Pharmaceutical compositions containing hyaluronic acid for use in the treatment of black disc disease

Abstract

The present invention describes and claims pharmaceutical compositions in gel form for use in the treatment of intervertebral disc degeneration, in particular the forms of dehydration and emptying of the nucleus pulposus known as black disc disease. These compositions comprise a hyaluronic acid derivative which forms hydrogels with precise rheological characteristics that make it ideal for filling the nucleus pulposus.

Claims

1. Method of treating black disc disease, which comprises hydrating a nucleus pulposus by administering to a patient in need thereof an effective amount of a hydrogel of hyaluronic acid hexadecylamide; wherein the hyaluronic acid has an average molecular weight MW ranging between 500,000 and 730,000 Da and a degree of molar amidation ranging between 1% and 3%, and the concentration of the hyaluronic acid hexadecylamide is 8 mg/ml.

2. The method according to claim 1, wherein the hydrogel further comprises pharmaceutically or biologically active substances.

3. The method according to claim 2, wherein pharmaceutically or biologically active substances are chosen from steroidal and non-steroidal anti-inflammatory drugs, cytokine inhibitors and local anaesthetics.

4. The method according to claim 1, which comprises treating pain in black disc disease.

5. The method according to claim 1, correctively treating the pelvic angle of incidence and the postural alterations connected with black disc disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention relates to pharmaceutical compositions in the form of hydrogels for use in the treatment of intervertebral disc degeneration, in particular the forms of dehydration and emptying of the nucleus pulposus known as black disc disease. In particular the pharmaceutical compositions according to the present invention are effective in the treatment of pain in black disc disease and in the treatment required to correct the pelvic incidence angle and postural alterations associated with black disc disease.

(2) Said compositions comprise a hyaluronic acid derivative which forms hydrogels with precise rheological characteristics that make it ideal for filling the nucleus pulposus. Specifically, the HA derivative used is hexadecylamide.

(3) As previously stated, HA can have a MW ranging from 50 to 1310.sup.6 Da. The HA used in the present invention can derive from any source, such as extraction from rooster combs (EP138572), fermentation (from Streptococcus equi or zooepidemicus, EP716688), or biosynthesis (from Bacillus, WO2012032153, WO 2012032154), and have a weight average molecular weight ranging between 400 and 310.sup.6 Da, in particular between 10.sup.5 Da and 10.sup.6 Da, and even more particularly between 500,000 and 730,000 Da.

(4) The amide derivative used in the present invention is prepared from the latter fraction.

(5) Hyaluronic acid amides are known to the skilled person; for example, the preparation of a wide range of hyaluronic acid amides (benzyl, octyl, dodecyl, etc.) is disclosed in EP1095064.

(6) Only hexadecylamide prepared as described below is used according to the present invention. Briefly, hyaluronic acid pre-derivatised to a tetrabutylammonium salt (TBA) is solubilised in dimethylsulphoxide (DMSO), and methanesulphonic acid is added to the resulting solution. Carbonyldiimidazole is then added, and the mixture is left to react under stirring for one hour at room temperature. Hexadecylamine is then added, and the mixture is left to react for about 16-24 hours at 40-42 C. A saturated solution of NaCl is then added to stop the reaction, and absolute ethanol is added to isolate the derivative by precipitation. The precipitate is washed first with a mixture of water and ethanol and then with ethanol alone, and finally dried under high vacuum.

(7) The degree of derivatisation is modulated by varying the quantity of the reagents used and adapting the reaction times; the degree of derivatisation can be measured by methods known to the prior art, such as HPLC. In the ambit of the present invention, the degree of amidation used ranges from 0.1% to 10% molar, preferably from 1% to 3% molar, measured by HPLC after hydrolysis of the amide and conjugation of the hexadecylamine released with a fluorophoric substance.

(8) Starting with the derivative thus obtained, a hydrogel can be formulated at a concentration ranging from 0.1 to 30 mg/ml, preferably 3 to 20 mg/ml, and even more preferably 5 to 15 mg/ml. The carriers preferably used are saline solution or phosphate buffer.

(9) In the preferred pharmaceutical composition according to the present invention, the hyaluronic acid has an average MW ranging between 500,000 and 730,000 Da, the degree of molar amidation ranges between 1% and 3%, and the hyaluronic acid hexadecylamide concentration ranges between 5 and 15 mg/ml.

(10) The hydrogel obtained undergoes a sterilisation process according to known techniques, for example in an autoclave, after being introduced into prefilled disposable syringes.

(11) Regardless of the concentrations, the hydrogel obtained is easily extrudable, viscous to ensure that it remains in situ after application, elastic to ensure that it absorbs and redistribute loads, and highly hydratable; it also retains the biological characteristics of the starting polymer, hyaluronic acid, and is therefore biocompatible, bioresorbable and totally harmless to the body, both in its unmodified state and after enzymatic degradation.

(12) In view of its particular characteristics, hydrogel can be associated with biologically or pharmacologically active substances (such as steroidal and non-steroidal anti-inflammatory drugs, cytokine inhibitors and local anaesthetics) deemed able to improve the symptoms of black disc. These substances can be inserted in the syringe on an extempore basis before application to the patient, so that the hydrogel functions both as a treatment for black disc and as a carrier of active substances.

(13) Some preparation examples are set out below, for descriptive purposes only.

Example 1

(14) Preparation of Hexadecylamide Derivative of HA with a Weight Average Molecular Weight Ranging Between 500 and 730 kDa and a Molar Amidation Content Ranging Between 1 and 3%

(15) 2 g of HA pre-derivatised to a TBA salt is solubilised in 200 ml of DMSO, and 64 A of methanesulphonic acid is added to the resulting solution; 52 mg of 1-1-carbonyldiimidazole is then added and left to react under gentle stirring for one hour at room temperature. 544 mg of hexadecylamine is then added, and the amidation reaction is conducted for 16-24 hours at 42 C. A saturated solution of NaCl is then added to stop the reaction, and 1.5 volumes of absolute ethanol are added after 15-30 minutes to isolate the derivative by precipitation. The precipitate is washed several times in 80:20 ethanol/water and then in ethanol alone, and finally dried under high vacuum at 40 C.

(16) 1.2 g of hexadecylamide derivative is obtained, whose degree of amidation, measured by HPLC, is about 2-3% molar.

Example 2

(17) Preparation of a Hydrogel of the HA Hexadecylamide Derivative at the Concentration of 8 mg/ml, Obtained as Described in Example 1

(18) 2 g of HA hexadecylamide derivative, obtained as described in Example 1, is placed in a suitable recipient, and 250 ml of phosphate buffer (PBS) at pH 6.9 is added. The buffer contains 8.5 mg/ml NaCl, 0.45 mg/ml Na.sub.2HPO.sub.412H.sub.2O, and 0.11 mg/ml NaH.sub.2PO.sub.42H.sub.2O.

(19) The mixture is left under stirring for at least 2 hours at room temperature, and for about 1 hour at 60 C. After this time, the T is returned to 20-25 C., and the mixture is left under stirring for 2-4 hours. The resulting mixture, which contains 8 mg/ml of hyaluronic acid hexadecylamide, is divided between glass syringes, which then undergo a damp heat sterilisation cycle (10 minutes) at about 121 C.

Example 3

(20) Treatment of Patients Suffering from Black Disc Disease with HA Hexadecylamide vs. Ozone: Pilot Study

(21) By means of an NMR scan used for other diagnostic purposes, 11 patients with black disc disease at lumbar level (L3) were identified and treated according to the following protocol.

(22) Materials and methods: anaesthetic solution (1% carbocaine); ozone 27 ; HA hexadecylamide hydrogel prepared as described in Example 2 (8 mg/ml); technique: guided radioscopic infiltration, after cutaneous anaesthesia by infiltration; the puncture site was identified by observing the lumbar region in the two orthogonal projections;

(23) The patients were divided into two groups:

(24) A) 6 patients treated with 0.5 cc of hydrogel;

(25) B) 5 patients treated with 3 cc of ozone 27 (control).

(26) After treatment the patients were made to rest for several hours, and discharged with a paracetamol-based painkilling treatment if necessary. All the patients underwent a disability evaluation (RMDQRoland Morris Disability Questionnaire) and a pain evaluation (VASVisual Analogue Scale) and, after 6 months, a sagittal NMR scan to evaluate any variations in the pelvic incidence angle.

(27) Results:

(28) RMDQ: all patients in both groups reported a reduction in disability in terms of mobility and an improved quality of life, of comparable extents;

(29) VAS scale: once again, all patients reported a definite reduction in pain, and this result, while expected for the group treated with ozone, is certainly surprising for Group A, which was treated with the derivatised HA hydrogel;

(30) X-ray evaluation: in the patients in Group A, normalisation of the disc was observed; it appeared paler, and therefore hydrated, on the NMR scan, and very similar to the untreated healthy discs. The patients in Group A also presented a definite improvement in the pelvic incidence angle.

(31) However, none of these functional effects were observed in the patients in Group B, treated with ozone: not only was there no improvement (normalisation) in the appearance of the disc, which actually worsened in 3 cases, but the treatment had no effect on the pelvic incidence angle.

CONCLUSIONS

(32) Although this is just one pilot study with a limited number of patients, it is evident that treatment with hyaluronic acid hexadecylamide has a surprisingly favourable effect not only on the symptoms, but above all on the functions, of patients suffering from black disc disease.

(33) The data presented here unequivocally demonstrate that treatment with hyaluronic acid hexadecylamide not only has a painkilling effect comparable with that of ozone, but above all has an unexpected curative effect, which restores the hydration of the nucleus pulposus and corrects the postural defect manifested by all patients suffering from black disc disease.