EP1940449A2

EP1940449A2 - Wound-healing pharmaceutical compositions comprising at least one soluble dextran and at least one platelet-derived growth factor

Info

Publication number: EP1940449A2
Application number: EP06808895A
Authority: EP
Inventors: Guy Dubreucq; Latifa Dahri-Correia; José CORREIA
Original assignee: Biodex SARL
Current assignee: Biodex SARL
Priority date: 2005-09-26
Filing date: 2006-09-26
Publication date: 2008-07-09
Also published as: US20070254828A1; KR101506593B1; ZA200803500B; EP1940448B1; CN103203023A; US20090221805A1; CN101631804B; BRPI0616439A2; EP1940448A2; PL1940448T3; RU2424824C2; US20110301086A1; KR20080080278A; FR2891149B1; CN101316607A; CA2623529C; WO2007034320A3; ES2406229T3; JP5438968B2; WO2007034320A2

Abstract

The invention concerns a wound-healing pharmaceutical compositions comprising at least one soluble dextran and at least one platelet-derived growth factor. The invention concerns a pharmaceutical composition having a wound-healing activity comprising at least one soluble dextran and at least one platelet-derived growth factor as well as its use for preparing a medicine with wound-healing activity, in particular designed to treat ulcers.

Description

Pharmaceutical composition with healing activity comprising at least one soluble dextran derivative and at least one platelet-derived growth factor

The present invention relates to a pharmaceutical composition with healing activity comprising at least one soluble dextran derivative and at least one platelet-derived growth factor as well as its use for the preparation of a medicament with healing action, in particular for the treatment of ulcers. Growth factors are a class of polypeptides with regulatory properties of many parameters of cell life (such as proliferation, differentiation, survival). These factors are secreted by multiple cell types. Growth factors exert their various effects by binding and activating a distinct subfamily of surface cell receptors with intrinsic tyrosine kinase activity.

PDGFs are growth factors released by platelets during blood coagulation, capable of promoting the growth of different cell types (Ross R. et al, Proc Natl Acad Sci USA, 1974, 71, 1207; Kohler N. & Lipton A., Exp. CeIl Res., 1974, 87, 297). It is now known that PDGF is produced by a number of cells other than platelets and that it is mitogenic for most cells derived from mesenchyme, ie blood, muscle, bone and cartilage cells, as well as cells of the brain. connective tissue (Raines EW, in "Biolog") of Platelet-Derivated Growth Factor, 1993, Westermark, B. and C. Sorg, Ed. Basel, Kerger, p. 74). Numerous articles also tend to show that PDGF derived from macrophages behaves as a chemotactic and mitogenic agent for smooth muscle cells, that it contributes to the myointimal thickening of the arterial walls characteristic of arteriosclerosis (Ross R. et al. Science, 1990, 248, 1009). PDGF activities further include, in particular, the stimulation of the release of granules by neutrophil monocytes (Tzeng DY et al, Blood, 1985, 66, 179), the facilitation of steroid synthesis by Leydig cells (Risbridger GP, Mol., E. Endocrino, 1993, 97, 125), stimulation of neutrophil phagocytosis (Wilson E. et al, Proc Natl Acad Sci. USA, 1987, 84, 2213), modulating the expression and secretion of thrombospondin (Majak RA et al, J Biol Chem, 1987, 262, 8821), and post-regulation of the ICAM-I gene. in vascular smooth muscle cells (Morisaki N. et al., Biochem Biophys Res Commun, 1994, 200, 612). Given its various properties, the use of recombinant PDGFs in the pharmaceutical field has already been considered.

Ulcer healing, like scarring in general, is a process that occurs primarily in three major phases. The healing process begins with a first inflammatory phase during which blood flow and flow increase around the site of ulceration. If bleeding from damaged blood vessels occurs, platelets invade the site of ulceration and allow coagulation to stop bleeding. Platelets also release PDGFs that will send signals to neighboring cells that trigger their proliferation, ie the second phase of the healing process. The third, final phase of the healing process is the remodeling phase. However, diabetic ulcers have the characteristic of healing very slowly and sometimes incompletely because the process of healing does not proceed normally, certainly because the blood flow to the skin is reduced in diabetics. This can lead to serious bacterial infections in ulcers, which can spread and sometimes require amputation of the foot or leg.

Therefore, there is currently on the market, particularly in the US market, a recombinant human PDGF-BB drug corresponding to the international nonproprietary name "becaplermin", sold under the trade name Regranex®. This medicine is indicated for the treatment of ulcers of the lower limbs of diabetics. It is in the form of a topically applied gel and promotes the healing of ulcers. In particular, it allows, just like endogenous PDGF, to promote cell proliferation and thus the formation of new tissues. The recombinant human PDGF-BB or beclapermin is obtained according to a preparation method implementing a recombinant DNA technology by insertion of the gene coding for the B chain of PDGF-BB in yeast, Saccharomyces cerevisiae. This technique is delicate, long and expensive and has the consequence of leading to the obtaining of a drug which is itself expensive for the patients. Regranex® is available in gel sold in tubes of 2, 7.5 or 15 grams, each gram of gel containing 100 μg of becaplermin. The amount of gel to be applied depends, of course, on the surface of the ulcer, however, the average cost of a treatment lasting twenty weeks is excessively high (in the order of US $ 1,400).

The inventors have therefore set themselves the goal of finding an excipient which makes it possible to increase the activity of PDGF-BB and to reduce the doses administered and, consequently, the toxicological risks and thus to access less expensive treatments for diabetic patients suffering from ulcers of the lower limbs. They have solved this problem, as is demonstrated hereinafter in the examples illustrating the present application, by the use of certain suitably selected soluble dextran derivatives. Indeed these allow to increase the activity of PDGF-BB, particularly in the context of the treatment of ulcers of the lower limbs of diabetic patients.

This result is obtained by the stabilization of the PDGF with respect to the chemical or physical degradations that can occur at physiological pH in vitro and in vivo by the development of a complex between a soluble dextran derivative and the

PDGF.

The present invention therefore relates to a pharmaceutical composition characterized in that it comprises, in a pharmaceutically acceptable carrier: at least one platelet-derived growth factor (PDGF), and

at least one soluble dextran derivative corresponding to the following formula (I):

DMC ₈ BbSu ₀ (I) in which: D represents a polysaccharide chain, preferably consisting of chains of glucosidic units,

MC represents methylcarboxylic groups, B represents N-benzylmethylenecarboxamide groups,

- Su represents sulphate groups (sulphating of the free hydroxyl functions, carried by the glucosidic units),

a, b and c represent the degree of substitution (ds) respectively of the groups MC, B and Su with i) a strictly greater than 0; (ii) b is such that

. either b is greater than or equal to 0.3 and c is between 0.1 and 0.5; . either b is strictly less than 0.3 and c corresponds to the following equation (1): c> 8.5 b ² - 5.41 b + 0.86 (1).

These dextran derivatives of formula (I), as well as their process of preparation are described more generally in the patent application WO 99/29734. These dextran derivatives of formula (I) are trivially called DMCBSu and are considered to be copolymers consisting of R-OH and R-OX subunits, X being able to be a methylcarboxylic (MC), benzylamide (B) or sulfate ( Su). Thus, a methylcarboxylic dextran (DMC) with a degree of substitution (ds) of 0.6 in methylcarboxylic groups contains 0.6 substituted group (R-MC) and 2.4 hydroxyl groups (R-OH), per unit. Invention, D preferably has a molar mass of between 1000 and 2000 000 Da, and even more particularly less than 70 000 Da.

According to a preferred embodiment of the invention, the dextran derivatives are chosen from compounds of formula (I) in which b is greater than or equal to 0.35.

In this case, and according to a very particularly preferred embodiment of the invention, the dextran derivatives are chosen from compounds of formula (I) in which a is between 0.5 and 0.8, and c is included between 0.1 and 0.5. Among such dextran derivatives of formula (I), mention may be made in particular of compounds in which a = 0.66; b = 0.38 and c is between 0.2 and 0.4. Dextran derivatives of formula (I) wherein a = 0.66; b = 0.38 and c = 0.29 are particularly preferred.

The amount of dextran derivatives of formula (I) present in the pharmaceutical composition according to the invention is preferably between 0.5 and 100 mg / g and even more preferably between 5 and 50 mg / g of composition.

According to the invention, the PDGFs are preferably chosen from

Recombinant human PDGFs having two B chains (rhPDGF-BB). Such

PDGFs can be obtained according to conventional techniques well known to those skilled in the art or directly purchased commercially from, for example, Research Diagnostic Inc. (USA).

The amount of PDGFs present in the pharmaceutical composition according to the invention is preferably between 1 and 200 μg / g of composition and even more preferably between 10 and 100 μg / g of composition. According to a particular and preferred embodiment of the invention, the weight ratio derived from dextran of formula (I) ZPDGFs is between 100 and 1000 and even more preferably between 300 and 700.

The pharmaceutical composition according to the invention is preferably a composition for topical application which may be in the form of gels, creams, sprays or patches, the presentation in the form of gels being particularly preferred,

The nature of the excipients that may be present in the pharmaceutical composition according to the invention is chosen according to its presentation form according to the general knowledge of the skilled person, that is to say the galenist.

Thus, when the composition according to the invention is in the form of a gel, it is preferably a cellulose gel such as for example a carboxymethylcellulose (CMC) gel.

The pharmaceutical composition according to the invention may further contain one or more additives such as those chosen from fillers, preservatives such as methyl parahydroxybenzoate, parahydroxybenzoate of propyl or m-cresol, antioxidants, stabilizing agents such as L-lysine hydrochloride, acidifying and alkalizing agents, opacifiers, etc.

When administered topically, the pharmaceutical compositions according to the invention are particularly intended for the treatment of ulcers, preferably ulcers of the lower limbs of diabetic patients. The inventors have indeed found that the use of such a composition makes it possible to improve and accelerate the healing of ulcers with compositions containing less quantities of PDGFs than the compositions currently available on the market (such as by example the drug marketed under the name Regranex®).

Thus, another subject of the invention is therefore the use of at least one pharmaceutical composition as described above, that is to say containing at least one dextran derivative of formula (I) as described hereinabove. and at least one platelet-derived growth factor for the preparation of a medicament with healing action for the treatment of ulcers by the topical route, and in particular the treatment of ulcers of the lower limbs of diabetic patients.

In this case, said drug is preferably intended to be administered in a course of 2 to 10 weeks at a rate of 1 to 2 applications per day on the injured part. In addition to the foregoing, the invention also comprises other arrangements which will emerge from the description which follows, which refers to an example of demonstrating the increase in the proliferative effect of a PDGF-BB with a dextran derivative of formula (I) and in the attached FIG. 1, which represents the amount of tritiated thymidine incorporated by human half fibroblasts (in Dpm × 10 ³ ), after stimulation of proliferation by PDGF-BB alone at a concentration ranging from 0.1 to 100 μg / ml (dotted line curve) or in the presence of a dextran derivative of formula (I) (solid line curve) at a concentration of 1 μg / ml.

It should be understood, however, that this example is given solely by way of illustration of the subject of the invention, of which it does not in any way constitute a limitation. EXAMPLE 1 Demonstration of the Increase in the Proliferative Effect of a PDGF-BB with a Dextran Derivative of Formula (I)

1) Procedure A primary culture of fibroblasts of human half (Human

Dermal Fibroblast adult (HDFa), a Cascade Biologics company) was carried out at a temperature of 37 ^° C. in medium αMEM (Minimum Essential Medium, Gibco company) with Glutamax, without ribo / desoxyribonucleotides, supplemented with 10% fetal calf serum. (SVF, Dutscher company) and 1% penicillin-streptomycin (Gibco company) in a saturated atmosphere in moisture and enriched in CO ₂ (5%). The medium was renewed every 4 days. Human fibroblasts were used between passages 1 and 5. A dilution of the cell suspension in the culture medium was then performed to seed culture dishes at a density of 5000 cells / well for 196 well plates ( Nunc company). For each lot of cells, the increase of the effect of PDGF-BB by a DMCBSu of molar mass 52,000 g / mol in which a = 0.66; b = 0.38 and c = 0.29 (prepared according to the method described in the international application WO 99/29734), at different concentrations, was verified by incorporation of tritiated thymidine (5000 cells / well in 100 .mu.l). To do this, after 24 hours of weaning, the fibroblasts were stimulated by addition of PDGF-BB (Research Diagnostic hic), at different concentrations ranging from 0.1 to 100 ng / ml, in the presence or absence of a derivative of dextran of formula (I) wherein a = 0.63; b = 0.38 and c = 0.29, at a concentration of 1 μg / ml. Incorporation of the tritiated thymidine (at 52 Ci / mmol, Amersham Biosciences company) was carried out 18 hours after stimulation with PDGF-BB in the presence or absence of the dextran derivative, by addition of a 50 μCi / solution. ml, 0.5 μCi / well. After 6 hours of incubation (ie 24 hours of stimulation), the culture medium was aspirated, the cells were fixed for one hour at 4 ^° C. by addition of 100 μl / well of trichloroacetic acid at 10% (ATCA ). The wells were then rinsed three times with 100 μl of ultrapure water and the cells were lysed overnight at ^{40 °} C. per 100 μl of 100 mM NaOH. The radioactivity was recovered in counting flasks, the wells were rinsed with 100 μl of 100 mM NaOH and the radioactivity was counted after addition of 1 ml of scintillation liquid (Zinsser Analytic), on a automatic meter sold by Beckman (LS 6000 TA). For all experiments, each stimulation condition was performed in triplicate.

2) Results

The results obtained are shown in the appended FIG. 1, in which the amount of tritiated thymidine incorporated by the fibroblasts (in Dpm × 10 ³ ) is expressed as a function of the concentration of PDGF-BB in μg / ml.

The solid curve represents the results in the presence of the dextran derivative at a concentration of 1 μg / ml and the dotted line curve the results in the absence of the dextran derivative, L ¹ ED ₅₀ corresponds to the concentration of PDGF-BB for have 50% proliferation of human fibroblasts. The ratio R is the ratio of the ED50 calculated as follows:

R = ED ₅₀ (PDGF-BB) / ED ₅₀ (PDGF-BB + DMCBSu)

These results show that when PDGF-BB is used alone, it is necessary to use 6 μg / ml to obtain 50% proliferation, whereas when PDGF-BB is complexed with a dextran derivative of formula (I) just 2 μg / ml to reach 50% proliferation. The ratio R, in this case, is equal to 3.

EXAMPLE 2 Demonstration of the Absence of the Increase in the Proliferative Effect of a PDGF-BB with a Dextran Derivative Not Included in Formula I

According to a protocol identical to that described in Example 1, the increase of the effect of PDGF-BB by a DMCBSu of general formula in which a = 0.81; b = 0.14; c = 0.19 (prepared according to the method described in the international application WO 99/29734), at different concentrations, was verified by incorporation of tritiated thymidine (5000 cells / well in 100 .mu.l).

The results obtained are shown in FIG. 2, the ratio R is equal to 1.2.

There is no increase in the effect of PDGF-BB. EXAMPLE 3 Demonstration of the Increase in the Proliferative Effect of a PDGF-BB with a Dextran Derivative Included in Formula I

According to a protocol identical to that described in Example 1, the increase of the effect of PDGF-BB by a DMCBSu of general formula in which a = 0.66; b = 0.38; c = 0.95 (prepared according to the process described in international application WO 99/29734), at different concentrations, was verified by incorporation of tritiated thymidine (5000 cells / well in 100 μl).

The results obtained are shown in FIG. 3, the ratio R is equal to 1.6. An increase in the effect of PDGF-BB is observed.

Claims

A pharmaceutical composition characterized in that it comprises, in a pharmaceutically acceptable carrier; at least one platelet-derived growth factor (PDGF), and

DMC _a B _b Su _c (I) in which: - D represents a polysaccharide chain,

MC represents methylcarboxylic groups,

B represents N-benzylmethylenecarboxamide groups,

- Su represents sulfate groups,

2. Composition according to claim 1, characterized in that D is constituted by chains of glucosidic units.

3. Composition according to claim 1 or 2, characterized in that D has a molar mass of between 1,000 and 2,000,000 Da,

4. Composition according to any one of the preceding claims, characterized in that the dextran derivatives are chosen from compounds of formula (I) in which b is greater than or equal to 0.35.

5. Composition according to Claim 4, characterized in that the dextran derivatives are chosen from compounds of formula (I) in which a is between 0.5 and 0.8, and c is between 0.1 and 0.5.

6. Composition according to any one of the preceding claims, characterized in that the dextran derivatives of formula (I) are chosen from compounds in which a = 0.66; b = 0.38 and c is between 0.2 and 0.4.

7. Composition according to Claim 6, characterized in that the dextran derivatives of formula (I) in which a = 0.66; b = 0.38 and c = 0.29.

8. Composition according to any one of the preceding claims, characterized in that the amount of dextran derivatives of formula (I) is between 0.5 and 100 mg / g of composition.

9. Composition according to Claim 8, characterized in that the amount of dextran derivatives of formula (I) is between 5 and 50 mg / g of composition.

10. Composition according to any one of the preceding claims, characterized in that the PDGFs are chosen from recombinant human PDGFs comprising two B chains.

11. Composition according to any one of the preceding claims, characterized in that the amount of PDGF is between 1 and 200 μg / g of composition.

12. Composition according to Claim 11, characterized in that the amount of PDGF is between 10 and 100 μg / g of composition.

13. Composition according to any one of the preceding claims, characterized in that the weight ratio derived from dextran of formula (I) ZPDGFs is between 100 and 1000.

14. Composition according to any one of the preceding claims, characterized in that it is a topically applied composition in the form of gels, creams, sprays or patches.

15. Composition according to claim 14, characterized in that it is in the form of a cellulose gel.

16. Composition according to any one of the preceding claims, characterized in that it contains one or more additives selected from fillers, preservatives, antioxidants, stabilizing agents, acidifying and alkalizing agents and opacifiers.

17. Use of at least one pharmaceutical composition as defined in any one of claims 1 to 16, for the preparation of a medicament with healing action.

18. Use according to claim 17, characterized in that the drug is intended for the treatment of ulcers topically.

19. Use according to claim 17, characterized in that the drug is intended for the treatment of ulcers of the lower limbs of diabetic patients.

20. Use according to any one of claims 17 to 19, characterized in that said medicament is intended to be administered in a course of 2 to 10 weeks at a rate of 1 to 2 applications per day on the injured part.