EA044480B1 - PHARMACEUTICAL COMPOSITION FOR TREATMENT OF POSTOPERATIVE PAIN AND METHOD FOR PREPARING SUCH COMPOSITION - Google Patents

Description

The invention relates to pharmaceutical compositions in the form of a gel containing a local amide anesthetic.

Prior Art

Postoperative pain is a complex response to tissue trauma caused by surgery, which causes hypersensitivity of the central nervous system. Postoperative pain can occur after any surgical procedure and increases the risk of postoperative complications, which also affects the patient's recovery and return to daily activities. The healing process is significantly slower if the patient is unable to move, eat or sleep without feeling pain, since pain is also felt in areas distant from the surgical site. In addition, pain causes a state of psychological prostration in the patient, which further worsens the overall clinical picture. Careful post-operative pain management is very important nowadays and the World Health Organization strongly supports it as it considers its provision as a fundamental right for patients.

The perception of pain depends not only on the type of surgery (abdominal and thoracic surgery are more painful than procedures involving the extremities, and incisions in the abdomen or chest cause even more pain when the peritoneum or pleura are involved) but also on subjective conditions , such as previous painful experiences, anxiety and genetic predisposition; For this reason, medical personnel tend to administer pain-relieving measures by adapting them to the intensity reported by each patient, which can be correctly measured using a pain rating scale. The most widely used of the various scales available are unidimensional scales, which are easy to administer and in which the only parameter considered is the amount of pain felt by the patient. The best known is the NRS (Downie et al. quantitative rating scale, Ann. Rheum. Dis., 1978, 378-381), where the operator asks patients to verbalize the amount of pain they experience on a scale from 0 to 10 (0 corresponds to no pain; 10 corresponds to maximum pain), and a scale (visual analogue scale - Scott, Huskisson, Pain, 1976, 2, 175-184), where the perceived pain is indicated by the patient with a mark on a line resembling a ruler, the length of which is 10 cm. This the latter scale is widely used due to its ease of understanding by patients, including pediatric patients. For patients who are unable to express their feelings, or patients who suffer from comorbid disorders that interfere with correct verbal and/or motor representation, other more complex scales are used.

Postoperative pain relief depends on pharmacological treatment. To relieve pain during and immediately after surgery, medications are usually given intravenously; The drugs in question are usually opioids, the use of which is limited to fairly short periods of time due to their serious side effects.

When the patient is able to take them, and the overall clinical picture allows this, then oral administration of drugs such as paracetamol, traditional non-steroidal anti-inflammatory drugs (NSAIDs) or cyclooxygenase-2 (COX-2), the side effects of which are well known, is carried out. Another approach to treating postoperative pain involves nerve blocks using local anesthetics. Depending on the type of surgery, regional analgesia can be peripheral, when the anesthetic is injected locally along the nerve, or central, when the anesthetic is injected close to the roots of the posterior spinal nerves. Peripheral analgesia relieves pain in a precisely defined area of the body innervated by a specific anesthetized nerve, such as an arm or leg, and is usually achieved by performing intradermal injections near the blocked nerve; central analgesia has a much broader effect and is achieved by injection into the epidural space through catheters or pumps, which can be left in situ for several days after surgery if necessary. However, such treatment always begins after surgery.

Typically, the most used local anesthetics in these cases are lidocaine, bupivacaine, etidocaine, ropivacaine and the like, generally called amide local anesthetics. These lipophilic molecules have pharmacological activity in their base form, which is able to penetrate nerve cell membranes, but are poorly soluble in water, and are therefore available on the market in their protonated and salt forms (usually hydrochloric acid), which are less active. Protonation occurs at the tertiary nitrogen atom, which acquires a partially positive charge and is therefore able to bind to an anion (usually a chloride, as already mentioned). The effect of local or central analgesia using local anesthetics has a very short duration due to the rapid absorption of the active ingredient into the bloodstream; The more vascularized the treated area, the faster the absorption. This can be corrected, especially in the case of peripheral blockade, by combining a local anesthetic with a vasoconstrictor. In this case, however, repeated administrations at relatively short intervals are necessary. In addition, due to their fat solubility, there is a tendency

- 1 044480 accumulation of local amide anesthetics in the fat present in the tissues, and therefore repeated administrations or higher doses can easily cause not only forms of dependence, but also significant side effects that can be neurological (drowsiness, feeling of intoxication, tinnitus , visual disturbances, agitation to the point of convulsions, coma and cardiorespiratory depression) or cardiac in nature (rhythm disturbances, ventricular extrasystole, ventricular and supraventricular tachycardia and conduction disturbances). Therefore, in general, to achieve effective, long-lasting pain suppression while minimizing the risks associated with the local anesthetic, it is usually combined with an oral NSAID in clinical practice.

To improve the duration of the analgesic effect, combinations of an anesthetic with a vehicle from which it is released have been investigated.

In particular, combinations with hydrophobic carriers are known due to their affinity for the active form of the active ingredient; for example, an injectable composition has been developed based on biodegradable lipid microgranules containing bupivacaine, which is gradually released (Exparel®). The administration of such a composition can only be carried out by injection and often causes redness, itching and erythema at the injection site, but, above all, has a short-term effect, not exceeding 24 hours. Other attempts have been made using a matrix based on polylactic acid, polyglycolic acid and corresponding copolymers, polyorthoesters, etc., which promote the release of local anesthetic, in combination with an NSAID. In this case, significant toxicity problems and side effects can easily occur due to the simultaneous administration of two active ingredients (US2017035777). In addition, there is the possibility that the local anesthetic may somehow be retained in a vehicle that is chemically similar to it, with the risk of undesirable accumulation and subsequent alteration of the pharmacokinetic profile. To address this aspect, other attempts have been made using hydrophilic matrices, where the local anesthetic is enclosed in its hydrochloride form, which is less active but is commercially available and is similar in nature to the matrix; for example, US2010266693 describes combinations of bupivacaine HCl with a matrix based on hyaluronic acid and fibrinogen. Lu et al. (Arch. Med. Sci., 2013, 9, 614-621) describe the use of ropivacaine HCl together with hydrophilic polymers. Despite the fact that the hydrophilic matrix exhibits better biocompatibility, is able to easily incorporate the ropivacaine salt and does not cause an accumulation effect, the results obtained in terms of the effectiveness and duration of analgesia do not meet the requirements: after a strong unwanted initial burst, the analgesic effect weakens in a fairly short time. reasons already described.

Description of the invention

The present invention provides a pharmaceutical composition in the form of a gel for use in the treatment of postoperative pain, containing or consisting of a hydrophilic matrix consisting of a hyaluronic acid (HA) derivative containing a local amide anesthetic in the form of a base, and capable of releasing the local anesthetic in lipophilic and basic form in a prolonged manner, wherein the hyaluronic acid derivative is selected from:

amide derivative obtained by amidation of the carboxyl groups of hyaluronic acid and the amino groups of hexadecylamine, with an average degree of amidation varying in the range from 1 to 3 mol.%, in a concentration from 5 to 40 mg/ml and obtained from HA with average molecular weight (MW) , varying in the range from 500 to 730 kDa;

and the local amide anesthetic is selected from bupivacaine, etidocaine and ropivacaine at a concentration ranging from 10 to 35 mg/ml.

In one embodiment, the composition of the invention further contains pharmaceutically acceptable excipients.

In yet another embodiment of the invention, the local amide anesthetic is ropivacaine.

In yet another embodiment of the invention, the hyaluronic acid derivative is a hexadecylamide derived from hyaluronic acid with a weight average molecular weight ranging from 500 to 730 kDa, and having an average degree of amidation ranging from 1 to 3 mol%, and a concentration of 5 to 15 mg/ml, and the local anesthetic is ropivacaine base form at a concentration of 10 to 35 mg/ml.

In yet another embodiment of the invention, the concentration of hyaluronic acid hexadecylamide is in the range of 7 to 12 mg/ml.

In yet another embodiment of the invention, the concentration of hyaluronic acid hexadecylamide is 8 mg/ml.

In one embodiment, the composition of the invention is capable of releasing the local anesthetic in lipophilic and base forms in a sustained manner over a period of up to five days.

The present invention also provides a method for preparing a composition according to the invention, comprising:

- 2 044480 (a) solubilizing the local anesthetic in its salt form in a single portion or in several portions in an aqueous vehicle, preferably phosphate buffered saline (PBS);

(b) precipitating the local anesthetic in its base form by treating the solution obtained in step (a) with the base until a pH value ranging from 6.5 to 8 is achieved;

(c) adding a hyaluronic acid derivative to the resulting suspension. In one embodiment, the base is NaOH.

The present invention also provides the use of a composition according to the invention for the treatment of post-operative pain.

Detailed Description of the Invention

It has now been discovered that slow release of local amide anesthetic can be achieved by incorporating it in a lipophilic, basic form into a hydrophilic matrix containing a hyaluronic acid derivative; in this context, the base form means the unprotonated form.

Thus, the subject of the invention are pharmaceutical compositions in gel form containing a hydrophilic matrix consisting of a hyaluronic acid derivative and containing a local amide anesthetic in a basic form, and optionally containing known pharmaceutically acceptable excipients. The compositions of the invention are particularly useful for the treatment of postoperative pain resulting from surgery of any kind, especially after orthopedic surgery, and exhibit their analgesic activity for up to 5 days after application.

They can be administered during surgery in various ways, for example, by injection and/or infiltration into the tissue surrounding the surgical wound, by direct instillation into an open wound, or by simple local application and distribution. The analgesic effect persists for an unexpectedly long period of time (up to 5 days inclusive), especially if the composition is injected into the tissue surrounding the wound, in particular, wounds after orthopedic surgery.

The invention also relates to a method for preparing said pharmaceutical compositions, which involves adding a hyaluronic acid derivative to an aqueous suspension of a local anesthetic in an unprotonated base form obtained by neutralizing an aqueous solution of salts, in particular the hydrochloride salt of the local anesthetic, using suitable bases.

The method, which can be performed in a one-pot technique without isolating intermediates, retains the local anesthetic in its active, lipophilic and basic form in a hydrophilic matrix network consisting of a hyaluronic acid derivative.

Another subject of the invention is a controlled release system (drug delivery system) containing a matrix consisting of a hyaluronic acid derivative capable of releasing the local anesthetic in its base form in a controlled manner, and further containing pharmaceutically acceptable excipients known to the skilled person.

Local amide anesthetics include lidocaine, bupivacaine, etidocaine and ropivacaine. Ropivacaine is particularly preferred due to its pharmacotoxicological characteristics.

Hyaluronic acid (HA) is a straight-chain heteropolysaccharide consisting of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues, having a molecular weight (MW) ranging from 50,000 to 13x106 Da, depending on the source. from which it is obtained, and the methods of production used. As used herein, the term average molecular weight means the average molecular weight calculated by the intrinsic viscosity method (Terbojevich et al., Carbohydr. Res., 1986, 363-377).

Hyaluronic acid is ubiquitously present in the human body, where it exhibits a variety of activities, ranging from mechanical support of cells in many tissues such as skin, tendon, muscle and cartilage, to modulation of various numerous cellular physiological and biological processes (proliferation, migration, cell differentiation and angiogenesis), tissue hydration and joint lubrication. More recently, it has been demonstrated that HA also acts as an anti-inflammatory agent by modulating the release of pro-inflammatory cytokines, particularly interleukin-1 (IL-1), and is also able to bind to specific opioid receptors, mimicking the analgesic effect.

Due to its special chemical structure, HA can be subjected to various transformations through suitable chemical reactions to obtain derivatives that retain the biological characteristics of the parent polymer, but differ from it from a physicochemical point of view and are therefore suitable for numerous applications.

According to the invention, the hyaluronic acid derivative is selected from derivatives of the following groups:

auto-cross-linked derivatives obtained by internal esterification, with a degree of esterification not exceeding 20%, preferably from 0.05 to 10% and even more preferably

- 3 044480 specifically from 4 to 5%, and derived from HA with a mass average MW varying in the range from 160 to 230 kDa, preferably 200 kDa, hereinafter referred to as ACP® (EP341745);

cross-linked derivatives obtained by using cross-linking agents such as BDDE (1,4-butanediol diglycidyl ether), with a degree of derivatization ranging from 2.5 to 25 mol%, preferably from 5 to 15 mol% . (relative to the hyaluronic acid repeat unit), and derived from HA with a mass average MW varying in the range from 500 to 730 kDa, hereinafter referred to as HBC (EP2470230);

amide derivatives, namely, amides formed by the interaction between the carboxyl group of HA and the amino group of amines of the aliphatic, arylaliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series, with the degree of amidation varying in the range from 0.1 to 50%, and obtained from HA with a mass average MW varying from 500 to 730 kDa, as described in EP1095064.

Preferred are amide derivatives of the aliphatic series, in particular hexadecyl-, octadecyl- or dodecyl- amides, more preferably hexadecylamide derived from HA with a mass average MW varying in the range from 500 to 730 kDa, and having an average degree of derivatization (amidation) varying in in the range of 0.1 to 10 mol%, preferably 1 to 3 mol%, as determined using high performance liquid chromatography (HPLC) after hydrolysis of the amide and conjugation of the released hexadecylamine to the fluorophore. In the context of the present invention, a hexadecylamide having the above characteristics and an average degree of derivatization (amidation) ranging from 1 to 3 mol% is designated HYADD®-4, and its preparation is described in EP1853279.

The derivatives described above, especially hexadecylamide, are characterized by the ability to gel in aqueous solution even at low concentrations to form a stable gel. The resulting gel is extremely effective in holding the local anesthetic in base form within its network and releasing it gradually; the released anesthetic is very quickly converted to its protonated form (the surgical site is rich in acidic fluids as a result of inflammation caused by the surgical procedure and physiological tissue repair mechanisms) and as its activity wanes, it is gradually replaced by another base form of anesthetic released from the gel network . In addition, the rheological characteristics of the gel facilitate extrusion of the final product, making it possible to apply it using small gauge needles and reasonable force.

Combining a hydrophilic HA derivative, especially the amide derivative HYADD®-4, and a local amide anesthetic, particularly ropivacaine, in its basic lipophilic form without the addition of other active agents results in a surprisingly effective controlled release system; the local anesthetic in base form is released from the hydrophilic matrix continuously and continuously, creating an effective analgesic effect that is much longer lasting than that obtained using the saline form. In particular, the release of ropivacaine base was assessed using in vitro testing illustrated below, compared with an equivalent composition containing the hydrochloride form. The comparison demonstrates that the composition according to the invention exhibits its analgesic effect for up to 5 days inclusive.

The compositions of the invention are prepared by converting salts of the amide anesthetic, in particular ropivacaine hydrochloride, into the corresponding bases, which thus remain stably suspended in the HA derivative matrix in the form of a fine precipitate and are released continuously and gradually.

In schematic form, the following steps are used to prepare the compositions of the invention:

solubilizing the local anesthetic in its salt form in an aqueous vehicle such as saline or phosphate buffered saline (PBS), preferably PBS, in a single portion or multiple portions; this lowers the pH of the solution, which becomes corresponding to the acidic region; the pH value obtained depends on the anesthetic used;

precipitating the local anesthetic in its base form by treating the solution prepared in step a) with a base until a pH value in the range of 6.5 to 8 is reached. Such base may be a base containing an alkali metal or alkaline earth metal ion, preferably an alkali metal, and even more preferably, the base is NaOH;

adding a selected crushed derivative of hyaluronic acid to the resulting suspension, leading to the formation of a gel.

In particular, with regard to ropivacaine specifically, after solubilization of the hydrochloride form in an aqueous vehicle, followed by a decrease in pH to values below 6 and treatment with a base, the salt form is converted into a basic, lipophilic, water-insoluble form, which is thus precipitated as a finely divided form . Stages of solubilization of ropivacaine and after

- 4 044480 blowing precipitation can be completed in one step (for ropivacaine HCl concentrations up to approximately 15-18 mg/ml) or can be divided into several steps when higher concentrations are required. In the second case, the amounts of base, usually NaOH, that are added at each stage are close to stoichiometric with respect to ropivacaine HCl (to cause almost complete precipitation) with pH values maintaining in the range of 6.5 to 8. Regardless of the number of stages performed, the resulting the precipitate is left in suspension in its mother liquor, to which the selected hyaluronic acid derivative, preferably HYADD®-4 amide, as defined above, is added in powder form; a stable, sterilizable gel is formed in which this sediment is enclosed in a network and from which it is gradually released. This formation is carried out in a one-pot reaction mode which is extremely simple and short-lived, without loss of reagents and therefore economical from an industrial point of view and ideally reproducible for all HA derivatives and all local anesthetics according to the invention.

The correct selection of administered doses will be made depending on the extent of the surgical intervention and the general health of the patient, obviously taking into account the necessary restrictions to prevent overdoses. In general, the hyaluronic acid derivative, in particular HYADD®-4 hexadecylamide, is prepared at a concentration ranging from 5 to 15 mg/ml, preferably 7 to 12 mg/ml and even more preferably at a concentration of 8 mg/ml. Other HA derivatives according to the invention, namely auto-cross-linked ACP® derivatives and cross-linked derivatives prepared using BDDE (HBC), are preferably used in concentrations ranging from 10 to 40 mg/ml, preferably from 20 to 30 mg /ml. HA derivatives in concentrations ranging from 5 to 40 mg/ml form a stable gel that releases the active ingredient in a constant, prolonged manner. From the point of view of a sustained release system, the local anesthetic may be included in the carrier at different concentrations, which can be adjusted as necessary. In particular, in the case of ropivacaine, the preferred anesthetic used, as well as in the case of other local anesthetics described, the concentrations range from 10 to 35 mg/ml relative to the final composition, preferably 10 mg/ml, 15 mg/ml, 25 mg/ml or 35/mg/ml. As such, in relation to the described pharmaceutical composition based on HYADD®-4 at a concentration of 8 mg/ml and ropivacaine base at a concentration of 35 mg/ml, each ml of the final composition will contain 8 mg of HYADD®-4 and 35 mg of ropivacaine base.

Considerations similar to those expressed regarding the HA derivatives and the local anesthetic used in base form and their concentrations can also be extended to the controlled release system described above containing a matrix, which will thus preferably consist of hyaluronic acid hexadecylamide HYADD ®-4 prepared in concentrations ranging from 5 to 15 mg/ml, preferably from 7 to 12 mg/ml and even more preferably 8 mg/ml, and ropivacaine base in concentrations ranging from 10 up to 35 mg/ml relative to the final composition, preferably 10 mg/ml, 15 mg/ml, 25 mg/ml or 35 mg/ml. As an alternative to HYADD®-4, the controlled release system matrix may consist of hyaluronic acid derivatives ACP® or HBC, as already described, in a concentration ranging from 10 to 40 mg/ml, preferably from 20 to 30 mg/ml, and a local anesthetic, preferably ropivacaine, in base form, in the concentrations specifically indicated above.

The compositions of the invention and the controlled release systems described above can be used in the treatment of postoperative pain and have the following characteristics:

they are all biocompatible and biodegradable;

they consist of a hydrophilic matrix that prevents abnormal accumulation of the active ingredient;

they can be sterilized using classical methods (in an autoclave);

they are stable at room temperature and even at 40°C for at least 6 months;

they can be easily extruded through needles up to 27 G gauge when applying reasonable pressure;

they can be administered by injection and/or infiltration into the tissue surrounding a surgical wound, by direct instillation into an open wound and by simple local application, for example by spreading along the edge of the prosthesis during its implantation; Routes of administration can also be combined depending on the type of surgery performed;

they are prepared in a very simple way in a one-pot reaction mode and are therefore economical from an industrial point of view;

the analgesic effect lasts for up to 5 days inclusive, especially when administered into the tissue surrounding the wound, in particular, wounds after orthopedic operations;

therefore, the compositions of the invention can be administered in a single dose during surgery, and the simultaneous use of NSAIDs or other oral painkillers

- 5 044480 may be significantly reduced or even eliminated completely.

The compositions of the invention are suitable for use as analgesics for numerous surgical applications, including:

orthopedic surgery: such as total or partial knee or hip replacement, shoulder, ankle, hand, or hallux valgus surgery, or general foot surgery, spine surgery, etc.;

abdominal surgery: such as hernia repair, appendectomy, colectomy, gastrectomy, colorectal surgery, etc.;

surgery of the genitourinary tract (prostatectomy, nephrectomy, hysterectomy, oophorectomy, cesarean section), etc.;

vascular surgery (hemorrhoidectomy).

The following examples illustrate the present invention in more detail.

Example 1: preparation of a gel based on HYADD®-4 (8 mg/ml) and ropivacaine base (15 mg/ml).

79.6 ml of 3 mM PBS, pH 7.3 ± 0.1, was poured into a 250 ml reactor equipped with a stirrer; 1.70 g of ropivacaine HCl was then added which, once dissolved, brought the pH of the mixture to approximately 5.7 and the mixture was left to stir for at least 20 minutes. At this point, 0.4 M NaOH solution was added slowly with stirring until a pH of 7.2-7.4 was achieved; this change in pH caused the precipitation of ropivacaine in base form, resulting in the formation of a very fine suspension.

After bringing the suspension volume to 100 ml using PBS with a pH of 7.3 ± 0.1, 0.8 g of crushed HYADD®-4 was added to the reactor with stirring; stirring was continued for approximately one hour and then the mixture was allowed to stand for at least 3 hours. The resulting gel-like suspension was stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter and stirred again for about at least another 10 minutes. The gel-like suspension was then used to fill syringes and sterilized in an autoclave.

Example 2: preparation of a gel based on HYADD®-4 (8 mg/ml) and ropivacaine base (25 mg/ml).

68 ml of 3 mM PBS, pH 7.3 ± 0.1, was poured into a 250 ml reactor equipped with a stirrer; 1.40 g of ropivacaine HCl was then added which, once dissolved, brought the pH of the mixture to approximately 5.8 and the mixture was left to stir for at least 20 minutes.

At this point, 9.6 mL of 0.4 M NaOH solution was added slowly with stirring, resulting in the first precipitation of ropivacaine base.

A further 1.43 g of ropivacaine HCl was added with a corresponding decrease in pH to approximately 5.8 and stirring was continued for 20 minutes. At this point, 0.4 M NaOH solution was added slowly with stirring until a pH of 7.2-7.4 was achieved, obtaining a very fine suspension of ropivacaine base.

After bringing the suspension volume to 100 ml using PBS with a pH of 7.3 ± 0.1, 0.8 g of crushed HYADD®-4 was added to the reactor with stirring; stirring was continued for approximately one hour and then the mixture was allowed to stand for at least 3 hours. The resulting gel-like suspension was stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter and stirred again for about at least another 10 minutes. The gel-like suspension was then used to fill syringes and sterilized in an autoclave.

Example 3: preparation of a gel based on HYADD®-4 (8 mg/ml) and ropivacaine base (35 mg/ml).

64 ml of 3 mM PBS, pH 7.3 ± 0.1, was poured into a 250 ml reactor equipped with a stirrer; 1.40 g of ropivacaine HCl was then added which, once dissolved, brought the pH of the mixture to approximately 5.7 and the mixture was left to stir for at least 20 minutes.

9.6 ml of 0.4 M NaOH solution was added slowly with stirring, resulting in the first precipitation of ropivacaine base.

Another 1.40 g of ropivacaine HCl was added, which again reduced the pH to acidic values (approximately 5.8), and stirring was continued for 20 minutes; then 9.6 ml of 0.4 M NaOH solution was added slowly with stirring.

An additional 1.16 g of ropivacaine HCl was added and the resulting acidic solution (pH approximately 5.8) was continued to stir for 20 minutes.

At this point, 0.4 M NaOH solution was added slowly with stirring until a pH of 7.2-7.4 was achieved, obtaining a very fine suspension of ropivacaine base.

After bringing the suspension volume to 100 ml using PBS with a pH of 7.3 ± 0.1, 0.8 g of crushed HYADD®-4 was added to the reactor with stirring; stirring was continued for about one hour and then the mixture was left to stand for at least 3 hours. The resulting gel-like suspension was stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter, and stirred again for at least another 10 minutes. The gel-like suspension was then used to fill syringes and sterilized in an autoclave.

- 6 044480

Example 4: preparation of a gel based on HYADD®-4 (8 mg/ml) and ropivacaine base (10 mg/ml).

80 ml of 3 mM PBS, pH 7.3 ± 0.1, was poured into a 250 ml reactor equipped with a stirrer; 1.134 g of ropivacaine·HCl was then added which, once dissolved, brought the pH of the mixture to approximately 5.8 and the mixture was left to stir for at least 20 minutes. At this point, 0.4 M NaOH solution was added slowly with stirring until a pH of 7.2-7.4 was achieved; this change in pH caused the precipitation of ropivacaine in base form, resulting in the formation of a very fine suspension.

After bringing the suspension volume to 100 ml using PBS with a pH of 7.3 ± 0.1, 0.8 g of crushed HYADD®-4 was added to the reactor with stirring; stirring was continued for approximately one hour and then the mixture was allowed to stand for at least 3 hours. The resulting gel-like suspension was stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter and stirred again for about at least another 10 minutes. The gel-like suspension was then used to fill syringes and sterilized in an autoclave.

Example 5: preparation of a gel based on HYADD®-4 (8 mg/ml) and bupivacaine base (15 mg/ml).

79.6 ml of 3 mM PBS, pH 7.3 ± 0.1, is poured into a 250 ml reactor equipped with a stirrer; 1.69 g of bupivacaine HCl is then added and the mixture is left to stir for at least 20 minutes.

The pH value of the mixture is 5.8. At this point, a 0.4 M NaOH solution is added slowly with stirring until a pH of 7.2-7.4 is achieved; this change in pH causes bupivacaine to precipitate in base form, resulting in a very fine suspension.

After bringing the suspension volume to 100 ml using PBS with a pH of 7.3 ± 0.1, 0.8 g of crushed HYADD®-4 is added to the reactor with stirring; stirring is continued for approximately one hour and then the mixture is allowed to stand for at least 3 hours. The resulting gel-like suspension is stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter and stirred again for about at least another 10 minutes. The gel-like suspension is then used to fill syringes and sterilized in an autoclave.

Example 6: preparation of a gel based on HYADD®-4 (8 mg/ml) and bupivacaine base (35 mg/ml).

64 ml of 3 mM PBS, pH 7.3 ± 0.1, are poured into a 250 ml reactor equipped with a stirrer; 1.40 g of bupivacaine HCl is then added and the mixture is left to stir for at least 20 minutes.

The pH value of the mixture is 5.9. 9.1 ml of 0.4 M NaOH solution was added slowly with stirring to obtain the first precipitate of bupivacaine base.

A further 1.40 g of bupivacaine HCl is added and the mixture is left to stir for 20 minutes; the pH value of the mixture is 5.9.

Then slowly add 9.1 ml of 0.4 M NaOH solution with stirring.

A further 1.14 g of bupivacaine ΉΟ is added, with a subsequent reduction in pH to approximately 5.9, and the mixture is left to stir for 20 minutes.

At this point, 0.4 M NaOH solution is added slowly with stirring until the pH is 7.27.4, obtaining a very fine suspension of bupivacaine base.

After bringing the suspension volume to 100 ml using PBS with a pH of 7.3 ± 0.1, 0.8 g of crushed HYADD®-4 is added to the reactor with stirring; stirring is continued for approximately one hour and then the mixture is allowed to stand for at least 3 hours. The resulting gel-like suspension is stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter and stirred again for about at least another 10 minutes. The gel-like suspension is then used to fill syringes and sterilized in an autoclave.

Example 7: preparation of a gel based on ACP® (20 mg/ml) and ropivacaine base (15 mg/ml).

80.0 ml of 1.5 mM PBS, pH 6.9 ± 0.1, is poured into a 250 ml reactor equipped with a stirrer; 1.70 g of ropivacaine HCl is added and the mixture is left to stir for at least 20 minutes. The pH value of the mixture is 5.7.

At this point, 0.4 M NaOH solution is added slowly with stirring until the pH reaches 6.87.0; this pH change causes ropivacaine to precipitate in base form, resulting in a very fine suspension.

After adjusting the volume of the suspension to 100 ml using PBS pH 6.9±0.1, 2.0 g of ACP® in powder form with a degree of esterification of 4.3% is added to the reactor with stirring.

Stirring is continued for approximately one hour and then the mixture is left to stand for at least 3 hours.

The resulting gel-like suspension is stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter, and stirred again for at least another 10 minutes. The gel-like suspension is then used to fill syringes and sterilized in an autoclave.

Example 8: preparation of a gel based on HBC (20 mg/ml) and ropivacaine base (25 mg/ml).

75 ml of 3 mM PBS, pH 7.3 ± 0.1, is poured into a 250 ml reactor equipped with a stirrer; add

- 7 044480 add 1.4 g of ropivacaine HCl and leave the mixture to stir for 20 minutes; the pH value of the mixture is 6.0.

9.6 ml of 0.4 M NaOH solution was added slowly with stirring to obtain the first precipitate of ropivacaine base.

A further 1.43 g of ropivacaine HCl is added and the mixture is left to stir for 20 minutes; the pH value of the mixture is 6.0.

At this point, 0.4 M NaOH solution is added slowly with stirring until the pH is 7.27.4, obtaining a very fine suspension of ropivacaine base. The volume is adjusted to 100 ml using 3 mM PBS, pH 7.3 ± 0.1 and then 2.0 g of HBC is added in powder form with a degree of derivatization of approximately 5.5 mol%. The mixture is heated to 60°C and then left to stir for 5 hours, allowing the HBC powder to gel; it is then cooled to 20°C.

The resulting gel-like suspension is stirred for at least 10 minutes, filtered through a 105 μm stainless steel filter, stirred again for at least another 10 minutes, finally used to fill syringes and sterilized in an autoclave.

Release test of ropivacaine base versus ropivacaine·HCl from HYADD®-4 vehicle.

This test was carried out using a classical release test apparatus consisting of a donor compartment and an acceptor compartment. In this particular case, the donor compartment is a container (Spectra/Por Float-A-Lyzer) that holds 1 ml of the sample to be analyzed and is covered with a dialysis membrane with a cut-off threshold of 100 kDa; The acceptor compartment is a 50 ml test tube containing 15 ml of extraction solvent (PBS, pH 7.0±0.1) and a magnetic stirring rod. After loading the composition to be analyzed into the donor compartment and introducing the donor compartment into the acceptor compartment, this structure is placed in a chamber thermostatically controlled at 37°C with gentle stirring. The analyzed samples were:

composition A: ropivacaine base, 10 mg/ml, in HYADD®-4, 8 mg/ml, prepared as described in example 4;

Composition B: ropivacaine HCl, 11.34 mg/ml (equivalent to ropivacaine base, 10 mg/ml), in HYADD®-4, 8 mg/ml, prepared in accordance with Example 4 simply by eliminating the alkalization step using NaOH.

This test was performed under extreme dilution conditions; in other words, at given points in time, the extraction solvent was completely replaced with fresh solvent of an equal volume to prevent the effect of increasing the concentration of the substance to be tested in the extraction solvent on its further release, and therefore on the change in the test results. In this particular case, the extraction solvent was changed after 2, 4, 6, 8, 24, 48, 72 and 144 hours and the concentration of ropivacaine was measured at each volume change; the cumulative results are shown in Fig. 1.

As will be seen, there is a significant difference in behavior between the two compositions.

From composition A of the invention, ropivacaine, the base is released in a gradual, constant, continuous, continuous manner until its effect is exhausted, i.e. Release of essentially all of the original ropivacaine occurs in approximately 140 hours (5 days). In contrast, from composition B the release of ropivacaine HCl occurs very quickly; after 8 hours, more than 90% of this anesthetic is released, and after 24 hours, at least 98%, so that its effect is practically exhausted.

Considering that the matrix of these two compositions is identical (HYADD®-4 in the same concentration), and the concentration of the anesthetic is the same, this test clearly demonstrates that the combination of a hydrophilic matrix consisting of an amide derivative of hyaluronic acid and a lipophilic Ropivacaine base form is extremely effective in releasing local anesthetic in a gradual, continuous, sustained manner, thereby providing long-lasting analgesic effect.

The experimental data thus obtained were confirmed by the results of preclinical efficacy tests carried out on animals, as described below.

Evaluation of the analgesic effect of ropivacaine base in the HYADD®-4 vehicle compared to ropivacaine HCl.

This study was conducted on pigs that were processed in accordance with the requirements of the Committee on the Ethical Use and Care of Laboratory Animals. The model used is the Brennan model of postoperative pain assessment (Brennan TJ et al., Pain, 1996, 64, 493-501). According to this model, a 1 cm longitudinal incision is made on the skin of the animal, as a result of which the skin, fascia and underlying muscle are cut; after suture placement, postoperative pain measurements are taken at certain time intervals in accordance with the von Frey method by touching the animal's skin with hairs of a known

- 8 044480 diameters, to which force is applied until the perceived pain causes a reaction in the animal. The lower the perceived pain, the greater the force applied, expressed in grams/mm ² (g); thus, at the initial moment (before making the cut), the magnitude of the force will be high; After making the cut, less force will be required to cause a pain reaction in the animal. Thus, the greater the force that must be applied to the von Frey hair to elicit a response in the animal, the greater the analgesic effect of the test substance.

The doses to be administered for each test substance were established based on the know-how of a qualified specialist, as follows:

the maximum dose of ropivacaine-HCl that can be administered without causing side effects is approximately 2.5 mg/kg (Srichartz GR, Berde CB in: Miller RD, ed. Miller's Anesthesia. 6th ed. Philadelphia: Elsevier Churchill Livingstone, 2005: 573 -603). Therefore, in this particular case, based on the fact that the weight of each pig taken in this study was approximately 10 kg, 2.5 ml of Naropin® (ropivacaine-HCl, 10 mg/ml), equivalent to 25 mg of ropivacaine per animal, was used;

in comparative tests reported in the literature (Davidson E.M. et al., Anesth. Analg., 2016, 122, 1663-72), ropivacaine in a lipophilic carrier was prepared in a ratio of 8:1 relative to the hydrochloride form in aqueous solution. Such high doses are necessary for controlled release testing because the local anesthetic, in its protonated form, is known to be metabolized very quickly and ceases to be effective. Therefore, this indication was followed by administering to each animal a dose of the pharmaceutical composition to be tested containing 8 times the amount of ropivacaine-HCl, i.e. an amount equivalent to 200 mg of ropivacaine-HCl. Because the pharmaceutical compositions tested contained different concentrations of ropivacaine base, the volume of each composition was calculated to exactly match the prescribed dose for ropivacaine-HCl (200 mg), and the volumes administered were obviously different.

30 pigs were used, divided into 5 groups of 6 animals, as follows:

group 1: animals were treated with vehicle, namely 11.76 ml of HYADD®-4 (8 mg/ml), and they constitute a negative control;

group 2: animals were treated with 11.76 ml of HYADD®-4, 8 mg/ml, containing ropivacaine base, 15 mg/ml, in an amount equivalent to 200 mg of ropivacaine-HCl, prepared as described in example 1;

group 3: animals were treated with 7.06 ml of HYADD®-4, 8 mg/ml, containing ropivacaine base, 25 mg/ml, in an amount equivalent to 200 mg of ropivacaine-HCl, prepared as described in example 2;

group 4: animals were treated with 5.05 ml of HYADD®-4, 8 mg/ml, containing ropivacaine base, 35 mg/ml, in an amount equivalent to 200 mg of ropivacaine-HCl, prepared as described in example 3;

group 5: animals were treated with 2.5 ml of Naropin® (ropivacaine-HCl, 10 mg/ml) aqueous solution, equivalent to 25 mg of ropivacaine-HCl, and constituted a positive control.

Such treatments were carried out before suturing the wound, injecting half the volume into the wound area and half into the tissue surrounding the wound at three different levels.

The von Frey test was performed at the initial moment, i.e. before the incision, and then 3, 8, 12, 24, 36, 48 and 72 hours after the operation. The test results are shown in Fig. 2. The values shown are statistically significant.

It is quite obvious that:

as expected, the vehicle has no effect;

the effect of Naropin® is relatively good after 3 hours, very weak after 8 hours and absent in the following hours;

The tested pharmaceutical composition containing HYADD®-4, at all concentrations of ropivacaine used, has a strong analgesic effect, which for some concentrations is much stronger (group 2) than the effect of Naropin® 3 hours after treatment, or in other cases is similar to this action, and much stronger than for later points in time. The analgesic effect of the compositions weakens over time in the case of lower concentrations of ropivacaine (groups 2 and 3) and increases in the case of higher concentrations of this substance (group 4). This further demonstrates the effectiveness of the controlled release system described in this invention.

Although the von Frey test was stopped after 72 hours (3 days) for ethical reasons, the data shown in FIG. 2, analyzed in conjunction with the release curve data in FIG. 1 indicate that the effectiveness of analgesia under the influence of the tested samples will be further maintained for a long time, since, for example, samples with the maximum concentration of ropivacaine (group 4) have a very strong analgesic effect 72 hours after treatment: to cause a painful stimulus in animal, an effort is required that is approximately 80% greater than that required at the initial moment. This aspect is important

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Claims (10)

since it means that the amount of ropivacaine can be adjusted based on the duration of the desired analgesic effect, depending on the surgical procedure to which the patient is to undergo, and other clinical considerations. In addition to these results, which clearly demonstrate the long-term effectiveness of the pharmaceutical compositions of the invention, it should be noted that none of the treated animals had adverse reactions caused by an overdose of ropivacaine, although the administered dose of the active ingredient was generally much higher than the standard dose in the hydrochloride form . This means that the pharmaceutical compositions tested in this application: act as a controlled release system (drug delivery system) for the local anesthetic, prolonging its analgesic effect over time; do not cause overdose and/or accumulation and therefore prevent well-known toxic side effects; eliminates the need for repeated administrations; allow you to adjust the duration of the analgesic effect; and thus represent a definite improvement on the state of the art in the treatment of postoperative pain. CLAIM 1. A pharmaceutical composition in gel form, intended for use in the treatment of postoperative pain, containing or consisting of a hydrophilic matrix consisting of a hyaluronic acid (HA) derivative containing a local amide anesthetic in base form, and capable of releasing the local anesthetic in lipophilic and basic form in a prolonged manner, wherein the hyaluronic acid derivative is selected from: amide derivative obtained by amidation of the carboxyl groups of hyaluronic acid and the amino groups of hexadecylamine, with an average degree of amidation varying in the range from 1 to 3 mol.%, in a concentration of 5 to 40 mg/ml and obtained from HA with average molecular weight (MW) , varying in the range from 500 to 730 kDa; and the local amide anesthetic is selected from bupivacaine, etidocaine and ropivacaine at a concentration ranging from 10 to 35 mg/ml. 2. The composition according to claim 1, further containing pharmaceutically acceptable excipients. 3. The composition according to claim 1 or 2, wherein the local amide anesthetic is ropivacaine. 4. The composition according to any one of claims 1 to 3, wherein the hyaluronic acid derivative is a hexadecylamide obtained from hyaluronic acid with a weight average molecular weight varying in the range from 500 to 730 kDa, and having an average degree of amidation varying in the range from 1 to 3% mol., and a concentration of 5 to 15 mg/ml, and the local anesthetic is ropivacaine in base form at a concentration of 10 to 35 mg/ml. 5. The composition according to claim 4, where the concentration of hyaluronic acid hexadecylamide is in the range from 7 to 12 mg/ml. 6. The composition according to claim 4, where the concentration of hyaluronic acid hexadecylamide is 8 mg/ml. 7. A composition according to any one of claims 1 to 6, capable of releasing the local anesthetic in lipophilic and basic form in a prolonged manner over a period of time up to five days. 8. A method for preparing a composition according to any one of claims 1-7, including: (a) solubilizing the local anesthetic in its salt form in a single portion or in multiple portions in an aqueous vehicle, preferably phosphate buffered saline (PBS); (b) precipitating the local anesthetic in its base form by treating the solution obtained in step (a) with the base until a pH value ranging from 6.5 to 8 is achieved; (c) adding a hyaluronic acid derivative to the resulting suspension. 9. The method according to claim 8, where the base is NaOH. 10. Use of a composition according to any one of claims 1 to 7 for the treatment of postoperative pain. -