition of 1 ml of dimethyl sulfoxide (DMSO), the reaction mixture is introduced into a preheated to 65 ⁰ C water bath. After a reaction time of about 30 minutes, a clear, colorless solution of indomethacinsuccinimidyl ester is present, which after cooling to room temperature is introduced very slowly into a 5% albumin solution. At the entry point, a cloudiness forms for a short time, but it dissolves quickly. The control chromatogram can be made directly after the coupling. The coupling yield is over 80%.

The separation of undesirable in the final product by-products DMSO, NHS, N- (3-dimethylaminopropyl) -N'-ethyl-urea and unbound indomethacin by ultrafiltration (YM 30, Millipore).

Quality Control (HPLC / SEC):

Guard column: Reprosil 200 SEC 5 x 4 mm, 5 μm (Dr. Maisch GmbH) Column: Reprosil 200 SEC 300 x 4.6 mm, 5 μm (Dr. Maisch GmbH)

Eluent: 0.18 M Na ₂ HPO ₄ ; pH 7.4; 5% methanol

Flow: 0.3 mL / min

Pressure: about 50 b

UV-vis: 280 nm

Retention times: oligomeric albumin fraction 5.93 min dimeric albumin fraction 8.19 min monomeric albumin fraction 8.98 min free indomethacin 29.45 min

The proportion of dimeric albumin is <3%.

Claims

claims

An NSAID-albumin conjugate comprising a non-steroidal anti-inflammatory drug (NSAID) and albumin, characterized in that in the conjugate the NSAID is directly covalently bound to the albumin and the molar ratio of NSAID: albumin is 2: 1 to 0.1: 1 ,

2. NSAID-albumin conjugate according to claim 1, characterized in that the albumin is human albumin.

3. NSAID-albumin conjugate according to one of the preceding claims, characterized in that the albumin is present in native form.

4. NSAID-albumin conjugate according to one of the preceding claims, characterized in that the NSAID is selected from acetylsalicylic acid, diclofenac,

Indomethacin, naproxen, flufenamic acid, mefenoic acid, tolmetin, ibuprofen, fenoprofen, ketoprofen, acemetacin or niflumic acid.

5. NSAID-albumin conjugate according to one of the preceding claims, characterized in that the molar ratio of NSAID: albumin is 1.1: 1 to 0.5: 1.

6. Medicament containing as active ingredient an NSAID-albumin conjugate according to one of the preceding claims.

7. Use of an NSAID-albumin conjugate according to any one of the preceding claims for the manufacture of a medicament for the treatment of inflammatory processes.

8. Use according to claim 7 for the manufacture of a medicament for the treatment of rheumatoid arthritis.

9. A process for the preparation of a conjugate comprising reacting an NSAID with albumin by a direct covalent coupling, characterized in that the molar ratio of NSAID: albumin is 2: 1 to 0.1: 1.

10. The method according to claim 9, characterized in that first from the NSAID a Succinimidylester is formed by means of a carbodiimide and then the succinimidyl ester of the NSAID is reacted with albumin.

11. The method according to claim 10, characterized in that the succinimidyl ester of NSAIDs by reaction of an NSAID with N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide and N-hydroxysuccinimide is formed.

12. A process for the preparation of a conjugate comprising reacting an NSAID with albumin by a direct covalent coupling, characterized in that an NSAID and albumin in the presence of N- (3-

Dimethylaminopropyl) -N'-ethyl-carbonyldiimide as activating reagent and that the molar ratio of NSAID: albumin is 2: 1 to 0.1: 1.

13. The method according to claim 12, characterized in that the NSAID in an organic solvent, in particular in an anhydrous organic solvent, with N- (3 Dimethylaminopropyl) -N'-ethyl-carbocliimid reacted, activated by heating and then the NSAID is reacted with albumin.