RO132702B1 - Process of preparation of pharmaceutical formulation comprising complex systems based on lidocaine, cyclodextrine derivatives and liposomes - Google Patents

Description

RO 132702 Β1

The invention refers to a process for obtaining new complex systems based on lidocaine and derived from esterified cyclodextrin with analgesic properties.

The complex systems obtained by incorporating lidocaine in liposomes by encapsulation, in cyclodextrin derivatives as inclusion complexes or in innovative combinations between liposomes and inclusion complexes with cyclodextrins modified with oligoester sequences, are an approach to create controlled release systems, easily adaptable in operating configurations of pain therapy. The lidocaine encapsulation forms are then included in polymer matrices in the form of gels based on: chitosan, poloxamer, modified polyxyloxanes, as single components or in combinations. The introduction of the lidocaine complex in these formulations gives the created assembly the pelliculogenic properties and the appropriate hydrophilic-hydrophobic ratio for controlled, efficient and sustained release. The use of inclusion complexes in reservoir-type systems facilitates the transfer of the substance through the skin, thus improving the pharmacokinetic profile at the skin level. The formulations thus synthesized meet the chemical, physical, technological and biomedical criteria necessary for successful use in the field of transdermal pain therapy.

There are a number of topical analgesics with local surface application in the form of gel, cream, spray, dressing, drops, etc. used depending on the procedure to be followed [L. Brannon-Peppas, Polymers in Controlled Drug Delivery, Medical Plastics and Biomaterials Magazine, 1997], The first commercially available product was TAC (Trad Analgesic Cocktail), a combination of tetracaine, adrenaline (epinephrine) and cocaine, used in d i I a c e r a analgesia (face and scalp) [Web page: http://en.wikipedia.org/wiki/Lidocaine/prilocaine], Later, lidocaine replaced cocaine, thus resulting in the product LET (lidocaine, epinephrine and tetracaine), alternative to TAC with low toxicity and low cost [R.F.Wolff, ActaNeurol. Scand., 2011,123(5), 295-309], currently the only local anesthetic available worldwide for intact skin is EMLA (Eutectic Mixture of Local Anesthetics) [S. Kundu, Am. Fam. Physician., 2002, 66, 99-102] marketed as a 5% emulsion containing a eutectic mixture of lidocaine/prilocaine in equal amounts. since skin absorption of topical products is highly variable, and available preparations are absorbed superficially through intact skin, penetration has been improved by using eutectic mixtures (they melt at lower temperatures compared to those of the individual components), liposomal preparations (lidocaine liposomally encapsulated: Ela-Max available in the US), percutaneous patches (lidocaine/tetracaine percutaneous patch: Synera) [K. Miller, AANAJ., 2001, 69, 185-187] or by sequential layered application (topical lidocaine with epinephrine: TLE) [S. S. Davis, L. Uium, Int. J. Pharm., 176, 1998, 1-8], lontophoresis can also be applied, but the procedure is expensive and may cause discomfort due to the electrical sensation.

Local anesthetic agents belong to the class of aminoamides and include compounds such as: lidocaine, bupivacaine, prilocaine, mepivacaine and etidocaine. Lidocaine (LID) is the most commonly used local anesthetic due to its efficiency and rapidity of action, lack of toxicity and sensitivity. Lidocaine blocks voltage-dependent sodium channels, stops ectopic discharges of fine afferent fibers, slows peripheral nociceptive sensitization and central hyperexcitability. Lidocaine is available in multiple forms of administration, for example, 5% patches (Lidoderm®, Endo Pharmaceuticals Inc.) for the treatment of postherpetic neuralgia and other focal neuropathic syndromes (mononeuropathies, intercostal neuralgias, and postamputation pain) in which traditional antidepressant treatment tricyclics, anticonvulsants and opioids fail [R.M.Ottenbrite, K.Park, T.Okano, Biomedical Applications of Hydrogels Handbook, Ist Ed., Springer, 2010]. More

RO 132702 Β1 pharmacokinetic studies have shown that the systemic absorption of the lidocaine patch in 1 healthy adults is minimal when up to 4 units are applied in 12-24 h; the rate of absorption is lower in patients with postherpetic neuralgia who receive a patch with 3 lidocaine pure or combined with other agents. No interactions with other drugs were observed and its use does not lead to beta-mediated sensory loss at the site of 5 application [K.MilIer, AANAJ.,2001,69, 185-187], Topical anesthetics, for example, ointment composed of lidocaine 5%, pramoxine hydrochloride 1% with hydrocortisone acetate 1%, 7 alcohol lignocaine spray 5%, lignocaine gel 1-5%, and cinchocaine 2%, have been studied for the relief of perineal pain after vaginal delivery. However, evidence of effectiveness and 9 long-term effects was inconclusive.

in recent years, the administration of adjuvant agents along with the local anesthetic such as epinephrine, clonidine and dexamethasone has been reported as a viable way to prolong the action of anesthetics. Another measure involves the use of release matrices, 13 such as liposomes, microemulsions, microspheres, microcrystals, crosslinked and/or thermosensitive hydrogels, thermosensitive nanogels, nanoparticles, aqueous polymer solutions, 15 liquid or solid polymer matrices, bioadhesive films , interpenetrated polymer networks, lipid-protein-sugar particles, polymers based on fatty acid dimers 17 and ceramic granules [Y. J. Jang, J. H. Lee, T. B. Seo, S. H. Oh, Eur. J. Pharm. Biopharm., 2017, 115, 113-121], 19

According to specialized literature, the development of systems based on local anesthetics represents a field with significant development potential, due to their complexity, 21 with aspects still not elucidated.

Controlled release systems are devices capable of incorporating and delivering to the human body a certain amount of active substance, in order to obtain an increased therapeutic effect by controlling the rate and time of release, as well as the site of action. 25 The main objective of controlled release systems is to maintain a constant concentration of the biologically active compound, which should be between the minimum effective 27 and the toxic concentration over a long period of time.

in the development of percutaneous administration systems there are a number of factors that 29 modulate the release kinetics of the biologically active compound encapsulated in a controlled release vehicle, such as: the chemical structure of the immobilized molecule, the type of polymer network 31 that makes the vehicle, the additives used for obtaining the system and possible chemical interactions [A.F. Rogobete, M. Dragomirescu, O.H. Bedreag, D. Sandesc, CA. 33 Cradigati, M. Sarandan, M. Papurica, S.E. Popovici, C. Vernic, G. Preda, Trends Anaesth. Crit. Care, 2016, 9, 27-34], Thus, due to increased clinical standards, for 35 a controlled release system to be viable, it must meet the requirements of selectivity, bioavailability, biodegradability, reduced toxicity, increased bioactivity, extended time 37 of action and low costs.

Liposomes, the most widely used controlled release systems, are lipid vesicles that fully enclose an aqueous volume. These colloidal particles are typically constituted of phospholipids with or without additives and are formed of biomolecular concentric layers capable of encapsulating drugs. Liposomes work by penetrating the epidermis, transporting the drug through the layers of the skin into the peripheral circulation. To increase the degree of their use, the literature reports the use of a variety of polymers, the most used being poly-(ethylene glycol) (PEG), poly-(N-vinyl-pyridodone) (PVP), poly-(vinyl alcohol ) (PVA), 45 polyoxazoline (Pox) or poly-(acrylic acid) (PAA).

RO 132702 Β1

The polymers used in percutaneous administration systems must be biocompatible and chemically compatible with the drug and other components of the system, such as penetration enhancers, to ensure an efficient and constant delivery of the drug throughout the period of administration and shelf life of the preparation [C. Valencia, B.G. Auner, Eur. J. Pharm. Biopharm., 58, 2004, 279-289],

Chitosan, the diacetyl derivative of chitin, has many properties such as biocompatibility, biodegradability, nontoxicity and antimicrobial activity. Chitosan has the necessary properties to be used as a pharmaceutical excipient, being used in controlled and targeted release studies of most classes of bioactive molecules. These properties make chitosan one of the most promising biopolymers in tissue engineering, preparation of wound dressings, gene therapy and drug delivery [T. Aoyagi, R. Tadenuma, Y. Nagase, Macromol. Chem. Phys., 1996,197,677-686], considerable efforts have been made in recent years to improve the technical properties of chitosan by derivatization. For example, chitosan-laurate and chitostan-myristate hydrogels improve skin diffusion of the lyophilized drug relative to chitosan-palmitate and chitosan-stearate hydrogels. This fact can be explained by the interaction of hydrogels with the stratum corneum, thus increasing the solubility of the drug in the skin [J.J. Escobar-Chavez, M. Lopez-Cervantes, A. Naik, Y.N. Kalia, D. Quintanar-Guerrero, A. Ganem-Quintanar, J. Pharm. Pharmacist. Sci., 2006, 9, 339-345],

Polysiloxanes are synthetic polymers intensively studied for biomedical applications. Oligodimethylsiloxanes (ODMS) substituted with 2-pyrrolidone at one end of the chain have shown transdermal penetration enhancer abilities. The potentiation activity is influenced by the length of the siloxane macromolecular chain and the chemical structure of the end groups. The ratio between the hydrophobic chain and the polar end group is also a decisive factor for drug penetration-enhancing activity [C. He, S.W. Kim, D.S. Lee, J. Controlled Release, 2008, 127, 189-207],

Poloxamers, are poly(ethylene oxide-propylene oxide) (PEO-PPO-PEO) triblock copolymers, also known as Pluronic®, with surfactant properties, widely used in pharmaceutical systems. Concentrated aqueous solutions of Pluronic exhibit two thermo-reversible phase transitions with increasing temperature: the sol-gel and gel-sol transition. Pluronic-based hydrogels exhibit considerable viscosity, partial stiffness, and some persistence over time due to packing in ordered micellar structures and intermicellar interpenetrations. encapsulation of lidocaine in biodegradable Pluronic F-127-polycaprolactone nanoparticles causes, for example, prolonged infiltration anesthesia in rats without severe toxicity, indicating a possible way to develop local anesthetics that persist over time [E. Fishbeine, Y. Meshulam, R. Sahar, S. Chapman, H. Liani, I. Barness, A. Amir, J. Appl. Toxicol., 2000, 1, 133-136; T. Grațieri, G.M. Gelfuso, E.M. Rocha, V.H. Sarmento, O. de Freitas, R.F.V. López, Eur. J. Pharm. Biopharm., 2010,75, 186-193], Pluronic F-127-based lidocaine or pentamide ointments topically applied to pig skin have been shown to produce beneficial effects [G. Niu, F. Du, L. Song, H. Zhang, J. Yang, H. Cao, Y. Zheng, Z. Yang, G. Wang, H. Yang, S. Zhu, J. Control. Release, 2009, 138, 49-56],

The feasibility of poloxamer gel-based delivery strategies is limited by relatively rapid dissolution under physiological conditions. Various solutions have been explored to overcome this inconvenience, focusing on improving the strength, adhesion and permanence time of poloxameric systems, either by modifying their structures or by incorporating mucoadhesive polymeric substances such as chitosan that present a good

RO 132702 Β1 biocompatibility and biodegradation [A. Sosnik, D. Cohn, J.S. San Roman, G.A. Abraham, 1 J. Biomater. Sci. Polym. Ed., 2003, 14,227-239; T. Ur-Rehman, Int. J. Pharm., 2011, 409,19-29; R. New, Liposomes. A Practical Approach, Oxford University Press, 1990; 3

T. Loftsson, P. Jarho, M. Mâsson, T. Jărvinen, Expert Opinion on Drug Delivery, 2005, 2(2), 335-351],5

Cyclodextrins (CDs) are mainly used to improve the water solubility and stability of drugs. Cyclodextrins of pharmaceutical relevance contain 6, 7 or 87 dextrose molecules (α-, β-, γ-cyclodextrin) linked in a 1,4 configuration to form rings of various diameters or β-cyclodextrin derivatives with increased water solubility , such as hydroxypropyl-β-cyclodextrin (ΗΡβ-CD). Cyclic molecules have a hydrophilic exterior and a lipophilic cavity into which organic molecules of appropriate size can be included 11 to form noncovalent inclusion complexes with increased solubility in aqueous media. A recent literature review [B. McCormack, G. Gregoriadis, Int. J. Pharm., 1994, 112, 13

249-258] analyze the influence of CD concentration on the transdermal penetration capacity of CD-drug complexes. Penetration decreases with increasing CD concentration. 15 the improvement of skin penetration by cyclodextrins has also been attributed to the extraction of lipids from the stratum corneum [B. McCormack, G.J. Gregoriadis, Drug Target., 1994, 17

2, 449-454],

The concept of associating CDs with liposomes consists in encapsulating the CD-drug inclusion complex 19 in liposomes, thus combining the advantages offered by CD (increasing drug solubility) and liposomes (targeted action of drugs and increasing 21 permeability) in a single system, thus avoiding the problems associated with each individual system [H. Chen, J. Gao, F. Wang, W. Liang, Drug Delivery, 14 (4), 201-208; G. Ragnoa, 23 E. Cione, A. Garofalo, G. Genchi, G. loele, A. Risoli, A. Spagnoletta, Int. J. Pharm., 2003,265,125-132; F. Maestrelli, M.L. Gonzâlez-Rodrîguez, A.M. Rabasco, P. Mura, Int. 25 J. Pharm., 2005, 298(l), 55-67; N. Shufang, W.L. Hsiao, P. Weisan, Int. J.Nanomed., 2011, 6, 151-166], For example, the complexation of ketoprofen with CD increased the solubility of the drug, and improved the stability of the internal aqueous phase encapsulation of the vesicle, conferring a better control of its release without altering the permeability properties through the skin [M.T. Popescu, S. Mourtas, G. Pampalakis, S.G. Antimisiaris, C. Tsitsilianis, Biomacromolecules, 2011, 12, 3023-3030], A different approach involves 31 incorporating liposomal drugs into the F127 gel, thus increasing the amount of incorporated drug and slow release of the drug compared to the pure gel. Therefore, a system containing both F127 and liposome is of great interest to combine the thermogelling properties of F127 and the transport abilities of the liposome. First, the desired drug release rate from this liposomal gel formulation could be achieved by incorporating an optimal amount of liposomes contained in the gel, instead of increasing the concentration of F127. The liposome can represent not only a way to increase the loading of lipophilic drugs in gels, but also act as a reservoir for slow and steady drug release [M. Bonacucina, M.F. Spina, M. Cespi, S. Pucciarelli, M. Angeletti, G.F. Palm trees, Eur. J. Pharm. Sci., 2007, 3 (2), 115-122; L. 41 Nogueiras-Nieto, C. Alvarez-Lorenzo, I. Sandez-Macho, A. Concheiro, F.J. OteroEspinar, J. Phys. Chem. B, 2009, 113, 2773-2782], 43

The hydrophilic/hydrophobic character of the percutaneous administration systems determines a variation of the structure and the percentage of polymers in the composition of the films, for an effective control of the drug release characteristics. The presence of hydroxypropyl-P-cyclodextrin (ΗΡβ-CD) in poloxamer gels affects both gelation temperatures and 47

RO 132702 Β1 micellization as well as the elasticity of the final samples through the special change of the microstructure of the gelled samples. This mutual influence paved the way for the exploration of several poloxamer 407/HPP-CD systems to create various percutaneous delivery systems that bring together the well-known properties of cyclodextrin and the thermogelling behavior of poloxamer [F. Castiglione, M. Valero, CA. Dreiss, A. Mele, J. Phys. Chem. B, 2011, 115, 9005-9013; F. van de Manakker, T. Vermonden, C.F. van Nostrum, W.E. Hennink, Biomacromolecules, 2009, 10, 3157-3175; C. Peptu, A. Nicolescu, CA. Peptu, V. Harabagiu, B. CSimionescu, M. Kowalczuk, J. Polym. Sci.: Part A: Polym. Chem., 2010, 48, 5581-5592],

Among these options, the Bupivacaine multivesicular liposome-based Exparel® delivery system has been approved for human use. The conclusions of the study in the rat model proved the efficiency of the system: the duration of action of blocking the sciatic nerve was approximately double (4 h, compared to 2 h with bupivacaine), due to the sustained release of bupivacaine from liposomes [Y.J. Jang, J.H. Lee, T.B. Seo, S.H. Oh, Eur. J. Pharm. Biopharm., 2017, 115, 113-121],

The studies most relevant to the present invention are presented below. Abdelkader and others [D.H. Abdelkader, M.A. Osman, S.A. El-Gizawy, A.M. Faheem, P.A. McCarron, Int. J. Pharm.,2016,500,326-335] synthesized hydrogels with potential analgesic applications in traumatic wounds, composed of polyvinyl alcohol cross-linked with tetrahydroxyborate anions and different concentrations of chlorinated lidocaine. The obtained results showed that the hydrogels do not induce a rapid anesthetic effect due to the low degree of incorporation and low release kinetics. Anirudhan and others [T.S. Anirudhan, S.S. Nair, Anoop S. Nair, Carbohyd. Polym., 2016,152,687-698] obtained a new transdermal patch based on lidocaine uniformly dispersed in a complex polymer matrix containing hyaluronic acid covalently linked with 3-(dimethylamino)-l-propylamine and chitosan grafted with glycidyl methacrylate and methacrylate bottles. In vitro release studies have shown the possibility of using this medical device only at pH 5.5 with a release kinetics of up to 56% in the first 12 h, followed by a plateau up to 24 h. A more recent study is developing a local anesthetic injectable gel for post-operative pain management, based on lidocaine and a mixture of polyamine-b-poly(ethylene glycol)-b-polyamine with nitroxide radicals bound to the polyamine chains by electrostatic interactions in the form of micelles. The prolonged and enhanced effect resulting from in vivo studies demonstrated the effectiveness of the system [Y. Nagasaki, Y. Mizukoshi, Z. Gao, CP. Feliciano, K. Chang, H.Sekiyama, H. Kimura, ActaBiomaterialia, 2017,57, 127-135], A complex of lidocaine and multivalent ions was synthesized in order to obtain a long-acting anesthetic system. The results of in vitro and in vivo experiments indicated that this complex facilitates the prolonged release of lidocaine even without adjuvants, thus ensuring nerve blockade for a long duration of action (> 14 h) without further increase in neurotoxicity [Y.J. Jang, J.H. Lee, T.B. Seo, S.H. Oh Eur. J. Pharm. Biopharm., 2017, 115, 113-121],

Patent application EP 0838225 (A2) (23.10.1997) discloses an aqueous local anesthetic solution having a pH of 6.5-8.5, containing a local anesthetic included in cyclodextrin or a derivative thereof in a concentration of local anesthetic of at least 0.02%, a method for improving the solubility of the local anesthetic in water at a pH of 6.5-8.5, comprising including the local anesthetic in cyclodextrin or a derivative thereof, a local anesthetic agent comprising a compound of including the local anesthetic in cyclodextrin or a derivative thereof, a method for decreasing the neurotoxicity of the local anesthetic agent, comprising including the local anesthetic in cyclodextrin or a derivative thereof and a

RO 132702 Β1 local anesthetic inclusion compound in cyclodextrin or a derivative thereof. The local anesthetic agent 1 and aqueous local anesthetic solution of the present invention have low neurotoxicity, so the duration of anesthesia can be safely controlled clinically by 3 reducing/increasing its concentration or dose. in particular, the aqueous local anesthetic solution can be used at a pH close to that of a living body. Thus, even 5 can be used when living tissue has an acidic pH. Moreover, the pain caused by local acidosis at the injection site with a conventional local anesthetic dissolved in an acid solution can be reduced. 7

WO 95/05198 (23.02.1995) discloses a local anesthetic preparation comprising a local anesthetic agent and 2-hydroxypropyl-beta-cyclodextrin (CDEX). generally, the local anesthetic agent 9 is in the form of a conventional aqueous formulation and the CDEX is present in a concentration of 0.01-15%, preferably 0.02-10%. The use of 2-hydroxypropyl-beta-11 cyclodextrin for the preparation of a local anesthetic preparation, as well as a method of producing local anesthesia in a mammal, including a human, are also described. 13 despite the promising results in prolonging the duration of anesthesia through the use of adjuvants and delivery matrices, certain obstacles must be overcome before 15 these agents are widely applied. Such obstacles include insufficient duration of action and the fact that anesthetic effects are not reproducible due to different release patterns with each manufacturing batch.

The main disadvantages of complex topical lidocaine-based analgesics 19 reported to date limit therapeutic success by:

- variable efficiency (depending on dose and time); 21

- systemic effects and cognitive impairment;

- variable toxicity over a long period of time; 23

- reduced percutaneous impermeability due to the variability of the absorption of topical products; 25

- short duration of action and the need to administer repeated doses, which can cause infections, muscle pain, etc. 27

The most similar complex systems based on local anesthetics for transdermal pain therapy have been realized as follows:29

- local anesthetic with pronounced effect and extended duration of action composed of lidocaine (2-5% in saline solution) or bupivacaine (0.5-0.75% in saline solution) complexed with 2-31 hydroxypropyl-p-cyclodextrin (grade of substitution 40% and concentration of 0.02-10%) [F.M.

Borgbjerg, Local Anesthetic preparation, WO 95/05198, 1995];33

- aqueous solutions based on local anesthetics such as lidocaine, bupivacaine, dibucaine or tetracaine (in the form of acid or salt, concentration between 0.02-0.05%)35 complexed with: cyclodextrin or its derivatives and salicylic acid , incorporated with the aim of improving the anesthetic effect over a long period of time [Hiji, Yasutake, 37 Aqueous local anesthetic solution, EP 0838225 A2, 1997].

- complex based on the inclusion of benzocaine in γ-cyclodextrin in order to formulate 39 a local anesthetic for the relief of surface pain [J.P. Moldenhauer, New inclusion complexes of benzocaine in gamma-cyclodextrin, useful as non-volatile 41 local anesthetics, e.g. for alleviating surface pain, DE 10033059 A1, 2000].

The technical problem that the invention solves consists in obtaining lidocaine-based complex systems with analgesic properties, which allow improving patients' compliance with medical treatment by relieving pain, with fewer adverse effects on the central nervous system and a minimum drug regimen, the efficiency of the drug dose between safety and efficiency through individualized drug therapy 47, as well as the possibility of controlled administration.

RO 132702 Β1

Process for obtaining a pharmaceutical formulation with analgesic properties comprising complex systems based on lidocaine, cyclodextrin derivatives and liposomes, according to the invention, removes the disadvantages presented above in that:

- unilamellar/multilamellar liposomes are formed based on single phospholipids or in combinations of the glycerophospholipid type, chosen from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipins and sphingophospholipids and/or cholesterol and/or their derivatives, followed by hydration of these liposomes with a lidocaine solution incorporated in the cyclodextrin derivative obtained by:

- the modification of α-, or β-, or γ-cyclodextrin, preferably β-cyclodextrin with oligoester sequences through the opening reaction of the lactide cycle, with the formation of a product containing a number of lactide units between 1...21 , preferably 1...4 lactide units strung together;

- the inclusion of lidocaine through an open system mixing process with the complexation stoichiometry in the range of 0.5...1.5 with an optimal value of 1:1;

- it results in obtaining a complex system that increases the solubility of lidocaine in acidic media, increases bioavailability and a toxicity to a value below the legal threshold established in the case of use as a transdermal delivery system for external drugs.

By applying the invention, the following advantages are obtained:

- the controlled release of lidocaine ("burst effect - pronounced anesthetic effect in the interval 0-20 min followed by controlled release up to 100 min, demonstrated by the in vitro release curve") and the painless administration of various medicinal preparations (gels);

- improved ability to incorporate lidocaine (encapsulation in liposomes and CD);

- reduced toxicity due to the compounds forming the vehicles (screening animals used in the study);

- fast and long time of action compared to the base compound (in vitro release, analgesic/anesthetic effect determined by nociceptive stimulation with the help of tests: hot plate, cold plate and algesimetric);

- low costs of large-scale use (low-cost synthetic materials);

- innovative, easily adaptable lidocaine encapsulation methods.

The solution to the proposed technical problem consists in the development of medicinal formulations based on the combination of a multitude of penetration enhancers (cyclodextrins modified with oligoester sequences, liposomes, functionalized polysiloxanes) capable of increasing the bioavailability and diffusivity of lidocaine in the layers of the skin through different mechanisms. Lidocaine is initially encapsulated in liposomes, derived from cyclodextrins as inclusion complexes and also in an innovative combination of liposomes and inclusion complexes, which is then inserted into a polymer gel matrix (chitosan, polyxyloxane, poloxamer) designed specifically to ensure proper hydrophilic/hydrophobic balance and skin adhesion. the entire assembly will be able to transdermally deliver the analgesic compound in an effective and sustained manner. The novelty of the invention consists in the use of cyclodextrins modified with oligoesters for lidocaine encapsulation, a technique not used for this purpose until now.

According to the invention, the initial stage of obtaining lidocaine-based complex systems involves the incorporation of the drug into liposomes, into cyclodextrin derivatives (inclusion complexes) and into a combination of liposomes and inclusion complexes. These compounds represent the basis of the composition of pharmaceutical formulations that allow adhesion to the skin, controlled transport and non-toxicity, under the conditions of local diffusion of the active principle.

RO 132702 Β1

Description of lidocaine incorporation procedures: 1

- complex CDLA-LID systems:

Initially, the CD compound was modified with aliphatic oligoester sequences grafted on 3 hydroxyl groups by β-cyclodextrin-initiated ring-opening oligomerization reaction of lactide with a molar ratio of lactide/cyclodextrin units of 3, thus obtaining, 5 cyclodextrin modified with oligolactide units (CDLA) with molecular masses of 1566 g/mol. The oligomerization reaction was demonstrated by analyzing the ¹ H NMR and ESI-MS spectra 7 (fig. 1,2). It is observed that the cyclodextrin was functionalized with a variable number of lactide units from 1 (2 lactic acid units) [1296 = 1134(CD) + 144(LA) + 18(NH ₄ ⁺ )] to 6 (12 9 lactic acid units) [2016 = 1134 + 144 · 6 + 18], Thus, the average length of oligolactide chains is 6 lactic acid units. 11

The preparation of CDLA-LTD complexes involves the optimization of the mechanical mixing process, namely, open-system kneading and closed-system ball-milling 13 . According to the results obtained, the open system mixing procedure was considered optimal, because grinding in the ball mill in a closed system did not allow the successive evaporation of solvents during mixing, thus, additional complications appeared in the phase of separation of complexes, and the resulting products consisted of mixtures of 17 CDLA-LTD complexes and free components.

In a typical experimental procedure, solutions of CDLA (M = 1566 g/mol; 173.3 g in 19 mixture of 200 mL pharmaceutical ethanol and 66.7 mL distilled water) and LID (M = 234.34 g/ mol; 26.7 g in 135 mL of ethanol) were prepared in two separate vessels, by stirring (anchor stirrer, 200 rpm) for 10 min. The LID solution was then slowly added over 10 min with continuous stirring over the CDLA solution. The mixture was transferred to a mixer with a capacity of 1 L and mixed for 2 h at a temperature of 25°C, in an open system. After about 30 min of kneading, the solution becomes milky and at the end of the 25 kneading period it acquires the appearance of a semi-solid paste due to the progressive loss of solvents. This was followed by drying it in a vacuum oven (0.1 mm Hg) at 40°C for 6 h which led to the formation of a fine powder of CDLA-LTD complex, with a yield of 98% due to the partial hydrolysis of CDLA during the complexation process and the loss by evaporation of a quantity of the lactic acid thus formed, during the drying process. The parameters taken into account, namely, mixing time, temperature and vacuum drying period, for the successful realization of the process of obtaining CDLA-LTD complexes were optimized following the tests carried out. 33

The CDLA/LID composition was proven at the molecular level by NMR spectrometry (fig.

3) and by MS/MS fragmentation (Figs. 4A and 4B). Thus, in the NMR spectrum the resonance peaks of all components are visible, and from the MS/MS spectrum it is observed that the fragmentation of the parent ion with m/z = 1657 [1657 = 1134(CD) + 144 · 2(LA)234(LID ) + 1 (H ⁺ )] leads to 37 the appearance of a fragment having the mass LID [235 = 234(LID) + 1 (H ⁺ )] confirming the inclusion of LID in CDLA. 39

The solubility of LID in water increases considerably in the presence of CDLA, as can be seen from the solubility diagram in Fig. 5A. Also, the complexation stoichiometry was determined to be 1:1 as shown in fig. 5B constructed using the calibration curve (dependence of UV absorbance of LID on LID concentration in water, 43 R = 0.999). The stability constant of the inclusion complexes was determined to be ~1.8-10 ⁵ M' ¹ . 45

- complex systems of the LID-liposomes and CDLA-LID-liposomes type

RO 132702 Β1

CDLA-LID inclusion complexes were incorporated into reservoir-type systems (liposomes). Two types of liposomes, small unilamellar (SUV) and multilamellar vesicle (MLV) have been obtained and used due to the simplicity and reproducibility of the preparation procedures by hydration of lipid films followed by sonication or extrusion. CDLA-LID inclusion complexes were dispersed in the aqueous phase of both types of liposomes, proving the dependence of encapsulation of the complexes depending on the type of liposomes used. Both phospholipids (phosphatidyl choline) and combinations between phospholipids and cholesterol were used as lipids, the latter being introduced with the main purpose of improving the stability of liposomes over time. Both the obtained liposomes and the liposome/cyclodextrin-lidocaine complex systems were characterized physico-chemically, demonstrating the feasibility of the preparation process. Shape and lamellarity were determined by SEM microscopy techniques revealing an average diameter of 2,150 pm for MLV lysozymes (Fig. 6).

in a first step, multilamellar liposomes (MLV) were prepared by the method of hydration of thin lipid films. For this, 100 mg of lipids (60 mg of phospholipids (PC (phosphatidyl choline) or DSPC (1,2-distearoyl-sn-glycero 3-phosphocholine)) and 40 mg of cholesterol (Chol) are dissolved in 10 mL of a chloroform/methanol mixture ( 2/1, v/v), the solvent mixture is then evaporated under vacuum from a round bottom flask coupled to an evaporator until a thin lipid film is formed. The lipid film is then hydrated with a certain volume of phosphate buffer solution or with LID or CDLA-LID solution (10 mL LID-CDLA solution of concentration 52.5 mg/mL).The hydration of the lipid film is carried out at a temperature of 25°C in the case of use PC or 60°C for DSPC. After complete hydration, during which the liposomes are formed, the liposome suspension is then sonicated for 30 min to remove aggregates. The MLV liposomes are then purified by repeated cycles of centrifugation /washing.

Liposomes (SUVs) were prepared by sonicating the MLV liposome suspension with a sonication probe for 1–2 cycles until the suspension became completely transparent. The SUV liposome suspension was then centrifuged at 15000 rpm for ΙΟΙ 5 min in order to precipitate titanium fragments resulting from detachment from the ultrasound probe due to the high intensity used. finally the liposomes were incubated for 1-2 h at a temperature above the thermal transition temperature (25°C for PC and 60°C for DSPC) to normalize the structural defects in the liposomal membranes. Separation of liposomes from unencapsulated LID was performed by centrifugation in the case of MLV (3 cycles at 15,000 rpm for 40 min) and by molecular mass exclusion chromatography (SEC) using a 1 x 30 cm Sephadex G-50 column eluted with phosphate buffer solution, pH 7.4. The liposomes were then stored at 4°C before use.

The liposomes were studied from the point of view of size and size distribution and the results considered optimal are shown in table 1. Due to the solubility of the CDLA-LID complex in water and its distribution inside the aqueous compartment of liposomes, there are no size variations between simple liposomes and those loaded. Morphology studies were performed only on MLV-type liposomes, SUV-type liposomes could not be analyzed by scanning electron microscopy due to their instability. Fig. 6 and 7 compare the morphology of pure and CDLA-LID-loaded MLV liposomes.

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Size distribution of liposomes

Table 1

Lipid composition Average diameter (nm) SUV liposomes MLV liposomes PC-Chol 112 (±5.6) 2250 (± 45.8) DSPC-Chol 132 (±6.3) 3347 (± 73.2)

in the following, 4 examples of the invention are presented in connection with fig. 1...12 which represent:

- fig. 1, ¹ H-NMR spectrum of CDLA compound;

- fig. 2, ESI-MS spectrum of CDLA compound;

- fig. 3, Ή-NMR spectra of LID, CDLA and CDLA-LID,

- fig. 4, the ESI MS/MS spectrum in which the fragmentation of the parent ion with m/z = 1657 of the CDLA/LID complex system is observed;

- fig. 5. A, solubility diagram of LID in the presence of CDLA; B: Job diagram of the CDLA-LID system;

- fig. 6, SEM image of MLV liposomes;

- fig. 7, SEM micrographs of MLV-type liposomes loaded with CDLA-LID;

- fig. 8, lidocaine release efficiency in vitro\

- fig. 9, morphology of Pol/Chi gels;

- fig. 10, morphology of Pol/Chi/Psi gels;

- fig. 11, algesimetric tests on EMLA and Chi/CDP-LID;

- fig. 12, comparative study on rats treated with CDLA-LID derivatives in the Hot plate and Cold plate tests.

Example 1

Liquid systems of CDLA-LID complexes integrated in a chitosan gel

Transdermal administration and preclinical analysis studies involve the integration of CDLA-LID complexes into chitosan-based gels. Preliminary studies considered obtaining preparations in the form of a solution. LI D and CDLA-LID were dissolved in acidic pH solutions of 2% chitosan (Chi, Brookfield viscosity, 200000 cPs; degree of deacetylation approximately 80%) and 2% lactic acid. Solutions were prepared with LID in concentrations up to 5%. A total dissolution of LID as well as CDLA-type complexes was found.

Example 2

Solid systems of CDLA-LID complex integrated in chitosan gel (CHI/CDLA-LID) The transdermal administration of CDLA-LID complexes involves their incorporation into a pharmaceutical formulation that allows adhesion to the skin under conditions of local diffusion of the active principle and stability at storage of the preparation. The process of obtaining a final solid state product involves dispersing 200 g of CDLA-LID complex by stirring (anchor stirrer, 300 rpm), for 10 min, at room temperature in 540 g of an aqueous solution of 2% chitosan and 2% lactic acid. The obtained viscous dispersion is immediately subjected to the lyophilization process (pressure, 0.045 mbar; temperature, -50°C; time 24 h).

The product obtained in the form of a fine, white powder is stored at room temperature, protected from contact with atmospheric air. Tested after 12 months, it demonstrated that CDLA-LID retains its structural integrity following conditioning in chitosan, in the dry state. The mentioned conditioning method allows a gel to be quickly obtained by simply mixing with a predetermined amount of water.

RO 132702 Β1

These systems were tested in vitro to determine the efficiency of lidocaine release through the dialysis membrane using a Franz Cell type device. The rapid release kinetics up to 20 min and the maintenance of the analgesic effect for more than 100 min are shown in fig. 8 compared to the commercial product EMLA (cream in the form of an oil emulsion (eutectic mixture of base lidocaine and prilocaine in equal proportions) in water in a ratio of 1:1).

Example 3

CDLA-LID complex systems integrated in PCP (poloxamer-chitosanpolyxylosan) gels

A first stage is represented by the preparation of poloxamer-chitosan (Pol/Chi) and poloxamer-chitosan-polysiloxane (Pol/Chi/PSi) gels to benefit from the change in gel morphology and fluidity with temperature, induced by the presence of poloxamer and the increase in the ability to transdermal transport of the active principle from the gel, property provided by polysiloxane. The two types of gels were obtained using different mass ratios between the poloxamer, chitosan and polyxyloxane components (Table 2). Initially, two solutions were obtained: a 25% aqueous solution of poloxamer (Pol) (Pluronic F127) and a 2% chitosan (Chi) solution, (Brookfield viscosity, 200000 cPs, degree of deacetylation 76%) in aqueous mixture: 2% lactic acid, pH 4. The two solutions are cooled to 5°C, mixed on an ice bath by magnetic stirring (300 rpm) for one hour and then lyophilized. To obtain poloxamer-chitosan-polysiloxane gels, polysiloxane (PSi with molecular weight Mn = 1260 g/mol) is added in the mixing phase in an amount of 1% compared to chitosan.

Composition and appearance of POL/CHI and POL/CHI/PSi gels

Table 2

Sample code Mass ratio Pol/Chi (dry matter) appearance At t > 6°C At t < 6°C Pol/Chia 60/1 Fluid gel Pole/Chib 30/1 Fluid Viscous fluid Pole / Chic 20/1 Fluid Viscous fluid Pol /Chid 15/1 Fluid Viscous fluid Pole / Key 10/1 Fluid Viscous fluid Pol/Chi/PSia 60/1 Fluid Gel Pole /Chi/PSib 30/1 Fluid Gel Pole/Chi/PSic 20/1 Fluid Gel Pol/Chi/PSid 15/1 Viscous fluid Viscous fluid Pol/Chi/PSie 10/1 Viscous fluid Viscous fluid

After the optimization study of the formulation of PCP-type gels, followed the stage of preparation of PCP gels loaded with various formulations of lidocaine (CDLA-LID compound). The studies to obtain these types of gels were based on the maximum amount of CDLA-LID that can be solubilized in Pol/Chi and Pol/Chi/PSi solutions (table 3). An increase in the amount of the CDLA-LID complex that can be solubilized in the gel is observed with the decrease in the poloxamer content.

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As can be seen from the data in Table 2, the Pol/Chi samples retain their 1 gelling capacity at low temperatures only for poloxamer/chitosan ratios of 60/1. The decrease of this ratio in favor of chitosan causes the loss of this property. 3 The SEM technique highlights the fact that the Pol/Chi gels are porous (fig. 9), and the pore size varies depending on the Pol/Chi ratio. 5

The addition of very small amounts of hydrophobic polysiloxane results in widening the range of concentrations in which the temperature sensitivity of the gel is preserved (up to 7 at poloxamer/chitosan ratios of 20/1). The introduction of polysiloxane accentuates the microphase segregation of the mixture and further modifies the porosity of the gels (fig. 10). 9

Shrinkage of the roughly hexagonal pores characteristic of chitosan is evident, especially in gels with lower chitosan content.11

Composition of POL/CHI and POL/CHI/PSi gels loaded with CDLA-LID13

Table 3

Sample code Maximum amount of CDLA-LID (w/w, gel) Pol/Chi _a 0.238 Pole/Chi _b 0.197 Pol/Chi _c 0.183 Pol/Chi _a 0.467 Pol/Chi _e 0.470 Pol/Chi/PSi _a 0.567 Pol/Chi/PSi _b 0.688 Pol/Chi?PSi _c 0.751

Example 425

CDLA-LID-loaded ML V liposome complex systems embedded in a chitosan gel (Chi/MLV-CDLA-LID)27 in a typical experimental procedure, the multiple complex preparation step involves mixing CDLA-loaded MLV liposomes LID (9.7 mg) with 1 mL of 2%29 solution of Chi dissolved in lactic acid (2%). These systems were tested in vitro to determine lidocaine release efficiency through membrane dialysis using a 31 Franz Cell device. The rapid release kinetics up to 20 min and the maintenance of the analgesic effect for more than 100 min are shown in fig. 8 compared to the commercial product 33 EMLA (cream in the form of an oil emulsion (eutectic mixture of lidocaine base and prilocaine in equal proportions) in water in a ratio of 1:1) which does not have a burst effect and has a lower 35 release efficiency at 100 min.

Analyzing the release efficiency of the three formulations tested in vitro, we observe 37 the superiority under the given conditions of LI D formulations based on complexes and/or liposomes compared to the commercial product EMLA. 39

Demonstration of the Use of CDLA-LID Derivatives in Transdermal Pain Therapy Pharmacodynamic Animal Tests 41

Effects of CDLA-LID derivatives (CDLA-LID 2.5%, CDLA-LID-liposomes, Chi/CDLALID and Chi/MLV-CDLA-LID) compared with 2.5% lidocaine gel and EMLA 43 cream (components: lidocaine 2.5% and prilocaine 2.5%) were studied at different time intervals

RO 132702 Β1 using adult Wistar rats of similar weight maintained under the same conditions of light and humidity, food and water. All experiments were carried out in accordance with the ethical norms in force, according to the recommendations of the European Commission regarding the performance of preclinical studies with medicinal products, the analytical, pharmacotoxicological and clinical norms and protocols related to the testing of medicines (Order 906/25.07.2006), the Ethics Commission of International Association for the Study of Pain (IASP), and based on the opinion of the Ethics Commission of the University of Medicine and Pharmacy Gr. T. Popa, cowards.

In vivo studies were performed on the skin of rats without hair, removed by a combination of shaving and application of depilatory cream. Initially, the model of inflammatory hyperalgesia with carrageenan was developed according to the specialized literature [H. Wheeler-Aceto, A.Cowan, Psychopharmacology, 1991, 104(1), 35-44], followed by the step of administering the gels/cream to the depilated skin of the posterior thoracic region for 1 min every hour for 4 h. The anesthetic effect of the compounds tested together with the EMLA commercial cream was determined by nociceptive stimulation using the Hot Plate, Cold Plate and algesimetric tests. Rats were tested for response to these tests at baseline, 30 min after application of the cream/gel to the right hindpaw, 1 h, and hourly up to 4 h. The results of the in vivo tests demonstrate the analgesic effect of compounds derived from CDLA-LID, especially the compound Chi/CDLA-LID and Chi/MLV-CDLA-LID compared to EMLA. In algesimetric testing, the Chi/CDLALID complex comes into action slowly up to 15 min after administration and has a maximum effect at 1530 min, then the effect diminishes but does not disappear even at 2 h (fig. 11), net superior effect reported to the effect of EMLA cream. The results obtained with the analgesia meter were also verified by the Cold Plate test, where the effect of Chi/CDLA-LID is significantly higher than that of EMLA cream at 30 min after administration, then the effect is diminished, and at one hour it is similar to that of EMLA cream and then regresses (fig. 12). Hot Plate testing 5 min after administration reveals a significantly greater effect of Chi/CDLA-LID gel than EMLA cream (fig. 12), an effect that is significantly preserved when tested at 15 min, 30 min and the same as in the previous test, it had a similar effect to the EMLA cream one hour after administration, the effect decreasing towards 2 h. From an anatomopathological point of view, the changes found in the harvested fragments (the rats were euthanized according to the ethics in force) from the batch that was administered Chi/CDLA-LID are similar to the batch tested with EMLA. Behaviors similar to the Chi/CDLA-LID gel were also found for the Chi/MLV-CDLA-LID liposomal formulations, superior to the commercial EMLA product (fig. 12).

Claims (6)

RO 132702 Β1 Claims 1 1. Process for obtaining a pharmaceutical formulation with analgesic properties 3 comprising complex systems based on lidocaine, cyclodextrin derivatives and liposomes, characterized in that: 5 - unilamellar/multilamellar liposomes are formed based on single phospholipids or in combinations of the glycerophospholipid type, chosen from phosphatidylcholine, phosphatidylethanolamine, 7 phosphatidylserine, phosphatidylinositol, cardiolipins and sphingophospholipids and/or cholesterol and/or their derivatives, followed by hydration of these liposomes with a solution of lidocaine 9 incorporated in the cyclodextrin derivative obtained by: - the modification of α-, or β-, or γ-cyclodextrin, preferably β-cyclodextrin with 11 sequences of oligoesters through the opening reaction of the lactide cycle, with the formation of a product containing a number of lactide units between 1..21 , preferably 1...4 strung 13-lactide units; - the inclusion of lidocaine through an open system mixing process with the complexation stoichiometry in the range of 0.5...1.5 with an optimal value of 1:1; - it results in obtaining a complex system that increases the solubility of lidocaine in acidic media, increases bioavailability and toxicity to a value below the legal threshold established in the case of use as a transdermal delivery system of 19 drugs for external use. 2. Process according to claim 1, characterized in that the inclusion step 21 of the lidocaine solution incorporated in the cyclodextrin derivative in the lipozymes is optional. 3. Method according to claim 1, characterized in that, to improve23 the stability over time and increase the adhesiveness on the skin, the complex system based on lidocaine, cyclodextrin derivatives and liposomes or the system based on lidocaine and cyclodextrin derivatives are conditioned with selected polymeric systems between poloxamer-chitosan or poloxamer-chitosan-polysiloxane.27 4. Process according to claim 3, characterized in that, in the case of the poloxamer-chitosan polymer system, the chitosan has a molecular weight between 50000.. .40000029 Yes, viscosity between 20...2000 cP or -<100 mPa.s, degree of deacetylation between 75...85% or chitosan derivatives which preferably have a viscosity of 200 cP31 and degree of acetylation of 76% and the poloxamer has a variable number of polypropylene-glycol and polyethylene-glycol units, molecular mass between 1.9-15 kDa, optimally Pluronic F127, and the mixing mass ratio is between 80:1 and 5:1 . 5. Process according to claim 3, characterized in that, in the case of the poloxamer-chitosan polymer system or chirosan-polysiloxane derivatives, the polysiloxane is functionalized with terminal amino propyl groups and has a molecular weight between 37 1000...2500 Da, optimal 1 260, and the mixing ratio to chitoxan is from 0.5 to 1.5%, chitosan has a molecular mass between 50-400 kDa, viscosity between 39 20-2000 cP or -<100 mPa.s, grade of deacetylation between 75-85%, and the chitosan derivatives preferably have the viscosity of 200 cP and the degree of deacetylation of 76%, the poloxamer-chitosan or chitosan derivatives mixing mass ratio 41 is between 80:1 and 50:1. 43 6. Pharmaceutical formulation with analgesic properties obtained by the process defined in claims 1-5. 45