INJECTABLE COMPOSITION FOR SKIN AND SOFT TISSUE AUGMENTATION

Abstract

The present invention relates to the synergistic effect of calcium hydroxyapatite particles and hyaluronic acid on the bio-stimulation. The invention refers to an injectable composition comprising one or more types of calcium hydroxyapatite particles and hyaluronic acid and to a method for preparing such injectable composition. Furthermore, it relates to a cosmetic method for improving appearance of the skin and/or contour of a subject by means of the injectable composition. Moreover, the invention refers to the injectable composition for use in a method of treating a pathologic condition associated with pathologic deterioration of connective tissue.

Claims

1-15. (canceled)

16. An injectable composition comprising: (A) one or more types of calcium hydroxyapatite particles as component A; (B) hyaluronic acid as component B comprising: (B1) one or more types of high-molecular weight hyaluronic acid of a mean molecular weight in the range of from 1 to 5 MDa as component B1, or (B2) one or more types of low-molecular weight hyaluronic acid of a mean molecular weight in the range of from 5 to 100 kDa as component B2, or (B3) a combination of components B1 and B2; (C) one or more pharmaceutically acceptable carriers as component C comprising at least one viscous or liquid carrier; (D) optionally, one or more local anesthetics as component D; and (E) optionally, one or more pharmaceutically acceptable additives other than components A, B, C, and D as component E, wherein the weight ratio of components A:B, referred to the dry matters of components A and B, is in the range of from 1000: 1 to 1:10.

17. The injectable composition of claim 16, wherein the injectable composition comprises hyaluronic acid of components B1 and B2 and has a weight ratio of components B1:B2, referred to the dry matters of components B1 and B2, in the range of from 1000:1 to 1:1.

18. The injectable composition of claim 16, wherein the weight ratio of components A:B, referred to the dry matters of components A and B, is in the range of from 100: 1 to 1:10, or of from 50:1 to 1:5, or of from 20:1 to 1:4, or of from 10:1 to 1:3, or of from 5:1 to 1:2, or of from 2:1 to 1:1.

19. The injectable composition of claim 16, wherein the hyaluronic acid of component B1, if present, is non-crosslinked hyaluronic acid and component B2, if present, is non-crosslinked hyaluronic acid.

20. The injectable composition of claim 16, wherein component B1 has a mean molecular weight in the range of from 1.2 to 3.5 MDa, or of from 1.4 to 3.2 MDa, or of from 1.6 to 3.0 MDa, or of from 1.8 to 2.8 MDa, or of from 2.0 to 2.6 MDa, or of from 2.0 to 2.6 MDa, or of from 2.2 to 2.4 MDa.

21. The injectable composition of claim 16, wherein component B2 has a mean molecular weight of in the range of from 5 to 80 kDa, or of from 5 to 60 kDa, or of from 5 to 50 kDa, or of from 5 to 45 kDa, or of from 5 to 40 kDa.

22. The injectable composition of claim 16, wherein the calcium hydroxyapatite particles have a mean particle size of from 1 to 150 μm, or of from 2 to 100 μm, or of from 5 to 80 μm, or of from 10 to 60 μm, or of from 15 to 50 μm, or of from 20 to 45 μm.

23. The injectable composition of claim 16, wherein the injectable composition comprises one or more pharmaceutically acceptable carriers as component C.

24. The injectable composition of claim 16, wherein the injectable composition consists of: (A) 10 to 80% by weight, referred to dry matter, based on the composition, of one or more types of calcium hydroxyapatite particles having a mean particle size of 1 to 150 μm as component A; (B) 0.1 to 50% by weight, referred to dry matter, based on the composition, of hyaluronic acid consisting of: (B1) 50 to 100% by weight, referred to dry matter, based on the total content of hyaluronic acid, of one or more types of high-molecular weight hyaluronic acid of a mean molecular weight in the range of from 1 to 5 MDa as component B1; and (B2) 0 to 50% by weight, referred to dry matter, based on the total content of hyaluronic acid, of one or more types of low-molecular weight hyaluronic acid of a mean molecular weight in the range of from 5 to 100 kDa as component B2; (C) 1 to 80% by weight, based on the composition, of one or more pharmaceutically acceptable carriers as component C comprising at least one viscous or liquid carrier; (D) 0 to 10% by weight, based on the composition, of one or more local anesthetics as component D; and (E) 0 to 10% by weight, based on the composition, of one or more pharmaceutically acceptable additives other than components A, B, C, and D as component E.

25. A cosmetic method for improving appearance of the skin and/or contour of a part of interest of the face or body of a subject, the method including the following steps: (i) providing components A, B, C, optionally D, and optionally E, wherein the components are defined as in claim 16; and (ii) injecting the components into the skin of the part of interest of the face or body of a subject.

26. The cosmetic method of claim 25, wherein the method includes the following steps: (i) providing an injectable composition; and (ii) injecting the injectable composition into the skin of the part of interest of the face or body of a subject, wherein the injectable composition comprises: (A) one or more types of calcium hydroxyapatite particles as component A; (B) hyaluronic acid as component B comprising: (B1) one or more types of high-molecular weight hyaluronic acid of a mean molecular weight in the range of from 1 to 5 MDa as component B1, or (B2) one or more types of low-molecular weight hyaluronic acid of a mean molecular weight in the range of from 5 to 100 kDa as component B2, or (B3) a combination of components B1 and B2; (C) one or more pharmaceutically acceptable carriers as component C comprising at least one viscous or liquid carrier; (D) optionally, one or more local anesthetics as component D; and (E) optionally, one or more pharmaceutically acceptable additives other than components A, B, C, and D as component E, wherein the weight ratio of components A:B, referred to the dry matters of components A and B, is in the range of from 1000: 1 to 1:10.

27. The cosmetic method of claim 25, wherein the method is further characterized in that it is a method for a purpose selected from the group consisting of filling of wrinkles, improving facial lines, breast reconstruction or augmentation, rejuvenation of the skin, buttocks augmentation, remodeling of cheekbones, soft-tissue augmentation, filling facial wrinkles, improving glabellar lines, improving nasolabial folds, improving marionette lines, improving buccal commissures, oral commissures, improving peri-lip wrinkles, improving crow's feet, improving subdermal support of the brows, malar and buccal fat pads, improving tear troughs or nose, augmentation of lips, augmentation of cheeks, augmentation of peroral region, augmentation of scars such as acne scars, augmentation of infraorbital region, resolving facial asymmetries, improving jawlines, augmentation of chin, and combinations of two or more thereof.

28. The cosmetic method of claim 25, wherein the step (ii) is injecting the injectable composition in connective tissue of the subdermal skin and thereby stimulating the production of collagen.

29. The injectable composition of claim 16 for use in a method of treating a pathologic condition associated with pathologic deterioration of connective tissue.

30. A method for preparing an injectable composition, wherein the method comprising the steps of: (i) providing: (a) one or more solutions comprising one or more types of calcium hydroxyapatite particles as component a; (b) one or more solutions comprising one or more types of non-crosslinked hyaluronic acid as component b comprising: (B1) one or more types of non-crosslinked high-molecular weight hyaluronic acid of a mean molecular weight in the range of from 1 to 5 MDa as component B1, or (B2) one or more types of non-crosslinked low-molecular weight hyaluronic acid of a mean molecular weight in the range of from 5 to 100 kDa as component B2, or (B3) a combination of components B1 and B2; and (c) optionally, one or more further components selected from the group consisting of one or more pharmaceutically acceptable carriers as component C, one or more local anesthetics as component D, one or more other pharmaceutically acceptable additives as component E, and a combination of two or more thereof; and (ii) mixing components a and b, and optionally, c, to obtain the injectable composition, wherein the injectable composition induces collagen production in the skin cells than component a, calcium hydroxyapatite particles, or component b, non-crosslinked hyaluronic acid, alone.

31. The injectable composition of claim 23, wherein the one or more pharmaceutically acceptable carriers comprise one or more components selected from the group consisting of one or more polysaccharide derivatives other than hyaluronic acid or pharmaceutically acceptable salts thereof, one or more polysaccharides or pharmaceutically acceptable salts thereof, glycerol, and combinations or two or more thereof.

32. The cosmetic method of claim 27, wherein the method is a method for filling of wrinkles of interest of a subject comprising the following steps: (i) providing an injectable composition; and (ii) injecting the injectable composition subcutaneously or intradermally into the wrinkles of interest, wherein the injectable composition comprises: (A) one or more types of calcium hydroxyapatite particles as component A; (B) hyaluronic acid as component B comprising: (B1) one or more types of high-molecular weight hyaluronic acid of a mean molecular weight in the range of from 1 to 5 MDa as component B1, or (B2) one or more types of low-molecular weight hyaluronic acid of a mean molecular weight in the range of from 5 to 100 kDa as component B2, or (B3) a combination of components B1 and B2; (C) one or more pharmaceutically acceptable carriers as component C comprising at least one viscous or liquid carrier; (D) optionally, one or more local anesthetics as component D; and (E) optionally, one or more pharmaceutically acceptable additives other than components A, B, C, and D as component E, wherein the weight ratio of components A:B, referred to the dry matters of components A and B, is in the range of from 1000: 1 to 1:10.

33. The cosmetic method of claim 28, wherein the collagen is selected from collagen type III, collagen type I, or a combination of collagen type I and III.

34. The injectable composition of claim 29, wherein the pathologic condition is selected from the group consisting of urinary incontinence, vesicoureteral reflux, vocal cord augmentation, lipoatrophy, a pathologic condition associated with age-related or pathologic deterioration of connective tissue, and combinations of two or more thereof.

35. The injectable composition of claim 29, wherein the lipoatrophy is lipoatrophy in a patient suffering from human immunodeficiency virus (HIV).

Description

EXAMPLES

[0141] Materials

[0142] Calcium Hydroxyapatite Particles (CaHA):

[0143] CAS No. 1306-06-05 as used in the product RADIESSE®, K100086592, spherical microspheres, size distribution between >10 and <100 μm (as determined by laser diffraction), mean particle size 22-24 μm with a D-ratio of 0.93 (as determined by laser diffraction), material density approximately 3.1 g/cm.sup.3;

[0144] HA High:

[0145] High-molecular weight sodium hyaluronate having a molecular weight of 2.3 MDa, non-crosslinked, intrinsic viscosity 2.41 m.sup.3/kg (25° C., EP monograph method, Ubbelohde viscometer), HYALURONATE Na F100 (htl Biotechnology, France);

[0146] HA Low:

[0147] low-molecular weight sodium hyaluronate having a molecular weight range of 5 to 40 kDa, non-crosslinked;

[0148] Fibroblasts: fibroblastic cells (adult/single donor/breast (PromoCell, #412Z020-P3); Fibroblast growth medium: cell culture medium including 1 mM vitamin C and 1% by weight of PenStrep (penicillin-streptomycin);

[0149] Anti-Collagen III Antibody:

[0150] polyclonal antibody, rabbit, used as primary antibody (Invitrogen, PA5-34787);

[0151] Anti-Rabbit Antibody:

[0152] AlexaFluor488-labeled secondary antibody detecting the primary rabbit antibody (Invitrogen, A11034);

[0153] DAPI: 4′,6-diamidino-2-phenylindole (SIGMA, D9542); and

[0154] CellMask: deep red plasma membrane strain (Invitrogen, C10046).

[0155] Preparation of Hyaluronic Acid Solutions

[0156] A stock solution of low-molecular weight sodium hyaluronate (HA low) in fibroblast growth medium at a concentration of 20 mg/ml was prepared. Furthermore, a stock solution of high-molecular weight sodium hyaluronate (HA high) in fibroblast growth medium at a concentration of 2 mg/ml was prepared. At higher concentrations, undesired gelling occurred. These solutions were than further diluted in fibroblast growth medium to the desired concentrations and optionally mixed with each other.

[0157] Cell Culture and Sample Preparation

[0158] Fibroblasts were seeded at a density of 5000 cells per well. The cells were cultivated for 24 hours at standard conditions at 37° C. in fibroblast growth medium. After 24 hours, 200 μl of the hyaluronic acid-containing samples were added. The samples contained different amounts of hyaluronic acid. Some samples further contained 2 mg/ml calcium hydroxyapatite particles (CaHA). The treated cells were further incubated at standard conditions at 37° C. for 72 hours. After 72 hours, the medium was removed from the cells and the cells were fixed with cold methanol (−20° C.) for 10 minutes. Then, the fixed cells were washed three times with PBS (phosphate buffered saline) and stored at 4° C.

[0159] Collagen III Staining and Determination

[0160] The supernatant of the fixed cells was removed. Then, the cells were treated with 100 μl/well of a blocking buffer (5% by weight of albumin in PBS) for 2 hours at room temperature (RT). Subsequently, 70 μl/well of a solution of 6.7 μg/ml of the primary anti-collagen III antibody in Dako antibody solution (1:100) were added and incubated overnight in the dark at 4° C. on a horizontal mixer.

[0161] Subsequently, the treated fixed cells were washed three times with PBS. Subsequently, 70 μl/well of a solution of 10 μg/ml of the secondary AlexaFluo488-labeled anti-rabbit antibody in Dako antibody solution (1:200) were added and incubated for 1 hour at RT in the dark. The treated fixed cells were washed three times with PBS.

[0162] Subsequently, 70 μl/well of CellMask deep red plasma membrane strain was added in a dilution of 1:1000 in PBS (5 μg/ml). The fixed cells were incubated for 30 minutes at RT in the dark. Subsequently, 70 μl/well of a solution of 1 μg/ml DAPI solution in PBS (1:2000 dilution of an aliquot of 2 mg/ml) were added. The fixed cells were incubated for 10 minutes at RT in the dark. The treated fixed cells were washed three times with PBS.

[0163] The fluorescence signal was determined at an Imager for quantification of the signals. Furthermore, microscopic images were prepared. The results are depicted below.

[0164] Results

[0165] The results of collagen III production are shown in the following table. Herein, the synergistic effect of calcium hydroxyapatite particles (CaHA) and high-molecular weight hyaluronic acid (HA high) and low-molecular weight hyaluronic acid (HA low) was observed.

TABLE-US-00001 TABLE 1 Synergistic effect of the combination of CaHA with HA low or HA high, respectively (n > 3, percentage expression to an untreated control, rounded values) Collagen III Example/ cell count expression Comparative HA low HA high CaHA after 72 hours after 72 hours Example [mg/ml] [mg/ml] [mg/ml] [% of control] [% of control] 1 0.02 113.8 125.6 2 0.02 2 99.3 157.9 3 2 115.6 128.3 4 2 2 102.9 148.5 5 20 86.3 124.0 6 20 2 80.6 154.4 7 0.02 108.0 125.0 8 0.02 2 93.8 152.0 9 0.2 101.9 124.8 10 0.2 2 87.4 156.7 11 2 99.3 121.5 12 2 2 66.1 158.4 13 1 92.2 110.5 14 2 84.6 115.6 15 5 84.9 126.7

[0166] These data show the synergistic effect of hyaluronic acid (HA) and calcium hydroxyapatite particles (CaHA).

[0167] When adding HA alone, the collagen III Expression decreased with increasing HA concentration (cf. Comparative Examples 1, 3 and 5, as well as 7, 9 and 11). CaHA alone had a low effect with increasing CaHA concentration (cf. Comparative Examples 13-15). In contrast, the samples containing a combination of HA and CaHA showed a significant increase in collagen III expression.

[0168] Synergistic effects on collagen III expression were observed for the whole wide concentration range tested. Best synergistic effect on collagen III expression for HA low were observed for a combination of CaHA with 0.02 mg/ml of HA low. Best synergistic effect on collagen III expression for HA high were observed for a combination of CaHA with 2 mg/ml of HA high. The compositions of CaHA and HA maintained the vast majority of cells viable.

[0169] In a further independent experiment, lower concentrations of hyaluronic acid (HA) were tested. Thus, the CaHA:HA ratio was increased. The results are depicted in Table 2.

TABLE-US-00002 TABLE 2 Relative collagen III expression induced by a combination of CaHA with HA low or HA high, respectively (percentage expression to an untreated control, rounded values) Example/ Collagen III Comparative HA low HA high CaHA Expression Example [mg/ml] [mg/ml] [mg/ml] [% of control] 16 2 112.1 17 0.00002 95.7 18 0.00002 2 113.0 19 0.0002 95.1 20 0.0002 2 109.8 21 0.002 94.4 22 0.002 2 110.7 23 0.00002 99.5 24 0.00002 2 114.3 25 0.0002 97.0 26 0.0002 2 114.0 27 0.002 98.5 28 0.002 2 111.0

[0170] At such low concentrations of hyaluronic acid (HA), only a very low or no synergistic effect of HA and CaHA was achieved. HA low alone had a negative effect on collagen III expression. This result confirms the findings in the art (cf. Croce et al., Tissue & Cell, 2001, 33:326-331). This undesired effect was remedied by addition of CaHA.

[0171] Furthermore, different combinations of HA low and HA high were tested. The results are depicted in Table 3.

TABLE-US-00003 TABLE 3 Relative collagen III expression induced by a combination of CaHA with different combinations of HA low and HA high (percentage expression to an untreated control, rounded values) Collagen III Example/ Collagen III Expression Comparative HA low HA high CaHA Expression [% microspheres Example [mg/ml] [mg/ml] [mg/ml] [% of control] only of control] 29 0.002 0.002 97.9 87.9 30 0.02 0.002 96.5 86.6 31 0.2 0.002 99.7 88.9 32 2 0.002 99.3 89.2 33 0.002 0.02 94.8 85.2 34 0.02 0.02 97.9 87.9 35 0.2 0.02 98.0 88.0 36 2 0.02 96.0 86.2 37 0.002 0.2 98.8 88.7 38 0.02 0.2 100.4 90.1 39 0.2 0.2 99.1 88.9 40 2 0.2 97.1 87.2 41 0.002 2 93.6 84.0 42 0.02 2 97.0 87.0 43 0.2 2 98.9 88.8 44 2 2 99.1 89.0 45 0.002 0.002 2 107.8 96.8 46 0.02 0.002 2 103.9 93.3 47 0.2 0.002 2 103.9 93.3 48 2 0.002 2 102.6 92.1 49 0.002 0.02 2 100.5 90.2 50 0.02 0.02 2 106.1 95.2 51 0.2 0.02 2 109.9 98.7 52 2 0.02 2 104.8 94.1 53 0.002 0.2 2 108.6 97.5 54 0.02 0.2 2 111.1 100.0 55 0.2 0.2 2 112.0 100.6 56 2 0.2 2 110.3 99.0 57 0.002 2 2 123.0 110.4 58 0.02 2 2 129.1 115.9 59 0.2 2 2 131.6 118.2 60 2 2 2 133.1 119.5

[0172] This experiment confirmed the presence of a synergistic effect of a combination of CaHA and HA. The synergistic effect occured in wide concentration ranges. It was found that HA high has a higher synergistic effect than HA low. Highest synergistic effects were found for a combination of 2 mg/ml of HA high and 0.02 mg/ml to 2 mg/ml of HA low.

[0173] Furthermore, microscopic images with collagen staining were generated. It was found that collagen is deposited around fibroblasts which are in contact with the calcium hydroxyapatite particles. This indicated that the present composition is a particularly suitable dermal filler. Fibroblasts invading the dermal filler materials can deposit collagen in the filled area.

[0174] In summary, synergistic effects of a combination of CaHA and HA were shown in a number of experiments.

[0175] These results indicate that the compositions according to the invention are particularly well suitable for increasing expression of collagen and, thus, provide particularly suitable dermal and soft-tissue fillers.