BRPI1103250A2 - Nanostructured Compositions of Elastic Liposomes; Nanostructured compositions of elastic liposomes coated with any type of thermosensitive block copolymers; Nanostructured compositions of any type of elastic liposome for ocular application in the treatment of nanostructured elastic liposome compositions; Nanostructured compositions of elastic liposomes coated with any type of thermosensitive block copolymers; Nanostructured compositions of any type of elastic liposome for ocular application in the treatment of sub-retinal NEOVASCULAR EDEMAS AND RETINAL NEOVASCULAR MEMBRANES; Nanostructured compositions for ocular application in the treatment of subreetinal neovascular membranes and retinal neovascularization; MICRO AND Nanostructured compositions for ocular application in the treatment of keratoconus - Google Patents

Abstract

Nanostructured Compositions of Elastic Liposomes; Nanostructured compositions of elastic liposomes coated with any type of thermosensitive block copolymers; Nanostructured compositions of any type of elastic liposome for ocular application in the treatment of subreutinal neovascular membranes and retinal vasovascularization; Nanostructured compositions for ocular application in the treatment of non-vascular sub-retinal membranes and retinal neovascularization; MICRO AND NANO-STRUCTURED COMPOSITIONS FOR OCCULAR APPLICATION IN THE TREATMENT OF CERATOCONE The present invention relates to compositions containing the monoclonal antibody Bevacizumab and other pharmaceutically acceptable compounds with related chemical or similar therapeutic effect. Optionally associated with nanostructured pharmaceutical systems such as conventional polymer and copolymer-coated liposomes, liposomes, elastics and liposomes sensitive to any stimuli of the biological medium in which it is carried, especially thermosensitive block polymers and copolymers, as well as their presentation forms. pharmaceuticals for use in the treatment of subretinal neovascular membranes, macular edema and retinal neovascularization. The subject matter described in this paper reports the possible compositions and applications of elastic liposomes and liposomes coated with block copolymers for monoclonal antibody delivery, especially Bevacizumab, and other intraocular drugs applicable in the treatment of subretinal neovascular membranes, edema. macular and retinal neovascularizations.

Description

“Nanostructured compositions of elastic liposomes; Nanostructured compositions of elastic liposomes coated with any type of thermosensitive block copolymers; Nanostructured compositions of any type of elastic liposome for ocular application in the treatment of subreutinal neovascular membranes and retinal neuronal edema; Nanostructured compositions for ocular application in the treatment of subreutinal neovascular mem- bers Macular edema and retinal neovascularization; MICRO AND NANOTRUCTURED COMPOSITIONS FOR OCCULAR APPLICATION CERATOCONE TREATMENT ”

1- Summary

Vascular endothelial growth factor (VEGF) plays a key role in the development of various ocular pathologies associated with increased vascular permeability and / or neovascularization, such as macular edema, retinal vascular occlusions, diabetic retinopathy, related macular degeneration. age (AMD), prematurity retinopathy and neovascular glaucoma, among others.

Recent anti-VEGF drugs (bevacizumab, ranibizumab) have revolutionized the treatment of such pathologies over the past five years and especially exudative AMD, greatly improving the prognosis of patients and thereby achieving widespread and widespread dissemination. its use worldwide quickly.

However, its posiologies require administrations through monthly intra-vitreous injections indefinitely, which increases the risk of ocular complications such as retinal detachment, vitreous hemorrhage, endophthalmitis and uveitis. In addition to taking a high cost treatment and poor patient compliance. The development of slow-release anti-VEGF drug systems that reduce the need for intra-vitreous applications, reducing risks, cost and increasing patient adherence to treatment is of great interest in public medicine.

Drug delivery systems using nanovesicles such as liposomes and niosomes have a major advantage over conventional release systems because these systems act as drug reservoirs, so that a modification in the composition of structural lipids or properties can adjust the release rate or targeting of target tissues. The composition of nanovesicles is composed of mixtures of phospholipids and neutral surfactant, or 10 lipid, neutral surfactant and block copolymer, exhibiting a phase transition around 37 ° C. The lipid / surfactant mixture contains between 0.020 and 20 mol percent phospholipid associated with a hydrophilic polymer and additionally may also contain a thermosensitive block copolymer. Trapped in the aqueous phase of the liposomes will be the monoclonal antibody at a concentration of 1 to 5mg / mL, preferably 1.25mg / mL. The process of liposome preparation may be by reverse phase evaporation, consecutive dehydration-rehydration, ultrasound irradiation or, preferably, by the pressure homogenization technique, which provides better encapsulation efficiency and safety with respect to integrity. of the monoclonal antibody molecule. The process leads to the obtaining of structures with diameters suitable for the proposed application.

2- Field of the Invention

The present invention relates to compositions containing the monoclonal antibody Bevacizumab and other pharmaceutically acceptable compounds with related chemical structures or similar therapeutic effect. Optionally associated with nanostructured pharmaceutical systems such as liposomes, liposomes, elastics and liposomes coated with conventional polymers and copolymers sensitive to any stimuli from the biological medium in which it is delivered, especially thermosensitive block polymers and copolymers, as well as its pharmaceutical forms for use in the treatment of subretinal neovascular membranes, 5 macular edema and retinal neovascularization. The subject matter described herein reports the possible compositions and applications of elastic liposomes and liposomes coated with block copolymers for the delivery of monoclonal antibodies, especially Bevacizumab, and other intraocular drugs applicable in the treatment of this invention. - development of subretinal neovascular membranes, macular edema and retinal neovascularization.

3- Background of the Invention

3.1 Bevacizumab and related molecules

Angiogenesis is a process of developing new blood vessels from pre-existing blood vessels or bone marrow-derived endothelial precursor cells. It is essential for embryonic development, normal tissue growth, healing, and plays a key role in many diseases. Control of angiogenesis occurs through regulation between pro and anti-angiogenic factors in the body [TONINI et al., 2003]. When pro-angiogenic factors are produced in higher concentrations than anti-angiogenic factors, 20 new vessels are formed (angiogenesis). Under normal conditions, the body is able to maintain the balance between angiogenic modulators. In situations in which the organism loses the ability to modulate angiogenesis, pathologies related to increased vascular permeability and abnormal (neovascular) vessel formation arise [POLVERINI, 2002; TONINI et al., 2003].

Excessive angiogenesis occurs in various diseases, such as cancer (solid or hematological tumors), cardiovascular disease (atherosclerosis), chronic inflammation (rheumatoid arthritis, Crohn's disease), psoriasis (lesions characterized by increased vessel size and length). dermis), endometriosis and various eye diseases such as exudative age-related macular degeneration (AMD), proliferative diabetic retinopathy (RDP), choroidal neovascularization (CNV), prematurity retinopathy, vascular occlusion retinal and uveitis, among others [ALON ET AL., 1995; HANAHAN & FOLKMAN, 1996].

Several studies have shown that there is a correlation between neovascularization, which occurs during ophthalmic pathologies, and the presence of ocular ischemia, which continuously stimulates the production of VEGF, a key molecule in the development of neovascularization [AIELLO et al., 1994]. ; ADAMIS et al., 1994; CARMELIET & JAIN, 2000].

Bevacizumab competitively blocks VEGF receptors. The drug, a humanized monoclonal antibody obtained by recombinant DNA technology, was approved by the Food and Drug Administration (FDA) in 2004 and the European Community in 2005. When combined with conventional chemotherapy, it has prolonged patient survival. with lung, colon and breast cancer [HURWITZ et al., 2004],

In addition to the application of Bevacizumab in the treatment of various cancers, the drug has also been used to treat ocular pathologies resulting from excessive angiogenesis, such as Choroid Neovascularization (CNV), Proliferative Diabetic Retinopathy (RDP) and Age Related Macular Degeneration (AMD) [PANDYA et al., 2006; BARKIS, et al., 2006; 2007],

The first use of Bevacizumab in the treatment of neovascular age-related macular degeneration (AMD) was described in 2005 [MICHELS et al., 2005], when the authors intravenously infused the drug with improved visual acuity, OCT thickness and angiographic findings, with good tolerability.

Next, the intra-vitreous application of Bevacizumab in the treatment of neovascular AMD was described, providing a safer, more effective and less costly therapeutic option [ROSENFELD et al., 2005].

Following such studies many other authors have published hundreds of studies describing the benefit of using intra-vitreous Bevacizumab in the treatment of numerous eye diseases associated with increased vascular permeability and / or neovascularization such as: secondary subretinal neovascular membrane the AMD [AVERY et. al., 2006; SPAIDE et. al., 2006], myopia [SAKAGUCHI et. al., 2007] diabetic retinopathy [SPAIDE and FISHER, 2006], macular edema [MASON et. al., 2006] and retinal venous occlusion [ROSENFELD et.al., 2005].

Bevacizumab (Avastin®, Genentech, South San Francisco, CA) is a humanized VEGF monoclonal antibody that selectively inhibits all VEGF-A isoforms. It has been approved as a first-line treatment for metastatic colorectal cancer.

Considering that the intra-vitreous dose applied for local effect is about four hundred times lower than the intravenous dose used in conventional therapy, the potential toxicity of the drug is practically nonexistent.

Ranibizumab (Lucentis®, Genentech, South San Francisco, CA) is a humanized anti-VEGF fragment derived from Bevacizumab, which binds to all VEGF isoforms, was approved for treatment by FDA Age-Related Macular Degeneration in 2006 and ANVISA in September 2007.

However, the proposed treatment regimen is monthly intra-vitreous injections, requiring up to 24 applications in 2 years at a time, which increases the risks directly associated with the procedure, such as: endophthalmitis, vitreous hemorrhage, retinal detachment and uveitis [ROSENFELD et. al., 2006] [BRESSLER, 2009].

The cost of ranibizumab treatment is about 20 times more expensive than bevacizumab treatment, excluding, especially in underdeveloped countries, access to treatment by thousands of patients.

However, non-inferiority studies show that bevacizumab is at least as good as ranibizumab in the treatment of wet AMD [SUBRAMANIAN et. al., 2009].

Of great interest in public medicine [GARATTINI and BERTELE, 2010] is the development of slow-release anti-VEGF drug systems (especially bevacizumab) that reduce the need for intra-vitreous applications, reducing risks, cost. and increasing patient compliance with treatment.

The beneficial pharmacological effects of treating eye disease with these drugs can be greatly enhanced through the use of nanostructured drug delivery systems, especially nanostructured controlled eye release systems, reducing by at least one third to need for intra-vitreous injections due to prolonged therapeutic eye concentration.

3.2 Nanostructured Ocular Controlled Release Systems Controlled release systems based on the liposome platform have been

proposed as carriers for a wide variety of compounds, including pharmacologically active substances by any route of administration, diagnostic agents and cosmetics in general. Liposome structures typically may contain one or more hydrophobic bilayers (lipid or not) separated by aqueous phases and containing a central aqueous compartment. Substances of interest may be included in all aqueous phases or hydrophobic bilayers, or both, depending on their physicochemical nature. Water soluble compounds are not favorably incorporated into hydrophobic bilayers, but are suitable for encapsulation in internal aqueous spaces. Thus, water soluble substances tend to be encapsulated in the 5 aqueous phases, with certain advantages, because apart from being separated from the external environment by hydrophobic bilayers, they generally do not interfere with the stability of liposome structure [OLIVEIRA et al., 1992; OLIVEIRA, SCARPA & MILK, 1997; SCARPA, OLIVEIRA & CUCCOVIA, 1998; MOSQUEIRA et al., 2000; LIMA & OLIVEIRA, 2002; SAHOO et al., 2008]. Alternatively, the liposome structures may be unilamellar, containing a single central aqueous phase. These structures can be prepared by a wide variety of methods, giving rise to multilamellar vesicles (MLV); large unilamellar vesicles (LUV) and small unilamellar vesicles (SUV) [PUISIEUX, 1983; NEW, 1990].

In general, monoclonal antibodies are water soluble and can be encapsulated in the aqueous phases of liposomes making the structure of the nano-organized system (lipid bilayer) play a key role in drug release from the internal aqueous phase through hydrophobic bilayer. Thus, there remains a need for efficient biocompatible intraocular drug delivery systems with sufficient shelf life to respond to the stimulus of intraocular diseases and with the capacity to deliver therapeutic concentrations of drugs needed to treat these diseases.

4- DETAILED DESCRIPTION

4.1- Composition of elastic liposomes and coated liposomes and method of preparation

The composition of liposomes containing a water-soluble compound, such as monoclonal antibodies, encapsulated in the aqueous phase is desirable. Water soluble compounds are evidenced herein by a solubility of greater than 100 mg / ml at room temperature.

In the composition, liposomes are vesicles formed by phospholipids / surfactant or phospholipid / surfactant / block copolymer, which are formed by the use of high energy, such as ultrasound or high pressure homogenizer, and whose organization involves head contact. polar to the outside of the bilayer and the hydrophobic region to the inside of the lipid bilayer.

Liposomes have been successfully employed in the pharmaceutical field since 10 can compartmentalize drugs by modifying their bioavailability [OLIVEIRA et al., 2004; SILVA, 2004; OLIVEIRA et al., 2005; WANKZINSKI, 2005; OLIVEIRA, 2006]. They are microscopic vesicles formed by lipid bilayers alternated by aqueous compartments and containing a central aqueous compartment (Figure 1) [OLIVEIRA et al., 1992; OLIVEIRA et al., 1997; SCARPA & OLIVEIRA, 1998; MOS-15 QUEIRA et al., 2000; LIMA & OLIVEIRA, 2002; SAHOO et al., 2008].

The attached figure 1 shows the unilamellar liposome structure and number 1 therein identifies the central aqueous compartment; 2 bilayer; and 3 structural lipid.

They usually have as structural component phospholipids, mainly phosphatidylcholine of various origins (PC), or another component capable of forming bilayers, such as neutral surfactants derived from the sorbitan ring [GASPAR, 2006; FORMARIZ, TERRUGI, SILVA-JR, SCARPA & OLIVEIRA, 2006; OLIVEIRA et al .; 1997; TORCHILIN, 2005].

Other lipids with stable incorporation capacity within the bilayer may also be used. The vesicles are preferably formed of lipids typically containing two carbon chains and a polar head. Neutral surfactants are preferably formed by a carbon chain and a polar head. Block copolymers also have the characteristic hydrophilic and hydrophobic regions.

There are a wide variety of liposomes formed by natural or synthetic lipids, including phospholipids, such as: phosphatidylcholine, hydrogenated or unhydrogenated soybean phosphatidylcholine, phosphatidylserine, phosphatidyltannolamine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin and phosphate chain length, Carbon dioxide is typically between 14 and 22 carbon atoms, longitudinally and with varying degrees of unsaturation.

Liposomes are prepared by mixing a lipid with a neutral surfactant with a non-high transition temperature, but which may transition from the “sun” (low viscosity) state to a gel (high viscosity) state by the effect of a block copolymer, thermosensitive when in contact with eye tissues, at 36-37 ° C. This phase transition can be favored by slightly higher temperatures, if there are local inflammatory or infectious processes.

Examples of lipids that may be used in liposome composition include: hydrogenated, partially hydrogenated or unhydrogenated soybean phosphatidylcholine; distearoylphosphatidylcholine (DSPC); dimyristoylphosphatidylcholine (DMPC); dioctanoylphosphatidylethanolamine (DOPE); dipalmitoylphosphatidylethanolamine (DPPE); dipalmitioylphophatidylcholine (DPPC). In the liposomes proposed herein, it is not convenient to combine other lipids, such as cholesterol, which increase the rigidity of the lipid bilayer. TABLE I.

Physicochemical characteristics of some phospholipids used to obtain liposomes.

Carbon Phospholipid Ne: Temperature Electric charge Transition unsaturation at pH 7.4 Tm (0C) DSPC 18: 0 55 0 DMPC 14: 0 23 0 DOPE 18: 1 50 0 DPPE 16: 0 -16 0 DPPC 16: 0 41 0 As Deformable or elastic base liposome formulations should contain, in addition to structural phospholipid, another component capable of integrating the bilayers and forming the activating edge. Examples include single-chain ionic surfactants such as: cholate and deoxycholate, sorbitan ring derivatives such as polyoxyethylene sorbitan esters (water soluble) and sorbitan esters (hydrophobic), among others, whose main function is to form the liposome with an elastic structure about to deform without breaking its spherical structure.

The liposome formulation may also be comprised of block copolymers,

pharmaceutically acceptable, with the function of taking temperature-sensitive structures so that the preparations are liquid at room temperature, but with higher viscosity at the temperature of the human organism [CHANDDAROY et al., 2001], particularly for release. ocular [MA et al., 2008]. Examples of block copolymers useful in such preparations include: copolymers and block polymers derived from oxyethylene oxypropylene, polyoxypropylene polyoxyethylene and polyoxyethylene polyoxyethylene and polyoxyethylene polyethylene, all with molecular weights. giving different thermosensitivity and nanoorganization properties of the molecules. At a suitable temperature or when diluted in solvents, copolymer blocks can be freely mixed to generate disordered structures. However, the most common is a self-structuring with formation of a diversity of mesophases regulated by the size of the molecules. In mesophases there are different blocks in distinct microdomains which are highly enriched with blocks of the same type (eg polyoxyethylene or polyoxypropylene), sometimes to the point where essentially pure microdomains exist [LEE & REGISTER, 2004; LOO & REGISTER, 2004; DISCHARGE. et al., 19991.

As an example, the equilibria between the different known organized structures for the thermosensitive diblock copolymers are shown in Figure 2, in which the gradual migration from the orderly structure to the disordered structure and vice versa is dependent on the temperature of the structure. external environment or biological environment.

The attached figure 2 shows the structural organization of the coating polymer and in it the number 1 indicates the ordered structure; 2 refers to the double-headed arrow indicating that the organization is thermally reversible; and 3 indicates disordered structure.

The structure of the lipid bilayer is composed of the combination of phospholipid (soybean phosphatidylcholine) and a neutral surfactant derived from polyoxyethylene sorbitan, with hydrophilic characteristics sufficient to form a mixture with phospholipid, which induces the formation of an elastic bilayer with skill. deform without breaking the spherical structure.

TABLE 2

Composition of some formulations of elastic liposomes and liposomes coated with Pluronic F-127 thermosensitive block copolymer and empty liposomes. Formulation PC (mg) Tw 20 (mg) PL (mg) BZ (mg) EL 120 - - - ELTw 120 3 - - ELTwPL 120 3 6 - ELBZ 120 15 - 3,70 ELTwBZ 120 3 - 4,00 ELTwPLBZ 120 3 6 4.50 LEGEND: (EL) Empty Liposomes; (ELTw) Empty Elastic Liposomes; (ELTwPL) Elastic Coated Liposomes;

(ELBZ) Bevacizumab Liposomes; (ELTwBZ) Bevacizumab Elastic Liposomes; (ELTwPLBZ) Elastic Liposomes Coated with bezacizumab.

The amounts of drug detected in the various liposome formulations are shown in Table 2.

TABLE 3

Some physicochemical characteristics of elastic liposomes and thermosensitive polymer coated liposomes containing bevacizumab and empty liposomes.

Formulation Diameter (nm) Polydispersity Kcps Potential zeta (mV) Average (n = 10) EL 94.6 0.249 354.9 3.31 ELTw 93.1 0.235 332.3 3.15 ELTwPL 175.0 0.301 296.5 1, 01 ELBZ 91.3 0.274 319.2 3.73 ELTwBZ 90.1 0.265 313.6 0.95 ELTwPLBZ 207.0 0.313 315.1 0.61 LEGEND: (EL) Empty Liposomes; (ELTw) Empty Elastic Liposomes;

(ELTwPL) Elastic Coated Liposomes; (ELBZ) Bevacizumab Liposomes;

(ELTwBZ) Bevacizumab Elastic Liposomes; (ELTwPLBZ) Bezacizumab-coated Elastic Liposomes.

When included in the total lipid mixture, the surfactant compound (Tw) forms a

a homogeneous mixture with the phospholipid, taking the deformable lipid bilayer, plastic enough to make the structurally elastic and highly bioadhesive liposome. On the other hand, the thermosensitive polymer is incorporated into the liposomes together with the structural phospholipid, typically up to 15% relative to the phospholipid mass (Table 3). Due to its hydrophobicity the thermosensitive polymer is adhered to the external interface of the lipid bilayer, making it the most stable structure from the physical point of view and with the property of delaying the release of bevacizubam (Figure 3).

The attached figure 3 shows a schematic drawing of the thermosensitive polymer coated liposomes containing or not bevacizumab, in which BZ indicates bevacizumab.

When the active drug bevacizumab is nanoencapsulated in the structure of any of the described liposomes, it becomes partitioned within the aqueous phases if more than one peck is formed or only within the central aqueous phase when the formation of unilamellar structures is formed.

Typically the dispersion medium is an isotonic saline solution suitable for

ocular, periocular, intraocular or intravenous injection, such as 1% sodium chloride, with an osmolarity of about 350m0sm / kg. Alternatively, non-thermosensitive polymer coated elastic liposomes may also be dispersed in a structured carrier based on saline plus hydrophilic polymer in sufficient quantity to generate a hydrogel-like dispersion.

Dispersion of either liposome can be easily sterilized by sterile filtration or gamma radiation [PAUL et al., 2007], or by the two conjugated methodologies. These liposomes have excellent retention property of the active drug bevacizumab or other equivalent.

The phospholipid concentration was determined by the classical method of

phosphate measurement. The potency of bevacizumab in the formulations was determined by analysis of VEGEF gene expression in human fibroblast cell line. The drug release ability from liposome formulations was accomplished through in vivo pharmacokinetic profile with rabbits. Encapsulation efficiency was calculated from three determinations of the drug / polymer ratio. The diameters of elastic liposomes and thermosensitive polymer coated liposomes were measured by dynamic laser light scattering on Brookhaven Instruments Corporation model EMI 9863 with He-Ne IOmW laser light source with detector. at 90 ° and 532nm-HUGHES wavelength and 25 ° C temperature (Table 5 3). The characterization of the nanostructures was performed by low angle X-ray scattering (SAXS).

It is also understood that the determination of a given differentiated dose regimen for a specific patient treatment is also possible to meet a specific medical prescription. Because the required dose of treatment 10 may vary from person to person depending on age, general health, and the aggravation of the specific disease, doctors may prescribe some treatment that begins with a lower drug level to produce the response. desired dose and gradually increase the dose until the desired effect is achieved.

4.2 Differentiated biological and pharmacokinetic profiles of encapsulated Bevacizumab in elastic liposomes and coated liposomes relative to unencapsulated drug.

4.2.1 Analysis of VEGF gene expression with defibroblastoshuman cell lines.

Prior to evaluating the in vivo efficiency of the formulations obtained, the efficiency of the encapsulated drug was tested in cell culture (in vitro). The evaluation was performed by two distinct methods, called ELISA and Real Time PCR (Real Time 20 Polymerase Chain Reaction), which allowed the determination of the efficiency of Pluronic F-127 block copolymer elastic liposomes and liposomes. in vivo release (vitreous humor and aqueous humor) of the drug compared to the commercial formulation of bevacizumab. The evaluation was based on the ability of bevacizumab-containing elastic and coated liposome preparations to inhibit VEGF expression by human fibroblasts, compared to the commercial formulation of bevacizumab (Avastin aqueous solution for injection). The cell line used is well known for expressing and secreting a considerable amount of this growth factor. Petri dish cultured human fibroblasts were respectively incubated with bevacizumab in the commercial formulation, bevacizumab in elastic 5 liposomes and pluronic F-127 thermosensitive block copolymer coated bevacizumab liposomes at a concentration of 0.25 mg / mL. from bevacozumab. As control a culture of untreated cells was used. Real-time PCR allows the identification and quantification of genes expressed in the basal form (physiological condition), induction of increased gene expression (up regulatiori) or suppression of gene expression (down regulatiori). Results from Real-Time PCR analysis clearly show that bevacizumab preparations in elastic liposomes and in Pluronic F-127 thermosensitive block copolymer-coated liposomes were remarkably more efficient in inhibiting VEGF expression when compared to the formula. - Commercial bevacizumab (Avastin). The most intense inhibition of VEGF was with the liposomal formulation of the PLU F-127 thick block copolymer coated bevacizumab, followed by the less intense formulation of elastic liposomes revealed by the commercial bevacizumab. Relative quantification of gene expression was normalized by comparing amplification with an endogenous control using the β-actin gene (Table 4).

TABLE 4.

Primer sequence used in real-time PCR experiments.

Gene Primer Direct Reverse Primer Amplified Product (bp) VEGF CACATT GTT GGA-ACT C AC AC AC A- 165 AGAAGCAGCCCA CAACCAGGTCT β-actin CGTGGGCCGCCC- CGGAGG AAGAG-604 TAGGCACCAGGG GATa a figure showing a effect AGGGa a Figure 4 effect shown of elastic liposome (BEV LIP) formulations, plutonic thermosensitive block copolymer coated liposomes F-127 (BEV LIP PLU) containing bevacizumab and commercial bevacizumab (BEV) in the expression of VEGF in human fibroblasts.

The cells were incubated for 8 hours with the formulations at the indicated concentrations and then lysed for total RNA extraction. mRNA was transcribed into cDNA, used for real time PCR reactions with VEGF-specific primers.

The VGEF expression recorded in figure 4 is inversely proportional to the intensity of the arbitrary unit (ARBITRARY UNIT). Therefore, both bevacizumab liposome preparations were much more effective at inhibiting cell VEGF expression than the drug in commercial solution.

4.2.2 Ocular pharmacokinetics of bevacizumab in elastic liposomes, liposomes coated with Pluronic F-127 thermosensitive block copolymer and bevacizumab in the commercial formulation.

To determine the ocular pharmacokinetic profile of bevacizumab, female albino rabbits, with body mass between 1.5 and 2.0 kg, were fasted 12 hours before the start of the test. The experiment lasted 29 days with sample collection after 1, 3, 8, 15 and 29 days. The tests were performed in quadriplicate and for each day of sample collection 3 groups of animals containing 4 animals in each group were used (administration of commercial bevacizumab, 20 bevacizumab in elastic liposomes, bevacizumab in liposomes coated with Pluronic thermosensitive block copolymer). F-127 and control group, totaling 60 animals).

The animals were previously anesthetized by injecting the combination of ketamine hydrochloride and xylazine hydrochloride, 35 and 3 mg / kg respectively, intramuscularly. Subsequently, 0.2 ml of each preparation, equivalent to 1 mg bevacizumab, was administered intravitreally as a single dose. At 1, 3, 8, 15 and 29 days the animals were sacrificed, the eyes were nucleated, the collected vitreous humor (HV) and aqueous humor (HA) samples were analyzed by indirect colorimetric method. Sample preparation as well as bevacizumab quantification were performed 5 with the Easy-Titer® IgG Assay Kits, PIERCE kit according to the instructions provided by the kit manufacturer.

To determine Bevacizumab concentration in the HV and HA samples, a standard curve was constructed with the standard established by the Commercial Dilution Buffer Kit (Easy-Titer® IgG Assay Kits). Dilutions were prepared in triplicate at concentrations of 15.6 ng / mL, 31.2 ng / mL, 62.5 ng / mL, 125.0 ng / mL, 250.0 ng / mL, and 500.0 ng. / ml.

Due to the range of the kit used to quantify Bevacizumab in vitreous and aqueous humor (15-300 ng / mL), the samples were previously diluted in sodium phosphate buffer (PBS) to obtain a concentration of 100 μg / mL. Then each of the samples was further diluted in Dilution Buffer (Kit) to obtain a concentration of 250ng / mL. The concentrations found in the samples were subsequently corrected by the respective dilution factors used (1: 4 to 1: 500).

The accompanying Figure 5 is a graph showing the variation of Bevacizumab concentration in vitreous humor after intravitreal injection of 1.25 mg Bevacizumab into elastic liposomes, plutonium thermosensitive block copolymer coated liposomes. 127 and commercial bevacizumab (n = 3). In this figure, the Ocular Pharmacokinetics Profile of Bevacizumab in vitreous humor following intravitreal injection of 1.25 mg / mL commercial bevacizumab is indicated by (□); bevacizumab in elastic liposomes is indicated by (V); and Liposomes coated with Pluronic F-127 thermosensitive block copolymer is indicated by (O). Average of 3 determinations

The accompanying Figure 6 is a graph showing the variation of Bevacizumab concentration in aqueous humor following intravitreal injection of 1.25 mg of Bevacizumab into elastic liposomes, Liposomes coated with Plur 5 ronic F-127 thermosensitive block copolymer and commercial bevacizumab (n = 3). In this figure, the Ocular Pharmacokinetics Profile of Bevacizumab in aqueous humor following intravitreal injection of 1.25 mg / mL commercial bevacizumab is indicated by (Δ); bevacizumab in elastic liposomes by (□); and Plxironic F-127 thermosensitive block copolymer coated liposomes by

(THE). Average of 3 determinations.

The data in Figure 6 clearly show that the concentrations of bevacizulase

mabe in the formulations of bevacizumab in elastic liposomes and liposomes coated with Pluronic F-127 thermosensitive block copolymer remained higher than the concentration of bevacizumab in the commercial formulation throughout the pharmacokinetic follow-up. At the end of 29 days the concentration of 15 bevacizumab was still significantly higher, indicating that the formulations developed are completely adequate for the purposes of the intended clinical applications.

In many clinical cases intravitreal administration becomes indispensable and necessary, especially in the case of posterior eye diseases. On the other hand, the application of repeated intravitreal injections may lead to the appearance of ocular lesions (sequelae), hence the reason for the application of modified release systems for bevacizumab developed here and described which undoubtedly differ from commercial medicinal products. bevacizumab, because they increase the therapeutic efficacy and prolong the effect of bevacizumab, decreasing the patient's exposure to the possible toxic effects of the drug caused by higher doses and the potential trauma derived from the frequency of application required by the commercial four formulation. At the end of 29 days of pharmacokinetic follow-up no lesions were detected in the animal's ocular tissues, as well as any inflammatory response or other type of injury to the ocular tissues.

5- List of attached figures:

Figure 1 shows the unilamellar liposome structure;

Figure 2 shows the structural organization of the coating polymer;

Figure 3 shows a schematic drawing of thermosensitive polymer coated liposomes containing or not bevacizumab;

Figure 4 is a graph showing the effect of elastic liposome formulations.

cos (BEV LIP), Pluronic F-127 thermosensitive block copolymer coated liposomes (BEV LIP PLU) containing bevacizumab and commercial bevacizumab (BEV) in the expression of VEGF in human fibroblasts;

Figure 5 is a graph showing variation of Bevacizumab concentration in vitreous humor following intravitreal injection of 1.25 mg of Bevacizumab into elastic liposomes, Pluronic F-127 thermosensitive block copolymer-coated liposomes and commercial bevacizumab (n = 3 ); and

Figure 6 is a graph showing variation of Bevacizumab concentration in aqueous humor following intravitreal injection of 1.25 mg Bevacizumab into elastic liposomes, Pluronic F-127 thermosensitive block copolymer-coated liposomes and commercial bevacizumab (n = 3 ).

6- BIBLIOGRAPHICAL REFERENCES

ADAMIS A.P., MILLER J.W., BERNAL M.T., D5AMICO DJ., FOLKMAN J., YEO T.K., YEO K.T. Increased vascular endothelial growth factor in the vitreous of the eyes with proliferative diabetic retinopathy. Am. J. Ophthalmol., V. 118, no. 4, p.445-450, 1994.

AIELLO L.M., FERRARA N. Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders. The New Engl. J. Med. V.331, no.22, p. 1480-1487, 1994.

ALON T., HEMO I., ITIN A., PETER J., STONE J., KESHET E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med. P. 1024-1028, 1995.

AVERY R.L., DANTE J.P., MELVIN D.R., CASTELLARIN A.A., NASIR M.A., GIUST MJ. Intravitreal Bevacizumab (Bevacizumab) for Neovascular Age-Related Macular Degeneration. Ophthalmol. v. 113, no. 3, p. 363-372, 2006.

BAKRI S.J., CAMERON DJ., MACCANNEL C.A., PULIDO J.S., MARLER RJ. Absence of retinal histologic toxicity of intravitreal Bevacizumab in a rabbit model. Am. J. Ophthalmol. v. 142, no. 162-164, 2006.

BAKRI SJ., SNYDER M.R., REID J.M., PULIDO J.S., SINGH RJ. Pharmacokinetics of intravitreal Bevacizumab (Avastin). Ophalmology vol.14, no.5, p.855-859, 2007.

BRESSLER NM. Antiangiogenic approaches to age-related macular degeneration today.Ophthalmology, v. 116 (10 Suppl), p. S15-23, 2009.

CARMELIET, P. Mechanisms of angiogenesis and arteriogenesis. Nature Med., V. 6, p. 389-395,

2000

CHANDAROY P., WITHOUT HUI S.W. Temperature-controlled content release from encapsulating liposomes PLU F-127ronic F127. J Control Release, v.76, p.27-37, 2001.

DISCHER B.M .. WON Y.Y .. EGE D.S .. LEE J.C .. BATES F.S DISCHER D.E .. HAMMER D.A. Polymersomes: tough vesicles made from diblock copolymers. Science, v.14, no.28, pp. 143-1146, 1999.

FINGER P. Anti-VEGF Bevacizumab (Bevacizumabe®) for Radiation Optic Neuropathy. American J. Ophthal. v.143, p.335-338, 2007.

FORMIZE T.P., TERRUGI C.H.B., SILVA-JUNIOR A.A., SCARPA M.V., OLIVEIRA A.G. Biotechnological strategies for the controlled release of antigens and adjuvants in vaccines through liposomes. Acta Farm. Bonaerense v.25, no. 4, p.619-626, 2006.

GARATTINI S B.V. Bevacizumab and ranibizumab. A matter of public interest. BMJ., V. 13; P. 341,2010.

GASPAR OJ. Study of vancomycin encapsulation systems in unilamellar liposomes and delivery in medium containing block-sensitive pluronic F-127 block copolymer for intravitreal controlled release. 2003. Dissertation (Pharmaceutical Sciences) - Paulista State University Júlio de Mesquita Filho.

HANAHAN D .; FOLKMAN J. Pattems and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86: 353-64, 1996.

HURWITZ H .; FEHRENBACHER L .; NOVOTNY W. Bevacizumab PLU F-127s irinotecan, 5-fluorouracil, and leukovorin for the treatment of metastatic colorectal cancer. N. Engl. J. Med., V. 350, p.2335-2342, 2004.

KITZMANN AS, PULID JS, MOHNEY BG, BARATZ KH, GRUBE T., MARLER, RJ, DONALDSON MJ., ORNEILL5B-P., JOHNSTON PB, JOHNSON KM, DIXON, RD, SALOMAO Eye, p. 1-4, 2007. LEE L.B.W., REGISTER, R.A. "Equilibrium Control of Crystal Thickness and Melting Point through Block Copolymerization", Macromolecules, 37, 7278-7284 (2004).

LIMA E.M., OLIVEIRA A.G. Tissue tolerance of diclofenac sodium encapsulated in liposomes after intramuscular administration.Drug Dev. Ind. Pharm. v.28, p.673-680, 2002.

LOO Y.L., REGISTER R.A. “Crystallization Within Bloek Copolymer Mesophases,” Chapter 6 in Developments in Bloek Copolymer Science and Technology, I.W. Hamley, ed. (Chichester: John Wiley & Sons, 2004), pp. 213-243.

MA W.D., XU H., WANG C., NIE S.F., PAN W.S. Pluronic F-127-poly (acrylic acid) copolymers as in situ vehicle for ophthalmic drug delivery system. Int. J. Pharm. v.350, no. 1-2, p.247-256, 2008.

MASON J.O .; ALBERT J.R, VAIL R. Intravitreal bevacizumab (Avastin) for refractory pseudophakie macular edema. Retina, v. 26, no. 3, p. 356-7, 2006.

MICHELS S., ROSENFELD P.J., PULIAFITO C.A., MARCUS E.N, VENKATRAMAN A.S. Systemie bevacizumab (Avastin) therapy for neovascular age-related macular degeneration twelve-week results of an uncontrolled open-label clinical study. Ophthalmology. 2005 Jun; 12 (6): 1035-47.

VCF, LEGRAND P., PINTO-ALPHANDARY H., PUISIEUX F., BARRATT G. Poly (D, L-lactide) nanocapsules prepared by a solvent displacement process: Influence of the composition on physicochemical and structural properties J. Pharm . Sci, v.89, no.5, p.614-26, 2000.

NGUYEN Q.D., SHAH S., TATLIPINAR S., D.V., ANDEN E.V., CAMPOCHIARO P.A. Bevacizumab choroidal suppressions neovascularization caused by pathological myopia. Br. Opthalmol. v.89, p. 1368-1370, 2005.

NEW R.R.C. Preparation of sterile liposomes. In: - Liposomes - the Practical Approach. Oxford: University Press, 1990, 103p.

NEW R.R.C. Liposomes: a practical approach. Oxford: University Press, 1990.

OLIVEIRA A.G., SCARPA M.V., MILK C.Q.F. Liposomes: biotechnological strategy for controlled release and targeting of antimicobacterial drugs. Rev. Ciên. Farma v.18, p.109-121, 1997.

OLIVEIRA A.G., JORGE R., QUIRINO L.S., CARDILO J.A., WANCZINSKI B., BROCCHI M. Liposome-encapsulated vancomycin for endophthalmitis treatment inrabbit eyes. Invest. Ophthalmol. Vis. Know. v.45, p.515-515, 2004.

OLIVEIRA A.G., CARDILLO J.A., SCARPA M.V., WANCZINSKI B., SILVA-JÚNIOR A.A. Studies on controlled release and vectorization of drugs through liposomes Rev. Bras. Med. Sao Paulo, v.62, n.6, p.23 8-244, 2005.

OLIVEIRA A.G., SCARPA M.V. Liposomes: pharmaceutical and cosmetic applications, new perspectives. Infarma, Brasilia, v. 1, no. 3, p. 20-23,1992.

OLIVEIRA L.C. Liposomes containing ciprofloxacin contained in pluronic® F-127 thermosensitive block copolymer gel. 2006. Dissertation (Pharmaceutical Sciences) - Paulista State University Júlio de Mesquita Filho.

PANDYA N.M .; DHALLA N.S .; SANTANI D.D. Angiogenesis - a new target for future therapy. Vasc. Pharmacol., V. 44, p. 265-274, 2006.

PAUL T .. FINGER P.T., CHIN K. Anti-vascular endothelial growth factor bevacizumab (bevacizumab) for radiation retinopathy. Arch Ophthalmol. v.125, no. 6, p.751-6, 2007.

POLVERINI P.J. Angiogenesis in health and disease: insights into basic mechanisms and therapeutic opportunities. J. Dent. Educ. v.66, no. 8, p.962-975, 2002.

PUISIEUX, F. Les liposomes. Ann Pharm. Fr., v. 41, no. 3-13, 1983.

ROSENFELD P.J., MOSHFEGHI A.A., PULIAFITO C.A. Optical coherence tomography findings after intravitreal injection of Bevacizumab for neovascular age-related macular degeneration.Ophthalmic Surg. Imaging Lasers, v. 36, p. 331-335, 2005.

ROSENFELD PJ, BROWN DM, HEIER JS, BOYER DS, KAISER PK, CHUNG CY, KIM RY; MARINA STUDY GROUP. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med., 2006 v. 355, no. 14, p. 1419-31, 2006.

SAHOO S.K .; DILNAWAZ F .; KRISHNAKUMAR S. Nanotechnology in ocular drug delivery Drug Diseovery Today, v. 13, no. 3/4, p. 144-151, 2008.

SAKAGUCHI H, IKUNO Y, GOMI F, ΚΑΜΕΙ M, SAWA M, TSUJIKAWA M, OSHIMA Y, KUSAKA S, TANO Y. Intravitreal injection of bevacizumab for choroidal neovascularization associated with pathological myopia. Br J Ophthalmol., V.91, no.2, p.161-5, 2007.

SCARPA M.V., OLIVEIRA A.G., CUCCOVIA I.M. Structure and physicochemical properties of liposomes. Infarma, v.7, no. 1/2, p. 4-7, 1998.

SILVA A.R.A., Cyclodextrin-green indocyanine molecular complexes and liposome encapsulation for application in photodynamic therapy. 2004. Dissertation (Pharmaceutical Sciences) - Paulista State University Júlio de Mesquita Filho.

SPAIDE RF, LAUD K, HNE HF, KLANCNIK JM JR, MEYFFILLE CB, YANNUZZI LA, SORENSON J, SLAKTER J, FISHER YL, COONEY MJ. Intravitreal bevacizumab treatment of choroidal neovascularization secondary to age-related macular degeneration. Retina. 2006 Apr; v.26, no.4, p.383-90, 2006.

SPAIDE RF, FISHER YL. Intravitreal bevacizumab (Avastin) treatment of proliferative diabetic retinopathy complicated by vitreous hemorrhage. Retina. 2006 Mar; v. 26, no. 3, p. 275-8, 2006.

SUBRAMANIAN ML, NESS S, ABEDI G, AHMED E, DALY M, FEINBERG E, BHATIA S, PATEL P, NGUYEN M, HOURANIEH A. Bevacizumab vs age-related macular degeneration: early results of a prospective double-masked , randomized clinical trial. Am J Ophthalmol., V. 148, no. 6, p. 875-82, 2009.

TONINI T., ROSSI F., CLÁUDIO P.P. Molecular basis of angiogenesis and cancer. Oncogene v.22, p.6549-6565, 2003.

TORCHILIN, V.P. Recent advances with liposomes as pharmaceutical carriers. Nature Reviews Drug Discovery 4, 145-160, 2005

WANCZINSKI B. Development of vancomycin-containing liposomes delivered in Pluronic® F-127 thermosensitive polymer for intraocular application. 2005. 106f. Dissertation (Master in Pharmaceutical Sciences) - Faculty of Pharmaceutical Sciences, Paulista State University Júlio de Mesquita Filho, Araraquara, 2005.

Claims (22)

1. "Nanostructured compositions of elastic liposomes", characterized in that they comprise molecules created by encapsulation of bevacizumab and other constituent active drug monoclonal antibodies that are effective in the treatment of subretinal neovascular membranes, macular edema and retinal neovacularizations. 2. "Nano-structured Elastic Liposome Compositions" characterized by comprising molecules created by encapsulation of active drug constituting effective bevacizumab in the treatment of subretinal neovascular membranes, macular edema, and retinal neovacularizations. 3. "Nanostructured Elastic Liposome Compositions" characterized by comprising molecules created by encapsulation of any active drug constituent derivative effective in the treatment of subretinal neovascular membranes, macular edema, and retinal neovacularizations. 4. “NESRUCTURED COMPOSITIONS OF RE- ELASTIC LIPOSOMAS DRESSED WITH ANY KIND OF THERMOSENSITIVE BLOCK COPOLYMERS”, characterized by comprising molecules created by encapsulation of bevacizumab constituting an active active drug in the treatment of subrovascular edema and subrovascular membranes retinal neovacularizations. 5. “Nanostructured Elastic Liposome Compositions Coated with Any Type of Thermosensitive Block Copolymers”, characterized by molecules created by encapsulation of any active drug constituent substance effective in the treatment of neovascular macular edema and subretinal membranes. and retinal neovacularizations. 6. “NANO-STRUCTURED COMPOSITIONS OF ELASTIC LIPOSOMES COATED WITH ANY KIND OF THERMOSENSITIVE BLOCK COPOLYMERS”, characterized by constituting molecules created by encapsulation of bevacizumab constituting an active drug effective in the treatment of neovascu- lar neovascular edema and subreticular membrane membranes retina. 7. “NANO-STRUCTURED COMPOSITIONS OF ANY TYPE OF ELASTIC SOUND LIPOS FOR EYE APPLICATION IN THE TREATMENT OF SUBRETINIAL NEOVASCULAR AND NEOVASCULAR EDEMA MEMBRANES” according to claim 1 or 2; or other monoclonal antibody and / or derived molecules used for the purposes of treating subretininal neovascular membranes, macular edema and retinal neovacularizations. 8. “Nanostructured compositions for ocular application in the treatment of sub-retinal and retinal macular neovascular membranes and neovascularization” according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7, characterized by a hydrous substance or no, but with antitumor activity. 9. “Nanostructured compositions for ocular application in the treatment of sub-retinal and retinal macular neovascular membranes and neovascularization” according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7, characterized by drug encapsulation niosomes. 10. “Nanostructured compositions for ocular application in the treatment of sub-retinal and retinal macular neovascular membranes and neovascularization” according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7. whether or not suitable for ionic or nonionic hydrophilic gelling polymers. 11. "Nano-structured compositions for ocular application in the treatment of sub-retinal and retinal macular and neovascular membranes" according to claim 10, characterized by hydrophilic or non-ionic hydrophilic polymers methylcellulose, hydroxyethylcellulose, hydroxypropyl, carboxymethylcellulose, carboxyvinyl polymers, block copolymers, gelatins, resins, hydrophilic resins, collagen gels, collagen gels with solid or semi-solid elements, chitin and chitosan, hyaluronic acid and its salts, lignin , and the like. 12. “Nanostructured compositions for ocular application in the treatment of sub-retinal and retinal macular neovascular membranes and retinal neovascularization” comprising an active drug and organized small-scale combination structures characterized by a system containing one or more compartments. bilayers, lipidic or not, and containing a central aqueous compartment obtained by any method and by the effective active drug in promoting the treatment referred to in claims 1 to 9. 13. "Nanostructured compositions for ocular application in the treatment of sub-retinal and retinal macular and neovascular membranes" according to claim 12, characterized in that the active drug is a monoclonal derivative molecule (s). 14. "Nanostructured compositions for ocular application in the treatment of subretinal neovascular and retinal macular membranes and neovascularization" according to claim 12, characterized in that the substance has VGEF inhibitor activity. 15. "Nanostructured compositions for ocular application in the treatment of subretinal neovascular and retinal macular membranes and neovascularization" according to claim 12 or 13, characterized in that the substance has VGEF inhibitor activity. 16. “Nanostructured compositions for ocular application in the treatment of subretinal neovascular and retinal macular membranes and neovascularization” according to Claim 9 or 10 or 11 or 12, characterized by bevacizumab or other inhibitor activity VEGF may range from 0.01 to 100% purity. 17. “Nanostructured compositions for ocular application in the treatment of subretinal neovascular and retinal macular membranes and neovascularization” according to claim 2 or 3 or 4 or 5 or 6 or 7, characterized in that they are not added in conjunction with each other. hydrophilic gelling polymers, thermosensitive or not, ionic or nonionic. 18. “Nano-structured compositions for ocular application in the treatment of subretinal neovascular and retinal macular membranes and neovascularization” according to claim 17, characterized by hydrophilic or non-ionic hydrophilic polymers. methylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, carboxyvinyl polymers, block copolymers, gelatins, resins, hydrophilic resins, collagen gels, collagen gels with solid or semi-solid elements, chitin and chitosan, hyaluronic acid and its salts, lignin , and similar. 19. "Nano-structured compositions for ocular application in the treatment of sub-retinal and retinal macular and neovascular membranes" according to claim 18, characterized by the hydrogel-sensitive system, may be composed of block-sensitive polymers as pH-sensitive polymers. as the carboxyvinyl polymers. 20. "MICRO AND NANO-STRUCTURED COMPOSITIONS FOR OCCULAR APPLICATION IN THE TREATMENT OF CERATOCONE" according to claim 18 or 19, characterized in that they are arranged to increase the bioadhesion of nanostructures in the eye tissues and improve the yield profile of bevacizumab or another monoclonal antibody to the eye tissues of the anterior and posterior segments of the eyes. 21. “Nanostructured compositions for ocular application in the treatment of subretinal neovascular and retinal macular membranes” according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 characterized in that they are sterile or non-isotonic water soluble formulations for topical or intraocular application with compatible pH and osmolarity. 22. “NANO-STRUCTURED COMPOSITIONS FOR EYE APPLICATION IN THE TREATMENT OF SUBRETINARY NEOVASCULAR AND RETINAL NEOVASCULAR MEMBRANES” according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 9 10 or 11 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21, characterized by injectable eye use formulations.