WO2007114680A1 - Anti-inflammatory protein ophthalmic preparation - Google Patents

Anti-inflammatory protein ophthalmic preparation Download PDF

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Publication number
WO2007114680A1
WO2007114680A1 PCT/MX2007/000049 MX2007000049W WO2007114680A1 WO 2007114680 A1 WO2007114680 A1 WO 2007114680A1 MX 2007000049 W MX2007000049 W MX 2007000049W WO 2007114680 A1 WO2007114680 A1 WO 2007114680A1
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Prior art keywords
tnfα
ophthalmic
rejection
cornea
cytokines
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PCT/MX2007/000049
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Spanish (es)
French (fr)
Inventor
Juan LÓPEZ DE SILANES PÉREZ
Jorge Fernando PANIAGUA-SOLÍS
Alberto DÍAZ-QUIÑONEZ
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Laboratorios Silanes S.A. De C.V.
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Publication of WO2007114680A1 publication Critical patent/WO2007114680A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the technical field of immunology, in particular to the use of protein inhibitors involved in ocular inflammation or in ocular angiogenesis processes.
  • the invention relates to a topical treatment for the prevention of rejection of corneal tissue transplanted in mammals including humans, which is characterized as an acute inflammatory process.
  • the present invention is also of the technical field of the pharmacology of biotechnological products. It is a technology on the formulation of ophthalmic compositions of topical application, characterized in that its active ingredient is constituted by protein molecules with the capacity to block the activity of cytokines or factors involved in inflammation, neovascularization and transplant rejection.
  • Antitocytocin antibody or functional variants of said antibody is a neutralizing immunoglobulin of proinflammatory cytokines and the variants can be monoclonal or polyclonal, natural or recombinant or biologically active fragments thereof comprising Fv, Fab, F (ab ') 2 or scFv.
  • Bioactivity Is the level of specific biological activity, or potency, determined in an animal model in vivo or in in vitro biochemical tests.
  • Bioactivity of rhTNF ⁇ It is the biological activity of rh TNF ⁇ expressed as 50% Effective Dose (ED 50 ) determined in the cell line L929 (ATCC, CCL-1) exposed to different concentrations of rh TNF ⁇ .
  • Average Effective Dose (SD 50 ) of rhTNF It is the concentration of rhTNF required to cause cytotoxic death in 50% of a population of L929 cell cultures.
  • the ED 50 of the TNF corresponds to a unit of biological activity.
  • Bioactivity of F (ab ' ) 2 anti-rhTNF ⁇ The neutralizing activity of F (ab') 2 anti- rhTNF ⁇ expressed as 50% Neutralizing Dose (DN 50 ) determined in cell line L929 (ATCC, CCL-1). For the estimation of DN 50 , different concentrations of anti-TNF are challenged against "K" DE 50 of TNF, where "K” means the number of times sufficient of doses to distinguish quantifiable bioactivity.
  • DN 5 o It is the concentration of anti-TNF antibody (F (ab ' ) 2 anti-rhTNF ⁇ ) required, to inhibit 50% of the maximum observed cell death caused by TNF.
  • Bioactivity of VEGF It is the proliferation induction activity of VEGF expressed as Average Effective Dose (ED 5 o) determined in primary cultures of human umbilical vein endotheliums (HUVEC) exposed to different concentrations of VEGF.
  • ED 5 o Average Effective Dose
  • Average Effective Dose OF 50 of VEGF It is the concentration of VEGF necessary to induce 50% proliferation of HUVEC cells.
  • the ED 50 of the VEGF corresponds to a unit of biological activity.
  • Specific Biological Activity It is defined as the estimation of the Units of biological activity of TNF contained in a milligram of product, expressed by the ratio U / mg.
  • Cytokines Proteins that regulate the communication between the cells that produce them or other cell types. Its concentration in body fluids is normally in the range of picograms, during inflammation local concentrations can be much higher. Its fundamental action is the regulation of the mechanism of inflammation. There are pro-inflammatory and anti-inflammatory cytokines.
  • Eye drops Sterile preparation free of foreign particles containing one or more drugs generally dissolved in aqueous solution, whose purpose is the topical application in the eyes. It is chemically and biologically stable, and not irritating to the cornea. Stability: It is the ability of a drug or a drug to remain within the established quality specifications, in the package that contains it, during its useful life.
  • Stability studies These are the tests that are carried out on a drug or a medicine for a certain time, under the influence of temperature, humidity or light in the container that contains it.
  • Accelerated stability studies are studies designed under exaggerated storage conditions to increase the rate of chemical, biological degradation or physical changes of a drug or a drug.
  • Drug It is any natural, synthetic or biotechnological substance that has any pharmacological activity and is identified by its physical, chemical or biological actions.
  • F (ab ') 2 fragments of polyclonal anti-TNF ⁇ antibodies are considered the active principle of the ophthalmic solution that is claimed.
  • Batch It is the quantity of a drug or medication that is produced in a manufacturing cycle and whose essential characteristic is its homogeneity.
  • Methods of therapeutic treatment refers to the treatment that a patient who receives an ocular inflammatory process can receive, especially keratitis, uveitis, retinitis, cornea transplant rejection, blepharitis, diabetic retinopathy, macular degeneration, and which consists in the administration to said patient by topical route of an effective amount of a drug whose drug, or active ingredient, is an antibody or a biologically active variant or fragment thereof capable of neutralizing the activity of proinflammatory or angiogenic cytokines. , more specifically anti-TNF ⁇ and / or anti-VEGF.
  • Neutralizer It blocks the biological activity of a cytokine both in vitro and in vivo.
  • Placebo pharmacologically inert substance that is used as a control in clinical research. It serves to rule out cures due to unknown causes that would not be attributable to the therapy being investigated.
  • Stability protocol it is the study design related to tests and acceptance criteria, characteristics of the batch, sample handling, study conditions, analytical methods and packaging materials.
  • corneal allografts are the most successful type of transplants, rejection remains a major clinical problem, about 20% of patients (between 6,000 and 8,000 per year) receiving a transplant in the United States reject donated corneas (Arch Ophtalmol., 1992).
  • steroids and corticosteroids are the prophylactic treatment of choice to prolong the life of the transplanted corneal tissue, however, these steroids have severe side effects since they increase the susceptibility to opportunistic infections; steroids weaken the vascular walls, cause inhibition of cell proliferation in a non-selective manner and even apoptosis, cause elevation of intraocular pressure, can cause secondary glaucoma and induction of cataracts.
  • the more potent the anti-inflammatory action of the steroid is, the more severe the side effects they induce (BenEzra 1999).
  • CC chemokine genes or beta chemokines
  • MIP-1 ⁇ macrophages -1 alpha
  • MIP-1 ⁇ the inflammatory protein of macrophages -1 alpha
  • RANTES RANTES
  • TNF ⁇ is one of the main stimuli for the expression of a wide range of chemokines, including CC chemokines (Qian, Dekaris et al. 2000).
  • Local neutralization of TNF ⁇ activity can be an effective modality to suppress TNF ⁇ -mediated mechanisms in corneal transplantation.
  • neovascularization Another process that generates a poor prognosis of corneal transplantation is neovascularization, which is a severe complication particularly in corneal vascularization and in retinal diseases such as diabetic retinopathy, occlusion of the central retinal vein and branches, and Retinopathy of prematurity.
  • TNF ⁇ and VEGF Vascular Endothelial Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • neovascularization can occur during the chronic course of the disease.
  • the consequences of the neovascularization of the cornea are the severe reduction of visual acuity, with the inherent risk of bringing the patient to blindness.
  • the pathogenesis of corneal angiogenesis has not been clearly defined, however, VEGF is an angiogenic cytokine that plays a major role in vasculogenesis and pathological neovascularization, (Philipp, Speicher et al. 2000). It has been reported that there is concurrent lymphangiogenesis and hemangiogenesis, induced by VEGF, in individuals undergoing keratoplasty. The inhibition of VEGF activity after surgery improves long-term graft survival (Cursiefen, Cao e ⁇ al. 2004).
  • VEGF is involved in neovascularization processes in complications of diseases of the retina, in inflammatory diseases of the cornea, and in processes of rejection of corneal transplantation.
  • neutralizing agents such as antibodies or antibody fragments, could constitute a potential therapeutic strategy for the treatment of corneal transplantation (Yatoh, Kawakami ei a /. 1998).
  • compositions and Methods for treating hyperimmune response in the eye discloses a method to prevent rejection of corneal transplantation through the use of anti-IFN ⁇ F (ab ' ) 2 antibody fragments presenting data. It also claims, without results, the use of F (ab ') 2 anti-TNF ⁇ fragments. However, it does not propose specific pharmaceutical compositions and even less demonstrates its stability.
  • the invention relates to the new medical use of antibodies or their active fragments, in particular F (ab ') 2 characterized in that said activity refers to the neutralization of the biological activity of proinflammatory cytokines, the antibodies can be monoclonal or polyclonal type originated from mammals or birds, particularly horses, camels, sharks, or chicken eggs. Said medical use is also characterized by involving the topical ophthalmic administration.
  • F (ab ' ) 2 fragments used preferentially during the development of the present invention are those generated from complete antibodies of horses immunized with a recombinant cytokine or with immunogenic peptides belonging to said cytokine and subjected to the procedure described in the patents by López de Silanes J. et al. (López de Silanes, Mancilla Nava et al. 2005; López de Silanes, Paniagua-Sol ⁇ s et al.
  • ophthalmic solutions whose active principle are fragments are exemplified during the description F (ab ' ) 2 of neutralizing antibodies of human TNF ⁇ (hTNF ⁇ ) and of human VEGF (hVEGF).
  • compositions of the invention are pharmacologically stable in solution, under recommended handling and storage conditions.
  • the stability has been evaluated at 0, 90 and 180 days for the accelerated condition; and at 0, 90, 180, 270 and 365 days for the long-term condition within which the quality of the product is not modified and tolerates variable temperature and humidity conditions.
  • cytokine blockers are usually commercialized in lyophilized form, under these conditions, they are not an accessible product for patients to give them a topical use, in this case ophthalmic, and there is no indication of administration by topical route, in contrast, the ophthalmic compositions of the invention are offered in the form of solution as eye drops whose active ingredients are of a protein nature and with a functional activity that inhibits pro-inflammatory and angiogenic cytokines, which, in the state of the art prior to the present application, are not indicated for topical ophthalmic use.
  • a further advantage of the ophthalmic compositions of the invention is that they are an alternative to the use of steroids used for their anti-inflammatory and immunosuppressive capacity.
  • the ophthalmic compositions of the present invention have as active principle antagonistic molecules of the cytokine activity, the use of F (ab ') 2 fragments of antibodies with neutralizing capacity of the biological activity of TNF ⁇ and VEGF are presented as examples.
  • F (ab ') 2 fragments of antibodies with neutralizing capacity of the biological activity of TNF ⁇ and VEGF are presented as examples.
  • the use of these principles is advantageous in several aspects: They do not have the side effects of steroids, since they do not have the cytotoxic effects described by themselves. They are specific in blocking only the key cytokines.
  • their action is limited to the tissue where they are required, which reduces the risk of generating immunogenicity against the pharmacologically active molecule.
  • the active ingredient being F (ab ' ) 2 fragments of cytokine neutralizing antibodies
  • the present invention offers one more advantage, since the absence of the FC fragment prevents the induction of immune responses caused by the generation of anti-FC antibodies, such and as reported in the use of complete anti-antibody
  • TNF where immunogenicity is an important aspect to consider (Baert, Noman et al. 2003). This is of particular importance when the treatment must be administered for prolonged periods, both to avoid transplant rejection and for chronic immunological conditions.
  • Intraocular penetration of drugs that are applied topically in the eye is very limited due to tissue barriers: the cornea and the sclera.
  • the drug has an opportunity to reach the tissues affected by inflammatory or rejection processes.
  • F (ab ') 2 fragments have a molecular weight of 100 kDa vs. 150 kDa that have complete antibodies, which gives the first chance to penetrate the eye tissues.
  • Skurkovich reports the result of applying drops of F (ab ') 2 fragments of anti-IFN ⁇ antibodies in patients with cornea transplant rejection.
  • Skurkovich reports decreased edema and increased transparency of the transplant (Skurkovich, Kasparov et al. 2002), Skurkovich, B.
  • compositions and Methods for treating hyperimmune response in the eye (Skurkovich and Skurkovich 2003).
  • an ophthalmic composition is not formally disclosed and the experimental development of this antecedent does not imply that the management of a pharmacologically stable ophthalmic composition has been required.
  • Our new compositions have been formulated to prevent or minimize inflammatory processes of the cornea and rejection of corneal transplantation in humans, reducing the use of other potentially toxic immunosuppressive drugs.
  • the new compositions are presented as an alternative that, although it does not cure the basic pathology, it does manipulate the patient's immune response, so that the treating ophthalmologist may have as an option for consideration and careful choice.
  • the present invention proposes new ophthalmic formulations that solve several technical difficulties according to the state of the closest technique.
  • the technical difficulties are to achieve a pharmacologically stable ophthalmic composition with the particularity that the active ingredient is of a protein nature, and more particularly, the active ingredient is F (ab ') 2 fragments of polyclonal antibodies.
  • Another technical problem solved in this invention lies in verifying that the F (ab ' ) 2 fragments of polyclonal antibodies acting as active ingredient are capable of neutralizing cytokines. In particular, they are able to neutralize the biological activity of TNF ⁇ and VEGF and thus prevent the rejection of corneal transplants.
  • the present invention relates mainly to the ophthalmic use of neutralizing antibodies or their active fragments as neutralizers of the biological activity of proinflammatory and angiogenic cytokines, particularly the invention relates to pharmacologically stable eye drops or compositions, pharmacologically stable, non-pyrogenic and suitable for application topical, whose active ingredient is said antibodies or fragments thereof.
  • ophthalmic composition that meets the stability criteria and that is characterized in that the active ingredient is a neutralizing protein of the bioactivity of TNF ⁇ and / or VEGF is described as an example. This example is not intended to limit the use of TNF ⁇ and / or VEGF inhibitors.
  • said pharmacologically stable ophthalmic composition has as its active ingredient a fragment of TNF ⁇ neutralizing F (ab ' ) 2 antibody.
  • the formulation of the present invention contains the following components: 24 mg / mL of sodium dibasic phosphate, 66 mg / mL of monobasic sodium phosphate, 4.5 mg / mL of sodium chloride, 2 mg / mL of Glycerin, 5 mg / mL of Boric acid, 2 mg / mL of Propylene Glycol, all in ACS grade (American Chemical Society) dissolved in sterile distilled water, and contains as active substance fragments of neutralizing F (ab ' ) 2 antibodies of TNF ⁇ in a concentration of 5 mg / mL.
  • the invention relates to the use of said compositions in the topical ophthalmic treatment to avoid or minimize inflammatory processes of the cornea which consists of the administration of ophthalmic solutions whose active ingredient are blocking proteins of the bioactivity of pro-inflammatory and / or angiogenic cytokines.
  • the invention relates to the use of said compositions in the topical ophthalmic treatment to avoid or minimize acute rejection processes of corneal transplantation, which consists in the administration of ophthalmic solutions whose active ingredient are blocking proteins of the pro cytokine activity. -inflammatory and / or angiogenic. DESCRIPTION OF THE FIGURES
  • FIG. 1 Purity analysis of the active substance F (ab ') 2 anti-TNF ⁇ .
  • Figure 4 Power of the formulations A, B and C.
  • Figure 5 Identity of the active substance in formulations A, B and C.
  • An SDS-PAGE was run under reducing conditions with formulations A, B and C. Lines 1, molecular weight markers; Line 2-4, Active ingredient controls of different batches (before formulating); Formulation A (5 ⁇ g); Line 5, Formulation A (5 ⁇ g); Line 6, Formulation B (5 ⁇ g); Line 7, Formulation C (5 ⁇ g).
  • Figure 6 Protein recovery from pilot lots 211105-1, 211105-2 and 211105-3 during the stability study. The protein quantification results are shown by the Bradford method, at the conditions specified in the Stability Protocol. Results are shown at 0, 30, 60 and 90 days.
  • FIG. 7 Bioactivity recovery of pilot lots 211105-1, 211105-2 and 211105-3 in stability studies. The results of the recovered biological activity are shown, at the conditions specified in the Stability Protocol. Results are shown at 90, 180 and 365 days. For each specified time a control (active principle) run in parallel was included.
  • Figure 8 Determination of the neutralizing dose of F (ab ' ) 2 anti-VEGF.
  • the anti-TNF ⁇ F (ab ') 2 antibody fragments were obtained under Good Manufacturing Practices at Instituto Bioclon SA de CV according to previously described methods, and the product was characterized as active ingredient.
  • An electrophoresis was carried out in polyacrylamide gels under reducing conditions (SDS-PAGE run at 100 V for 1.5 h visualized with Coomasie G-250 bright blue) to determine the purity and estimate the molecular weights of the light (L) and heavy chains ( H) of F (ab ') 2 anti-TNF ⁇ .
  • rhTNF ⁇ human recombinant TNF ⁇
  • ED 50 mean effective dose
  • the assay of Figure 2A on the L929 cell line was modified using 5DE 50 (1250 pg / mL) of rhTNF ⁇ with different dilutions of the active ingredient [F (ab ') 2 anti -TNF ⁇ ].
  • the potency measured as 50% Neutralizing Dose (DN 50 ) of the active substance was 507 ng / mL and is the concentration of the product required to neutralize the bioactivity of 5DE 50 (1250 pg / mL) of rhTNF ⁇ in culture in 50% vitro.
  • Figure 2B is representative of three independent experiments performed in triplicate.
  • F (ab ') 2 fragments that neutralize the venom of Centruroides spp.
  • the control had no effect on the bioassay, and did not inhibit the biological activity of TNF ⁇ at any of the concentrations evaluated.
  • the neutralization test described was designed for the evaluation of the bioactivity of the active ingredient for the new pharmaceutical compositions of the invention. To avoid confusion when expressing protein concentration, the neutralizing bioactivity of F (ab ') 2 is expressed in Units / milligram (U / mg), according to the following formula: 5x lO fi
  • Formulation A was defined as the most appropriate formulation, considered as the most complete formulation with respect to the presence of viscous and anti-microbial agents. From this definition, three different batches (called 211105-1, 211105-2 and 211105-3) were formulated for accelerated stability studies. These new batches were evaluated Ia ocular irritability, according to the General Method of Analysis ⁇ 0516> Ocular Irritability (FEUM. 2004. 8 edition. VoI I, pp 470- 471). After observing the animals at 24, 48 and 72 hours, lots 211105-1, 211105-2 and 211105-3 (as expected from the previous results of eye irritability for formulation A) were NOT irritating.
  • the stability protocol to which the batches were subjected were 30 0 C + 2 0 C temperature with 65% + 5% relative humidity and 40 0 C + 2 0 C temperature with 75% + 5% relative humidity.
  • the time periods for the analyzes were established at 0, 30, 60 and 90 days.
  • the analyzes carried out included the evaluation of sterility, antimicrobial effectiveness, and more important for us was the determination of the protein concentration and the determination of the neutralizing bioactivity (potency) of F (ab ') 2 anti-TNF ⁇ .
  • EXAMPLE 6 The evaluation of sterility of lots 211105-1, 211105-2 and 211105-3 was carried out by incubation in culture media in Thioglycolate and Dextrose Sabouraud media for 14 days. The presence or absence of bacterial and fungal growth was determined, according to the General Methods of Analysis ⁇ 0381> Sterility. FEUM, 2004. pages 426-434; ⁇ 71> Sterility tests. USP, 2005. pages 2251-2256; ⁇ 81> Antibiotics. USP, 2005. pages 2256-2263.
  • the following bacterial agents were used as controls: Staphylococus aureus, Pseudomonas aureginosa, Clostridium sporogenes, Bacillus subtillis, in addition to fungi: Aspergillus niger and Candida albicans. In all cases the samples evaluated from lots 211105-1, 211105-2 and 211105-3 were reported sterile. The bacteria and fungi that were used as a control grew abundantly.
  • the antimicrobial effectiveness of the ophthalmic solutions of the invention was determined, according to the General Methods of Analysis ⁇ 0305> Effectiveness of antimicrobial condoms. FEUM, 2004. pages 401-402; ⁇ 51> Antimicrobial effectiveness testing. USP, 2005. pages 2242-2243. In all cases, the lots obtained evaluated at indicated times reduced the bacterial and fungal load according to the specifications described in the stability protocol.
  • EXAMPLE 8 Protein concentration and neutralizing bioactivity were performed according to what was described in the previous examples. A summary of the protein concentration and the neutralizing bioactivity recovered from lots 211105-1, 211105-2 and 211105-3, at the defined times and conditions, is summarized in the tables of Figures 6 and 7. In all cases Monitored protein was recovered ( Figure 6) according to the specifications described in the stability protocol, as well as specific neutralizing biological activity against TNF ⁇ ( Figure 7). The bioactivity for each of the particular conditions was evaluated against a control consisting of the active substance before formulating. Therefore, if there are variations in the bioactivity, it should be compared against the control in each particular case. All these data allow us to conclude that the proposed formulation confers stability to the active principle to the conditions and times described in the stability protocol.
  • Ophthalmic formulations whose vehicle contains formula A were evaluated, and as active ingredient it contains F (ab ' ) 2 fragments with neutralizing capacity of human recombinant rhVEGF.
  • F (ab ' ) 2 fragments with neutralizing capacity of human recombinant rhVEGF were evaluated, and as active ingredient it contains F (ab ' ) 2 fragments with neutralizing capacity of human recombinant rhVEGF.
  • the tetrazolium / formazan bioassay was used to measure the metabolic activity of primary cultures of human umbilical cord endothelial cells (HUVEC) obtained from the National Institute of Cardiology "Ignacio Chávez", and according to the methods described, (Cory, Owen et al. 1991).
  • Figure 8 A) shows the results expressed as mean effective dose (SD 50 ) of rhVEGF in ng / mL.
  • the ED 50 is defined as the concentration of rhVEGF in which the activity is 50% of the maximum response.
  • the figure is representative of three independent experiments and the ED 50 was 1,237 ng / mL.
  • Figure 8 B) shows the results expressed as the average neutralizing dose (DN 50 ) obtained by evaluating the bioactivity of F (ab ') 2 anti-VEGF fragments on the HUVEC cell proliferation assay using 3DE 50 (3.7 ng / mL) The DN 50 was 17.34 ⁇ g / mL EXAMPLE 10
  • a therapeutic indication of the ophthalmic solutions of the invention is the prevention of acute rejection of the cornea transplant.
  • a preclinical study was carried out that provided results on the efficacy of ophthalmic solutions that inhibit the biological activity of TNF- ⁇ , whose active substance consists of F (ab ') 2 fragments of polyclonal antibodies. This preclinical study was prospective, longitudinal, randomized double blind.
  • the objective of the study was to determine the effect of the drug under study on the frequency of rejection and the survival time of corneal transplantation in a murine model; for which the survival of orthotopic corneal allografts was evaluated.
  • mice were induced neovascularization with three nylon points in the cornea.
  • visit 1 day 1
  • the researcher according to a randomization table, separated two groups of 35 animals each, with induced neovascularization, to receive subsequent treatments.
  • Treatment 1 is the group in which the ophthalmic solution A already described (anti-TNF ⁇ eye drops), treatment 2 or placebo was used, is the group to which the vehicle solution was administered which is said ophthalmic formulation A but without active principle .
  • neovascularization was evaluated and transplantation was performed only to those animals that presented grade 4 or greater vascularization.
  • the mice received an antibiotic as prophylaxis (quinolone, an ophthalmic drop 3 times a day).
  • the mice received 1 drop of the drug or placebo (depending on the allocation group), three times a day for 8 weeks.
  • Two weekly visits were made for the evaluation of corneal opacity, the evaluation was masked by ophthalmologists, cornea experts, considering the concordance index.
  • Inclusion criteria As donors, 8-10 week old male mice, strain C57BL / 6, were used. As receptors, male mice of 8-10 weeks, strain BALB / C, were used.
  • Exclusion criteria donors with corneal opacity, and recipients with neovascularization less than 4 and post-operative complications.
  • the survival analysis was performed using a Kaplan-Meier test, taking into account the data of 18 visits in 9 weeks throughout the study. The data were grouped by degree of opacity.

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Abstract

The invention relates to novel ophthalmic compositions for topical use with proven stability whose active principle is a molecule of protein type capable of blocking the activity of cytokines or factors involved in ophthalmic processes of inflammatory type or of angiogenic type or acute transplant rejection. Amongst the pro-inflammatory cytokines, a special role is played by tumour necrosis factor alpha (TNFα) and vascular endothelial growth factor (VEGF) as potent angiogenic agent, and therefore particular preference is given to compositions whose active principle is proteins that neutralize the biological activity of these two cytokines. The active proteins may be natural or recombinant, monoclonal or polyclonal antibodies or biologically active fragments thereof that comprise Fv, Fab, F(ab')2 or scFv, and that are capable of preventing or minimizing the phenomenon of the rejection of biological tissue grafts, particularly ocular tissue, including the cornea.

Description

OFTÁLMICO PROTEICO ANTI-INFLAMATORIO.ANTI-INFLAMMATORY PROTEIC OPHTHALMIC.
CAMPO DE LA INVENCIÓN:FIELD OF THE INVENTION:
La presente invención se refiere al campo técnico de Ia inmunología, en particular al uso de inhibidores de proteínas involucradas en inflamación ocular o en procesos de angiogénesis ocular. De manera particular Ia invención se refiere a un tratamiento tópico para la prevención del rechazo del tejido corneal transplantado en mamíferos incluyendo humanos, que se caracteriza por ser un proceso inflamatorio agudo.The present invention relates to the technical field of immunology, in particular to the use of protein inhibitors involved in ocular inflammation or in ocular angiogenesis processes. In particular, the invention relates to a topical treatment for the prevention of rejection of corneal tissue transplanted in mammals including humans, which is characterized as an acute inflammatory process.
La presente invención es también del campo técnico de Ia farmacología de productos biotecnológicos. Es una tecnología sobre Ia formulación de composiciones oftálmicas de aplicación tópica, caracterizadas porque su principio activo está constituido por moléculas de carácter proteico con capacidad para bloquear Ia actividad de citocinas o factores involucrados en inflamación, neovascularización y rechazo de transplantes.The present invention is also of the technical field of the pharmacology of biotechnological products. It is a technology on the formulation of ophthalmic compositions of topical application, characterized in that its active ingredient is constituted by protein molecules with the capacity to block the activity of cytokines or factors involved in inflammation, neovascularization and transplant rejection.
DEFINICIÓN DE TÉRMINOS.DEFINITION OF TERMS.
Las siguientes definiciones se proveen para facilitar Ia interpretación de Ia presente solicitud de patente.The following definitions are provided to facilitate the interpretation of the present patent application.
Anticuerpo anticitocina o variantes funcionales de dicho anticuerpo: Es una inmunoglobulina neutralizante de citocinas proinflamatorias y las variantes pueden ser monoclonales o policlonales, naturales o recombinantes o fragmentos biológicamente activos de los mismos que comprenden Fv, Fab, F(ab')2 o scFv.Antitocytocin antibody or functional variants of said antibody: It is a neutralizing immunoglobulin of proinflammatory cytokines and the variants can be monoclonal or polyclonal, natural or recombinant or biologically active fragments thereof comprising Fv, Fab, F (ab ') 2 or scFv.
Bioactividad: Es el nivel de actividad biológica específica, o potencia, determinada en un modelo animal in vivo o en ensayos bioquímicos in vitro.Bioactivity: Is the level of specific biological activity, or potency, determined in an animal model in vivo or in in vitro biochemical tests.
Bioactividad de rhTNFα: Es Ia actividad biológica del rh TNFα expresada como Dosis Efectiva al 50% (DE50) determinada en Ia línea celular L929 (ATCC, CCL-1) expuesta a distintas concentraciones de rh TNFα. Dosis Efectiva Media (DE50) de rhTNF : Es Ia concentración de rhTNF requerido para ocasionar muerte ciíotóxica en el 50% de una población de cultivos de células L929. La DE50 del TNF, corresponde a una Unidad de actividad biológica.Bioactivity of rhTNFα: It is the biological activity of rh TNFα expressed as 50% Effective Dose (ED 50 ) determined in the cell line L929 (ATCC, CCL-1) exposed to different concentrations of rh TNFα. Average Effective Dose (SD 50 ) of rhTNF: It is the concentration of rhTNF required to cause cytotoxic death in 50% of a population of L929 cell cultures. The ED 50 of the TNF corresponds to a unit of biological activity.
Bioactivídad de F(ab')2 anti-rhTNFα: La actividad neutralizante de F(ab')2 anti- rhTNFα expresada como Dosis Neutralizante al 50% (DN50) determinada en Ia línea celular L929 (ATCC, CCL-1). Para Ia estimación de Ia DN50, se retan distintas concentraciones de anti-TNF frente a "K" DE50 de TNF, donde "K" significa el número de veces suficientes de dosis para distinguir bioactividad cuantificable.Bioactivity of F (ab ' ) 2 anti-rhTNFα: The neutralizing activity of F (ab') 2 anti- rhTNFα expressed as 50% Neutralizing Dose (DN 50 ) determined in cell line L929 (ATCC, CCL-1). For the estimation of DN 50 , different concentrations of anti-TNF are challenged against "K" DE 50 of TNF, where "K" means the number of times sufficient of doses to distinguish quantifiable bioactivity.
Dosis Efectiva Neutralizante Media (DN5o): Es Ia concentración de anticuerpo anti-TNF (F(ab')2 anti-rhTNFα) requerido, para inhibir el 50% Ia máxima muerte celular observada ocasionado por el TNF.Average Neutralizing Effective Dose (DN 5 o): It is the concentration of anti-TNF antibody (F (ab ' ) 2 anti-rhTNFα) required, to inhibit 50% of the maximum observed cell death caused by TNF.
Bioactividad de VEGF: Es Ia actividad de inducción de proliferación del VEGF expresada como Dosis Efectiva Media (DE5o) determinada en cultivos primarios de endotelios de vena umbilical humana (HUVEC) expuesta a distintas concentraciones de VEGF. |Bioactivity of VEGF: It is the proliferation induction activity of VEGF expressed as Average Effective Dose (ED 5 o) determined in primary cultures of human umbilical vein endotheliums (HUVEC) exposed to different concentrations of VEGF. |
Dosis Efectiva Media DE50 de VEGF: Es Ia concentración de VEGF necesaria para inducir el 50% de proliferación de células HUVEC. La DE50 del VEGF, corresponde a una Unidad de actividad biológica.Average Effective Dose OF 50 of VEGF: It is the concentration of VEGF necessary to induce 50% proliferation of HUVEC cells. The ED 50 of the VEGF corresponds to a unit of biological activity.
Actividad Biológica específica: Se define como Ia estimación de las Unidades de actividad biológica de TNF contenidos en un miligramo de producto, expresado por el cociente U/mg.Specific Biological Activity: It is defined as the estimation of the Units of biological activity of TNF contained in a milligram of product, expressed by the ratio U / mg.
Citocinas: Proteínas que regulan Ia comunicación entre las células que las producen u otro tipos celulares. Su concentración en fluidos corporales está normalmente en el rango de picogramos, durante Ia inflamación las concentraciones locales pueden ser mucho más altas. Su acción fundamental es Ia regulación del mecanismo de Ia inflamación. Existen citocinas pro-inflamatorias y anti-inflamatorias.Cytokines: Proteins that regulate the communication between the cells that produce them or other cell types. Its concentration in body fluids is normally in the range of picograms, during inflammation local concentrations can be much higher. Its fundamental action is the regulation of the mechanism of inflammation. There are pro-inflammatory and anti-inflammatory cytokines.
Colirio: Preparación estéril libre de partículas extrañas que contiene uno o más fármacos disueltos generalmente en solución acuosa, cuya finalidad es Ia aplicación tópica en los ojos. Es estable química y biológicamente, y no irritante a Ia córnea. Estabilidad: Es Ia capacidad de un fármaco o un medicamento de permanecer dentro de las especificaciones de calidad establecidas, en el envase que Io contiene, durante su periodo de vida útil.Eye drops: Sterile preparation free of foreign particles containing one or more drugs generally dissolved in aqueous solution, whose purpose is the topical application in the eyes. It is chemically and biologically stable, and not irritating to the cornea. Stability: It is the ability of a drug or a drug to remain within the established quality specifications, in the package that contains it, during its useful life.
Estudios de estabilidad: Son las pruebas que se efectúan a un fármaco o a un medicamento por un tiempo determinado, bajo Ia influencia de temperatura, humedad o luz en el envase que Io contiene.Stability studies: These are the tests that are carried out on a drug or a medicine for a certain time, under the influence of temperature, humidity or light in the container that contains it.
Estudios de estabilidad acelerada: Son los estudios diseñados bajo condiciones exageradas de almacenamiento para incrementar Ia velocidad de degradación química, biológica o los cambios físicos de un fármaco o de un medicamento.Accelerated stability studies: These are studies designed under exaggerated storage conditions to increase the rate of chemical, biological degradation or physical changes of a drug or a drug.
Fármaco (principio activo): Es toda sustancia natural, sintética o biotecnológica que tenga alguna actividad farmacológica y que se identifique por sus propiedades físicas, químicas o acciones biológicas. En el contexto de esta solicitud, los fragmentos F(ab')2 de anticuerpos policlonales anti-TNFα se consideran el principio activo de Ia solución oftálmica que se reclama.Drug (active substance): It is any natural, synthetic or biotechnological substance that has any pharmacological activity and is identified by its physical, chemical or biological actions. In the context of this application, the F (ab ') 2 fragments of polyclonal anti-TNFα antibodies are considered the active principle of the ophthalmic solution that is claimed.
Farmacológicamente estable: Son las condiciones cumplidas de conformidad con Io establecido en las guías para pruebas de estabilidad para medicamentos y productos nuevos emitidas por las agencias regulatorias de los países para Ia evaluación de productos medicinales.Pharmacologically stable: These are the conditions met in accordance with what is established in the guidelines for stability tests for drugs and new products issued by the regulatory agencies of the countries for the evaluation of medicinal products.
Lote: Es Ia cantidad de un fármaco o medicamento que se produce en un ciclo de fabricación y cuya característica esencial es su homogeneidad.Batch: It is the quantity of a drug or medication that is produced in a manufacturing cycle and whose essential characteristic is its homogeneity.
Métodos de tratamiento terapéutico: En el contexto de Ia invención se refiere al tratamiento que puede recibir un paciente que cursa un proceso inflamatorio ocular, en especial, queratitis, uveítis, retinitis, rechazo de trasplante de cornea, blefaritis, retinopatía diabética, degeneración macular, y que consiste en Ia administración a dicho paciente por vía tópica de una cantidad efectiva de un medicamento cuyo fármaco, o principio activo, es un anticuerpo o una variante o fragmento del mismo biológicamente activo con capacidad de neutralizar Ia actividad de - citocinas proinflamatorias o angiogénicas, más específicamente anti-TNFα y/o anti-VEGF.Methods of therapeutic treatment: In the context of the invention, it refers to the treatment that a patient who receives an ocular inflammatory process can receive, especially keratitis, uveitis, retinitis, cornea transplant rejection, blepharitis, diabetic retinopathy, macular degeneration, and which consists in the administration to said patient by topical route of an effective amount of a drug whose drug, or active ingredient, is an antibody or a biologically active variant or fragment thereof capable of neutralizing the activity of proinflammatory or angiogenic cytokines. , more specifically anti-TNFα and / or anti-VEGF.
Neutralizante: Que bloquea Ia actividad biológica de una citocina tanto ¡n vitro como in vivo. Placebo: sustancia farmacológicamente inerte que se utiliza como control en Ia investigación clínica. Sirve para descartar las curaciones debidas a causas desconocidas que no serían atribuibles a Ia terapia que se investiga.Neutralizer: It blocks the biological activity of a cytokine both in vitro and in vivo. Placebo: pharmacologically inert substance that is used as a control in clinical research. It serves to rule out cures due to unknown causes that would not be attributable to the therapy being investigated.
Protocolo de estabilidad: es el diseño del estudio relativo a pruebas y criterios de aceptación, características del lote, manejo de las muestras, condiciones del estudio, métodos analíticos y materiales de envase.Stability protocol: it is the study design related to tests and acceptance criteria, characteristics of the batch, sample handling, study conditions, analytical methods and packaging materials.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
Aunque los aloinjertos de cornea son el tipo de transplantes más exitosos, el rechazo sigue siendo un problema clínico importante, alrededor del 20% de los pacientes (entre 6,000 y 8,000 por año) que reciben un transplante en Estados Unidos rechazan las corneas donadas (Arch. Ophtalmol.,1992). El uso de esteroides y corticoesteroides son el tratamiento profiláctico de elección para prolongar Ia vida del tejido corneal transplantado, sin embargo, estos esteroides tienen efectos secundarios severos ya que incrementan Ia susceptibilidad a infecciones oportunistas; los esteroides debilitan las paredes vasculares, causan inhibición de Ia proliferación celular de manera no selectiva e incluso apoptosis, causan elevación de Ia presión intraocular, pueden ocasionar glaucoma secundario e inducción de cataratas. Desafortunadamente, entre más potente es Ia acción antiinflamatoria del esteroide, más severos son los efectos secundarios que ellos inducen (BenEzra 1999).Although corneal allografts are the most successful type of transplants, rejection remains a major clinical problem, about 20% of patients (between 6,000 and 8,000 per year) receiving a transplant in the United States reject donated corneas (Arch Ophtalmol., 1992). The use of steroids and corticosteroids are the prophylactic treatment of choice to prolong the life of the transplanted corneal tissue, however, these steroids have severe side effects since they increase the susceptibility to opportunistic infections; steroids weaken the vascular walls, cause inhibition of cell proliferation in a non-selective manner and even apoptosis, cause elevation of intraocular pressure, can cause secondary glaucoma and induction of cataracts. Unfortunately, the more potent the anti-inflammatory action of the steroid is, the more severe the side effects they induce (BenEzra 1999).
Rechazo de injertos y papel de las citocinas, en particular del TNF y del VEGF.Rejection of grafts and role of cytokines, in particular TNF and VEGF.
Si bien hasta ahora no se ha logrado identificar a todos los componentes celulares y moleculares que median el rechazo de los transplantes de cornea, se han encontrado evidencias de que Ia citocina proinflamatoria TNFα está involucrada en Ia respuesta inmune contra el trasplante (Yamada, Streilein et al. 1999). El rechazo del transplante de cornea está caracterizado patológicamente por Ia infiltración de leucocitos dentro del estroma del injerto así como Ia adherencia de células mononucleares (Larkin, Calder et al. 1997). El reclutamiento de células inmunes e inflamatorias hacia el tejido blanco, incluidos los injertos, se ha asociado con Ia función especializada de citocinas quimiotácticas conocidas como quimiocinas, (Luster 1998). Los estudios demuestran que el rechazo de transplante de cornea está asociado con un pronunciado aumento de Ia expresión de genes de quimiocinas CC (o beta quimiocinas), incluyendo Ia proteína inflamatoria de macrófagos -1 alfa (MIP-1α ), MIP-1β, y RANTES (Yamagami, Miyazaki eí al. 1999), en Ia activación y en el reclutamiento de linfocitos TH1 , (Ward, Bacon eí al. 1998). El TNFα es uno de los principales estímulos para Ia expresión de un amplio rango de quimiocinas, incluyendo las quimiocinas CC (Qian, Dekaris eí al. 2000). La neutralización local de Ia actividad de TNFα, puede ser una modalidad efectiva para suprimir los mecanismos mediados por TNFα en el transplante de cornea.Although until now it has not been possible to identify all the cellular and molecular components that mediate the rejection of corneal transplants, evidence has been found that the proinflammatory cytokine TNFα is involved in the immune response against transplantation (Yamada, Streilein et al. 1999). The rejection of the cornea transplant is pathologically characterized by the infiltration of leukocytes into the stroma of the graft as well as the adherence of mononuclear cells (Larkin, Calder et al. 1997). The recruitment of immune and inflammatory cells to white tissue, including grafts, has been associated with the specialized function of chemotactic cytokines known as chemokines, (Luster 1998). Studies show that corneal transplant rejection is associated with a pronounced increase in the expression of CC chemokine genes (or beta chemokines), including the inflammatory protein of macrophages -1 alpha (MIP-1α), MIP-1β, and RANTES (Yamagami, Miyazaki eí al. 1999), in the activation and recruitment of T H 1 lymphocytes, (Ward, Bacon eí al. 1998). TNFα is one of the main stimuli for the expression of a wide range of chemokines, including CC chemokines (Qian, Dekaris et al. 2000). Local neutralization of TNFα activity can be an effective modality to suppress TNFα-mediated mechanisms in corneal transplantation.
Otro proceso que genera un mal pronóstico del transplante de cornea es la neovascularización, Ia cual es una complicación severa particularmente en Ia vascularización corneal y en enfermedades retinianas como Io son Ia retinopatía diabética, Ia oclusión de Ia vena retiniana central y de las ramas, y Ia retinopatía del prematuro. Se ha propuesto que tanto el TNFα como el Factor de Crecimiento Vascular Endotelial (VEGF) pueden ser ambas moléculas blanco o apropiadas para intervenir en Ia prevención del rechazo del aloinjerto de cornea (Dekaris, Zhu et al. 1999; Yamada, Streilein eí al. 1999; Zhu, Dekaris et al. 1999; Cursiefen, Cao et al. 2004).Another process that generates a poor prognosis of corneal transplantation is neovascularization, which is a severe complication particularly in corneal vascularization and in retinal diseases such as diabetic retinopathy, occlusion of the central retinal vein and branches, and Retinopathy of prematurity. It has been proposed that both TNFα and Vascular Endothelial Growth Factor (VEGF) can both be white or appropriate molecules to intervene in the prevention of corneal allograft rejection (Dekaris, Zhu et al. 1999; Yamada, Streilein eí al. 1999; Zhu, Dekaris et al. 1999; Cursiefen, Cao et al. 2004).
En varias enfermedades inflamatorias de Ia cornea, Ia neovascularización puede ocurrir durante el curso crónico de Ia enfermedad. Las consecuencias de Ia neovascularización de Ia cornea son Ia reducción severa de Ia agudeza visual, con el riesgo inherente de llevar al paciente hasta Ia ceguera. La patogénesis de la angiogénesis corneal no se ha definido con claridad, sin embargo, el VEGF es una citocina angiogénica que juega un papel principal en Ia vasculogénesis y en Ia neovascularización patológica, (Philipp, Speicher eí al. 2000). Se ha descrito que existe linfangiogénesis y hemangiogénesis concurrente, inducida por VEGF, en individuos sometidos a keratoplastía. La inhibición de la actividad de VEGF después de Ia cirugía mejora Ia sobrevida del injerto a largo plazo (Cursiefen, Cao eí al. 2004).In several inflammatory diseases of the cornea, neovascularization can occur during the chronic course of the disease. The consequences of the neovascularization of the cornea are the severe reduction of visual acuity, with the inherent risk of bringing the patient to blindness. The pathogenesis of corneal angiogenesis has not been clearly defined, however, VEGF is an angiogenic cytokine that plays a major role in vasculogenesis and pathological neovascularization, (Philipp, Speicher et al. 2000). It has been reported that there is concurrent lymphangiogenesis and hemangiogenesis, induced by VEGF, in individuals undergoing keratoplasty. The inhibition of VEGF activity after surgery improves long-term graft survival (Cursiefen, Cao eí al. 2004).
En particular VEGF está involucrado en procesos de neovascularización en complicaciones de enfermedades de Ia retina, en enfermedades inflamatorias de Ia cornea, y en procesos de rechazo del transplante de Ia cornea. La inhibición de VEGF de manera tópica aplicando agentes neutralizantes, como anticuerpos o fragmentos de anticuerpo, podría constituir una estrategia terapéutica potencial para el tratamiento del trasplante de cornea (Yatoh, Kawakami eí a/. 1998).In particular, VEGF is involved in neovascularization processes in complications of diseases of the retina, in inflammatory diseases of the cornea, and in processes of rejection of corneal transplantation. Topically inhibiting VEGF by applying neutralizing agents, such as antibodies or antibody fragments, could constitute a potential therapeutic strategy for the treatment of corneal transplantation (Yatoh, Kawakami ei a /. 1998).
En el arte previo, se ha sugerido el uso de anticuerpos completos anti-TNF en padecimientos oftálmicos, sin embargo no se reportan composiciones farmacéuticas concretas, es decir cuyos componentes se definan con claridad y que contengan a dichos anticuerpos como su principio activo, menos aún se demuestra estabilidad ni efectividad (Wax and Tezel 2001).In the prior art, the use of complete anti-TNF antibodies in ophthalmic conditions has been suggested, however pharmaceutical compositions are not reported. concrete, that is to say whose components are clearly defined and that contain said antibodies as their active ingredient, even less stability or effectiveness is demonstrated (Wax and Tezel 2001).
Skurkovich Boris, ΘÍ al Appl: US 10/389.065 Pub. No. US 2003/0215448 A1 (Skurkovich and Skurkovich 2003) "Composition and methods for treating hyperimmune response ¡n the eye", divulgan el tratamiento de pacientes con transplante de cornea y de pacientes con uveítis, utilizando fragmentos F(ab')2 de anticuerpos anti-IFNγ. Sin embargo no propone composiciones farmacológicas en concreto y menos aún demuestra su estabilidad.Skurkovich Boris, ΘÍ al Appl: US 10 / 389.065 Pub. No. US 2003/0215448 A1 (Skurkovich and Skurkovich 2003) "Composition and methods for treating hyperimmune response in the eye", disclose the treatment of patients with corneal transplantation and of patients with uveitis, using F (ab ') 2 fragments of anti-IFNγ antibodies. However, it does not propose specific pharmaceutical compositions and even less demonstrates its stability.
Skurkovich, B. et al. 6,534,059 B2. "Compositions and Methods for treating hyperimmune response in the eye" (Skurkovich and Skurkovich 2003), divulga un método para prevenir el rechazo de transplante de cornea mediante el uso de fragmentos de anticuerpos F(ab')2 anti-IFNγ presentando datos. Reclama también, sin resultados, el uso de fragmentos F(ab')2 anti-TNFα. Sin embargo no propone composiciones farmacológicas en concreto y menos aún demuestra su estabilidad. Skurkovich, B. et al. 6,534,059 B2. "Compositions and Methods for treating hyperimmune response in the eye" (Skurkovich and Skurkovich 2003), discloses a method to prevent rejection of corneal transplantation through the use of anti-IFNγ F (ab ' ) 2 antibody fragments presenting data. It also claims, without results, the use of F (ab ') 2 anti-TNFα fragments. However, it does not propose specific pharmaceutical compositions and even less demonstrates its stability.
DESCRIPCIÓN DE LA INVENCIÓN:DESCRIPTION OF THE INVENTION:
La invención se refiere al nuevo uso médico de anticuerpos o de sus fragmentos activos, en particular F(ab')2 caracterizados porque dicha actividad se refiere a Ia neutralización de Ia actividad biológica de citocinas proinflamatorias, los anticuerpos pueden ser de tipo monoclonal o policlonal originados de mamíferos o de aves, en particular de caballos, camellos, tiburones, o de huevo de gallina. Dicho uso médico también se caracteriza por implicar Ia administración tópica oftálmica.The invention relates to the new medical use of antibodies or their active fragments, in particular F (ab ') 2 characterized in that said activity refers to the neutralization of the biological activity of proinflammatory cytokines, the antibodies can be monoclonal or polyclonal type originated from mammals or birds, particularly horses, camels, sharks, or chicken eggs. Said medical use is also characterized by involving the topical ophthalmic administration.
Los fragmentos F(ab')2 utilizados de manera preferencial durante el desarrollo de Ia presente invención son aquellos generados a partir de anticuerpos completos de caballos inmunizados con una citocina recombinante o con péptidos inmunogénicos pertenecientes a dicha citocina y sometidos al procedimiento descrito en las patentes de López de Silanes J. et al. (López de Silanes, Mancilla Nava et al. 2005; López de Silanes, Paniagua-Solís et al. 2005) De manera particular, y sin pretender limitar el alcance de Ia invención, durante Ia descripción se ejemplifican soluciones oftálmicas cuyo principio activo son fragmentos F(ab')2 de anticuerpos neutralizantes de TNFα humano (hTNFα) y de VEGF humano (hVEGF).F (ab ' ) 2 fragments used preferentially during the development of the present invention are those generated from complete antibodies of horses immunized with a recombinant cytokine or with immunogenic peptides belonging to said cytokine and subjected to the procedure described in the patents by López de Silanes J. et al. (López de Silanes, Mancilla Nava et al. 2005; López de Silanes, Paniagua-Solís et al. 2005) In particular, and without intending to limit the scope of the invention, ophthalmic solutions whose active principle are fragments are exemplified during the description F (ab ' ) 2 of neutralizing antibodies of human TNFα (hTNFα) and of human VEGF (hVEGF).
Durante el desarrollo de las soluciones oftálmicas y después de numerosas investigaciones, se lograron obtener formulaciones farmacológicamente estables aún cuando el principio activo es de naturaleza proteica, ya que se logró conservar su actividad biológica y eficacia durante el tiempo de almacenamiento y manejo..During the development of ophthalmic solutions and after numerous investigations, pharmacologically stable formulations were obtained even when the active ingredient is of a protein nature, since it was possible to conserve its biological activity and efficacy during the storage and handling time.
Las composiciones de Ia invención, son farmacológicamente estables en solución, bajo condiciones de manejo y almacenamiento recomendadas. La estabilidad ha sido evaluada a los 0, 90 y 180 días para Ia condición acelerada; y a los 0, 90, 180, 270 y 365 días para Ia condición a largo plazo dentro de los cuales Ia calidad del producto no se modifica y tolera condiciones variables de temperatura y humedad.The compositions of the invention are pharmacologically stable in solution, under recommended handling and storage conditions. The stability has been evaluated at 0, 90 and 180 days for the accelerated condition; and at 0, 90, 180, 270 and 365 days for the long-term condition within which the quality of the product is not modified and tolerates variable temperature and humidity conditions.
Los actuales bloqueadores de citocinas suelen comercializarse en forma liofilizada, en estas condiciones, no resultan ser un producto accesible para que los pacientes les den un uso tópico, en este caso oftálmico, y tampoco existe Ia indicación de administración por vía tópica, en contraste, las composiciones oftálmicas de Ia invención se ofrecen en forma de solución como colirios cuyos principios activos son de naturaleza proteica y con actividad funcional inhibidora de citocinas pro-inflamatorias y angiogénicas, los cuales, en el estado de Ia técnica previo a Ia presente solicitud, no se encuentran indicados para uso oftálmico tópico.The current cytokine blockers are usually commercialized in lyophilized form, under these conditions, they are not an accessible product for patients to give them a topical use, in this case ophthalmic, and there is no indication of administration by topical route, in contrast, the ophthalmic compositions of the invention are offered in the form of solution as eye drops whose active ingredients are of a protein nature and with a functional activity that inhibits pro-inflammatory and angiogenic cytokines, which, in the state of the art prior to the present application, are not indicated for topical ophthalmic use.
Una ventaja más de las composiciones oftálmicas de Ia invención es que son una alternativa al uso de esteroides empleados por su capacidad antünflamatoria e inmunosupresora.A further advantage of the ophthalmic compositions of the invention is that they are an alternative to the use of steroids used for their anti-inflammatory and immunosuppressive capacity.
Las composiciones oftálmicas de Ia presente invención tienen como principio activo a moléculas antagonistas de Ia actividad de citocinas, se presentan como ejemplos el uso de fragmentos F(ab')2 de anticuerpos con capacidad neutralizante de Ia actividad biológica del TNFα y del VEGF. Además de tener una efectividad comprobada, el uso de estos principios resulta ventajoso en varios aspectos: No tienen los efectos secundarios propios de los esteroides, ya que por sí mismos no tienen efectos citotóxicos descritos. Son específicos al bloquear únicamente a las citocinas clave. Además, al no ser aplicadas de manera sistémica su acción está limitada al tejido en donde se requieren, Io que disminuye el riesgo de generar inmunogenicidad contra Ia molécula farmacológicamente activa.The ophthalmic compositions of the present invention have as active principle antagonistic molecules of the cytokine activity, the use of F (ab ') 2 fragments of antibodies with neutralizing capacity of the biological activity of TNFα and VEGF are presented as examples. In addition to having proven effectiveness, the use of these principles is advantageous in several aspects: They do not have the side effects of steroids, since they do not have the cytotoxic effects described by themselves. They are specific in blocking only the key cytokines. In addition, as they are not applied systemically, their action is limited to the tissue where they are required, which reduces the risk of generating immunogenicity against the pharmacologically active molecule.
Siendo el principio activo fragmentos F(ab')2 de anticuerpos neutralizantes de citocinas, Ia presente invención ofrece una ventaja más, dado que Ia ausencia del fragmento FC previene Ia inducción de respuestas inmunes ocasionadas por Ia generación de anticuerpos anti-FC, tal y como se ha reportado en el uso de anticuerpos completos anti-The active ingredient being F (ab ' ) 2 fragments of cytokine neutralizing antibodies, the present invention offers one more advantage, since the absence of the FC fragment prevents the induction of immune responses caused by the generation of anti-FC antibodies, such and as reported in the use of complete anti-antibody
TNF donde Ia inmunogenicidad es un aspecto importante para considerar (Baert, Noman et al. 2003). Esto es de particular importancia cuando el tratamiento debe administrarse durante periodos prolongados, tanto para evitar un rechazo de transplante como para padecimientos inmunológicos crónicos.TNF where immunogenicity is an important aspect to consider (Baert, Noman et al. 2003). This is of particular importance when the treatment must be administered for prolonged periods, both to avoid transplant rejection and for chronic immunological conditions.
La penetración intraocular de fármacos que se aplican tópicamente en el ojo es muy limitada debido a las barreras tisulares: Ia cornea y Ia esclerótica. Sin embargo, en procesos de transplante de cornea o en lesiones de cornea, el fármaco tiene oportunidad de llegar a los tejidos afectados por los procesos inflamatorios o de rechazo. Los fragmentos F(ab')2 tienen un peso molecular de 100 kDa vs. 150 kDa que tienen los anticuerpos completos, Io que confiere mayor oportunidad a los primeros de penetrar los tejidos oculares. Skurkovich divulga el resultado de aplicar gotas de fragmentos F(ab')2 de anticuerpos anti-IFNγ en pacientes con rechazo al transplante de cornea. Al segundo día de tratamiento reporta disminución de edema y aumento de Ia transparencia del transplante (Skurkovich, Kasparov et al. 2002), Skurkovich, B. et al. 6,534,059 B2. "Compositions and Methods for treating hyperimmune response in the eye" (Skurkovich and Skurkovich 2003). Sin embargo, en estos antecedentes no se divulga formalmente una composición oftálmica y el desarrollo experimental de este antecedente tampoco implica que se haya requerido el manejo de una composición oftálmica farmacológicamente estable.Intraocular penetration of drugs that are applied topically in the eye is very limited due to tissue barriers: the cornea and the sclera. However, in corneal transplant processes or in corneal lesions, the drug has an opportunity to reach the tissues affected by inflammatory or rejection processes. F (ab ') 2 fragments have a molecular weight of 100 kDa vs. 150 kDa that have complete antibodies, which gives the first chance to penetrate the eye tissues. Skurkovich reports the result of applying drops of F (ab ') 2 fragments of anti-IFNγ antibodies in patients with cornea transplant rejection. On the second day of treatment reports decreased edema and increased transparency of the transplant (Skurkovich, Kasparov et al. 2002), Skurkovich, B. et al. 6,534,059 B2. "Compositions and Methods for treating hyperimmune response in the eye" (Skurkovich and Skurkovich 2003). However, in this background an ophthalmic composition is not formally disclosed and the experimental development of this antecedent does not imply that the management of a pharmacologically stable ophthalmic composition has been required.
Nuestras nuevas composiciones han sido formuladas para prevenir o minimizar procesos inflamatorios de Ia córnea y el rechazo del transplante de cornea en humanos disminuyendo el uso de otros fármacos inmunosupresores potencialmente tóxicos. Las nuevas composiciones se presentan así como una alternativa que, si bien no cura Ia patología básica, sí manipula Ia respuesta inmunológica del paciente, por Io que el oftalmólogo tratante Ia puede tener como una opción para su consideración y cuidadosa elección.Our new compositions have been formulated to prevent or minimize inflammatory processes of the cornea and rejection of corneal transplantation in humans, reducing the use of other potentially toxic immunosuppressive drugs. The new compositions are presented as an alternative that, although it does not cure the basic pathology, it does manipulate the patient's immune response, so that the treating ophthalmologist may have as an option for consideration and careful choice.
Basados en estos datos, Ia presente invención propone nuevas formulaciones oftálmicas que resuelven varias dificultades técnicas de acuerdo en el estado de Ia técnica más cercano. Las dificultades técnicas son, lograr una composición oftálmica farmacológicamente estable con Ia particularidad de que el principio activo es de carácter proteico, y más particularmente, el activo son fragmentos F(ab')2 de anticuerpos policlonales. Otro problema técnico resuelto en esta invención radica en comprobar que los fragmentos F(ab')2 de anticuerpos policlonales actuando como principio activo, son capaces de neutralizar a las citocinas. De manera particular, son capaces de neutralizar Ia actividad biológica de TNFα y de VEGF y así, de prevenir el rechazo de transplantes de cornea.Based on these data, the present invention proposes new ophthalmic formulations that solve several technical difficulties according to the state of the closest technique. The technical difficulties are to achieve a pharmacologically stable ophthalmic composition with the particularity that the active ingredient is of a protein nature, and more particularly, the active ingredient is F (ab ') 2 fragments of polyclonal antibodies. Another technical problem solved in this invention lies in verifying that the F (ab ' ) 2 fragments of polyclonal antibodies acting as active ingredient are capable of neutralizing cytokines. In particular, they are able to neutralize the biological activity of TNFα and VEGF and thus prevent the rejection of corneal transplants.
En el estado de Ia técnica se divulga el uso oftálmico de anticuerpos completos contra TNFα. Sin embargo, no se divulga una composición oftálmica formal, es decir, que se han utilizado preparaciones de inhibidores de citocinas únicamente en ensayos de investigación clínica o preclínica, pero no resuelven Ia estabilidad farmacológica. Tampoco resulta evidente que durante el desarrollo experimental esté implícito que se haya requerido el manejo de una composición oftálmica farmacológicamente estable. MODALIDADES DE LA INVENCIÓNThe ophthalmic use of complete antibodies against TNFα is disclosed in the state of the art. However, a formal ophthalmic composition is not disclosed, that is, cytokine inhibitor preparations have been used only in clinical or preclinical research trials, but they do not resolve the pharmacological stability. It is also not evident that during experimental development it is implicit that the management of a pharmacologically stable ophthalmic composition has been required. MODES OF THE INVENTION
La presente invención se refiere principalmente al uso oftálmico de anticuerpos neutralizantes o de sus fragmentos activos como neutralizantes de Ia actividad biológica de citocinas proinflamatorias y angiogénicas, particularmente Ia invención se refiere a colirios o composiciones oftálmicas farmacológicamente estables, estériles, no pirógenas y adecuadas para aplicación tópica, cuyo activo es dichos anticuerpos o fragmentos de los mismos.The present invention relates mainly to the ophthalmic use of neutralizing antibodies or their active fragments as neutralizers of the biological activity of proinflammatory and angiogenic cytokines, particularly the invention relates to pharmacologically stable eye drops or compositions, pharmacologically stable, non-pyrogenic and suitable for application topical, whose active ingredient is said antibodies or fragments thereof.
Se describe como ejemplo el desarrollo de una composición oftálmica que cumple con los criterios de estabilidad y que se caracteriza porque el principio activo es una proteína neutralizante de Ia bioactividad de TNFα y/o VEGF. Este ejemplo no pretende limitar al uso de inhibidores de TNFα y/o VEGF.The development of an ophthalmic composition that meets the stability criteria and that is characterized in that the active ingredient is a neutralizing protein of the bioactivity of TNFα and / or VEGF is described as an example. This example is not intended to limit the use of TNFα and / or VEGF inhibitors.
De manera más particular, dicha composición oftálmica farmacológicamente estable tiene como principio activo un fragmento de anticuerpo F(ab')2 neutralizante del TNFα.More particularly, said pharmacologically stable ophthalmic composition has as its active ingredient a fragment of TNFα neutralizing F (ab ' ) 2 antibody.
De manera preferente, Ia formulación de Ia presente invención contiene los siguientes componentes: 24 mg/mL de Fosfato dibásico de sodio, de 66 mg/mL de Fosfato monobásico de sodio, 4.5 mg/mL de Cloruro de sodio, 2 mg/mL de Glicerina, 5 mg/mL de Ácido bórico, 2 mg/mL de Propilenglicol, todo en grado ACS (American Chemical Society) disuelto en agua destilada estéril, y contiene como principio activo fragmentos de anticuerpos F(ab')2 neutralizantes del TNFα en una concentración de 5 mg/mL.Preferably, the formulation of the present invention contains the following components: 24 mg / mL of sodium dibasic phosphate, 66 mg / mL of monobasic sodium phosphate, 4.5 mg / mL of sodium chloride, 2 mg / mL of Glycerin, 5 mg / mL of Boric acid, 2 mg / mL of Propylene Glycol, all in ACS grade (American Chemical Society) dissolved in sterile distilled water, and contains as active substance fragments of neutralizing F (ab ' ) 2 antibodies of TNFα in a concentration of 5 mg / mL.
La invención se refiere al uso de dichas composiciones en el tratamiento oftálmico tópico para evitar o minimizar procesos inflamatorios de Ia córnea que consiste en Ia administración de soluciones oftálmicas cuyo principio activo son proteínas bloqueadoras de Ia bioactividad de citocinas pro-inflamatorias y/o angiogénicas.The invention relates to the use of said compositions in the topical ophthalmic treatment to avoid or minimize inflammatory processes of the cornea which consists of the administration of ophthalmic solutions whose active ingredient are blocking proteins of the bioactivity of pro-inflammatory and / or angiogenic cytokines.
De manera más particular Ia invención se refiere al uso de dichas composiciones en el tratamiento oftálmico tópico para evitar o minimizar procesos de rechazo agudo de transplante de cornea que consiste en Ia administración de soluciones oftálmicas cuyo principio activo son proteínas bloqueadoras de Ia actividad de citocinas pro-inflamatorias y/o angiogénicas. DESCRIPCIÓN DE LAS FIGURASMore particularly, the invention relates to the use of said compositions in the topical ophthalmic treatment to avoid or minimize acute rejection processes of corneal transplantation, which consists in the administration of ophthalmic solutions whose active ingredient are blocking proteins of the pro cytokine activity. -inflammatory and / or angiogenic. DESCRIPTION OF THE FIGURES
Figura 1. Análisis de pureza del principio activo F(ab')2 anti-TNFα. A) HPLC. Se muestra el perfil de filtración en gel de CIP A/l-0101 en una columna GF-250. B) SDS- PAGE bajo condiciones reductoras. Línea 1 , marcadores de peso molecular (250, 150, 100, 75, 50, 37, 25, 20 y 15 kDa); Línea 2, St. de IgG equina (10 μg); Línea 3, St. de ESA (10 μg); Línea 4, St. de F(ab')2 de caballo (10 μg); Línea 5, St. de Fab de caballo (10 μg); Línea 6, F(ab')2 anti-TNFα (15 μg). El tamaño esperado de Ia cadena ligera (L) y el fragmento de Ia cadena pesada (H) se indican con flechas.Figure 1. Purity analysis of the active substance F (ab ') 2 anti-TNFα. A) HPLC. The gel filtration profile of CIP A / l-0101 is shown on a GF-250 column. B) SDS-PAGE under reducing conditions. Line 1, molecular weight markers (250, 150, 100, 75, 50, 37, 25, 20 and 15 kDa); Line 2, Equine IgG St. (10 μg); ESA Line 3, St. (10 μg); Line 4, St. of F (ab ') 2 horse (10 μg); Line 5, Horse Fab St. (10 μg); Line 6, F (ab ') 2 anti-TNFα (15 μg). The expected size of the light chain (L) and the fragment of the heavy chain (H) are indicated by arrows.
Figura 2. Neutralización de Ia citotoxicidad mediada por TNFα con F(ab')2 anti-TNFα.Figure 2. Neutralization of the cytotoxicity mediated by TNFα with F (ab ') 2 anti-TNFα.
A) Bioactividad de rhTNFα. La actividad biológica de rhTNFα se evaluó por su efecto citolítico sobre células L929 en presencia de actinomycina D. B) Actividad neutralizante de F(ab')2 anti-TNFα. Se incubaron 5 DE50 de rhTNFα (125 pg/mL) con las concentraciones indicadas de F(ab')2 anti-TNFα. Inmediatamente se agregó esta mezcla al cultivo confluente de células L929. Se muestran los resultados con fragmentos F(ab')2 de anticuerpos no relacionados (Control). Las figuras son representativas de tres experimentos independientes.A) Bioactivity of rhTNFα. The biological activity of rhTNFα was evaluated for its cytolytic effect on L929 cells in the presence of actinomycin D. B) Neutralizing activity of F (ab ') 2 anti-TNFα. 5 OF 50 of rhTNFα (125 pg / mL) were incubated with the indicated concentrations of F (ab ') 2 anti-TNFα. This mixture was immediately added to the confluent culture of L929 cells. The results are shown with F (ab ') 2 fragments of unrelated antibodies (Control). The figures are representative of three independent experiments.
Figura 3. Componentes de las formulaciones A, B y C. Todas las cantidades especificadas en Ia tabla están expresadas en miligramos (a excepción del volumen de agua inyectable, que ya se especifica en mililitros).Figure 3. Components of formulations A, B and C. All amounts specified in the table are expressed in milligrams (except for the volume of injectable water, which is already specified in milliliters).
Figura 4. Potencia de las formulaciones A, B y C. Las formulaciones A, B y C, junto a sus respectivas matrices analíticas (excipientes sin principio activo), se evaluaron por triplicado en cuanto a Ia actividad biológica específica (potencia). Los resultados se expresan como millones de U/mg de proteína. "Y" es Ia respuesta analítica y corresponde a Ia media de los valores obtenidos.Figure 4. Power of the formulations A, B and C. The formulations A, B and C, together with their respective analytical matrices (excipients without active substance), were evaluated in triplicate in terms of the specific biological activity (potency). The results are expressed as millions of U / mg of protein. "Y" is the analytical response and corresponds to the average of the values obtained.
Figura 5. Identidad del principio activo en las formulaciones A, B y C. Se corrió un SDS-PAGE bajo condiciones reductoras con las formulaciones A, B y C. Líneas 1 , marcadores de peso molecular; Línea 2-4, Controles principio activo de diferentes lotes (antes de formular); Formulación A (5 μg); Línea 5, Formulación A (5 μg); Línea 6, Formulación B (5 μg); Línea 7, Formulación C (5 μg). Figura 6. Recuperación de proteína de lotes piloto 211105-1, 211105-2 y 211105-3 durante el estudio de estabilidad. Se muestran los resultados de cuantificación de proteína por el método de Bradford, a las condiciones especificadas en el Protocolo de Estabilidad. Se muestran resultados a los 0, 30, 60 y 90 días.Figure 5. Identity of the active substance in formulations A, B and C. An SDS-PAGE was run under reducing conditions with formulations A, B and C. Lines 1, molecular weight markers; Line 2-4, Active ingredient controls of different batches (before formulating); Formulation A (5 μg); Line 5, Formulation A (5 μg); Line 6, Formulation B (5 μg); Line 7, Formulation C (5 μg). Figure 6. Protein recovery from pilot lots 211105-1, 211105-2 and 211105-3 during the stability study. The protein quantification results are shown by the Bradford method, at the conditions specified in the Stability Protocol. Results are shown at 0, 30, 60 and 90 days.
Figura 7. Recuperación de Bioactivídad de lotes piloto 211105-1, 211105-2 y 211105-3 en estudios estabilidad. Se muestran los resultados de Ia actividad biológica recuperada, a las condiciones especificadas en el Protocolo de Estabilidad. Se muestran resultados a los 90, 180 y 365 días. Para cada tiempo especificado se incluyó un control (principio activo) corrido en paralelo.Figure 7. Bioactivity recovery of pilot lots 211105-1, 211105-2 and 211105-3 in stability studies. The results of the recovered biological activity are shown, at the conditions specified in the Stability Protocol. Results are shown at 90, 180 and 365 days. For each specified time a control (active principle) run in parallel was included.
Figura 8. Determinación de Ia dosis neutralizante de F(ab')2 anti-VEGF. A) Evaluación de Ia actividad proliferativa de rhVEGF sobre células HUVEC en cultivo primario. La proliferación celular se evaluó con Ia técnica de CellTiter96 (Promega) y se leyó Ia reacción colorimétrica a una Abs de 490 nm. La DE50 del rhVEGF165 fue de 1.23 ng/mL. B) Evaluación de Ia neutralización de Ia actividad proliferativa del VEGF. Figura representativa de tres ensayos independientes usando 3DE50 de rhVEGF-ι65 (3.7 ng/mL) y diferentes concentraciones de F(ab')2 anti-rhVEGF165 La Dosis neutralizante (DN50) fue de 17.34 μg/mLFigure 8. Determination of the neutralizing dose of F (ab ' ) 2 anti-VEGF. A) Evaluation of the proliferative activity of rhVEGF on HUVEC cells in primary culture. Cell proliferation was evaluated with the technique of CellTiter96 (Promega) and the colorimetric reaction was read at an Abs of 490 nm. The ED 50 of rhVEGF 165 was 1.23 ng / mL. B) Evaluation of the neutralization of the proliferative activity of VEGF. Representative figure of three independent tests using 3DE 50 of rhVEGF-ι 65 (3.7 ng / mL) and different concentrations of F (ab ' ) 2 anti-rhVEGF 165 The neutralizing dose (DN 50 ) was 17.34 μg / mL
Figura 9. Curva de Sobrevida. Curva generada tomando en cuenta el grado de opacidadFigure 9. Survival curve. Curve generated taking into account the degree of opacity
5 y por análisis de Kaplann-Meier. La probabilidad acumulada de sobrevida del transplante fue mayor en los animales que recibieron el tratamiento 1.5 and by Kaplann-Meier analysis. The cumulative probability of transplant survival was higher in the animals that received treatment 1.
EJEMPLOSEXAMPLES
La invención se describe a continuación con mayor detalle haciendo referencia a los siguientes ejemplos que corresponden a ensayos experimentales. Estos ejemplos se proveen únicamente con propósitos ilustrativos y no tienen Ia intención de limitar el alcance de Ia invención, a menos que se especifique Io contrario. Se presentan también para dar a conocer el mejor método para llevar a cabo Ia invención.The invention is described in more detail below with reference to the following examples corresponding to experimental tests. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention, unless otherwise specified. They are also presented to publicize the best method for carrying out the invention.
Todas las evaluaciones se llevaron a cabo siguiendo Métodos Generales de Análisis de Ia Farmacopea de los Estados Unidos Americanos (USP) y Ia Farmacopea de los Estados Unidos Mexicanos (FEUM), así como las guías ICH y CFR que aplican en cada caso. Las especificaciones para el desarrollo de las formulaciones se muestran en Ia tabla I. Estas especificaciones pueden variar de acuerdo a los resultados de los estudios deAll evaluations were carried out following the General Methods of Analysis of the Pharmacopoeia of the United States of America (USP) and the Pharmacopoeia of the United Mexican States (FEUM), as well as the ICH and CFR guidelines that apply in each case. The specifications for the development of the formulations are shown in Table I. These specifications may vary according to the results of the studies of
Figure imgf000015_0001
estabilidad, de Ia validación del proceso y de los estudios preclínicos que se realicen en el futuro.
Figure imgf000015_0001
stability, of the validation of the process and of the preclinical studies that are carried out in the future.
EJEMPLO 1EXAMPLE 1
Se obtuvieron los fragmentos de anticuerpo F(ab')2 anti-TNFα en condiciones de Buenas Prácticas de Fabricación en Instituto Bioclon SA de CV de acuerdo a métodos descritos previamente, y se procedió a Ia caracterización del producto como principio activo. Se realizó una electroforesis en geles de poliacrilamida en condiciones reductoras (SDS-PAGE corrimiento a100 V por 1.5 h visualizado con azul brillante de Coomasie G-250) para determinar Ia pureza y estimar los pesos moleculares de las cadenas ligera (L) y pesada (H) de F(ab')2 anti-TNFα. La pureza de F(ab')2 anti-TNFα se analizó en un HPLC Waters equipado con una bomba cuaternaria y detector de arreglo de diodos (Waters 2996) usando una columna de exclusión molecular Zorbax GF-250 (Agilent Technologies). Los resultados revelan una pureza mayor a 85% (figura 1A) y Ia masa molecular esperada (figura 1 B). Esto demuestra Ia ausencia de inmunoglobulinas completas (IgG's) y albúmina en Ia preparación.The anti-TNFα F (ab ') 2 antibody fragments were obtained under Good Manufacturing Practices at Instituto Bioclon SA de CV according to previously described methods, and the product was characterized as active ingredient. An electrophoresis was carried out in polyacrylamide gels under reducing conditions (SDS-PAGE run at 100 V for 1.5 h visualized with Coomasie G-250 bright blue) to determine the purity and estimate the molecular weights of the light (L) and heavy chains ( H) of F (ab ') 2 anti-TNFα. The purity of F (ab ') 2 anti-TNFα was analyzed in a Waters HPLC equipped with a quaternary pump and diode array detector (Waters 2996) using a Zorbax GF-250 molecular exclusion column (Agilent Technologies). The results reveal a purity greater than 85% (Figure 1A) and the expected molecular mass (Figure 1 B). This demonstrates the absence of complete immunoglobulins (IgG's) and albumin in the preparation.
EJEMPLO 2EXAMPLE 2
Inicialmente se evaluó Ia actividad citolítica de TNFα recombinante humano (rhTNFα), medida como dosis efectiva media (DE50), a través de un ensayo de bioactividad sobre Ia línea celular L929 (ATCC, CCL-1 número de lote 2869501), de acuerdo a métodos descritos (Hay and Cohén 1989). La DE50 para rhTNFα fue de 250 pg/mL (figura 2A) y es Ia concentración de rhTNFα requerido para matar el 50% de las células L929 en cultivo in vitro. Para evaluar Ia capacidad neutralizante (potencia) del principio activo, se modificó el ensayo de Ia figura 2A sobre Ia línea celular L929 utilizando 5DE50 (1250 pg/mL) de rhTNFα con distintas diluciones del principio activo [F(ab')2 anti-TNFα]. La potencia medida como Dosis Neutralizante al 50% (DN50) del principio activo fue 507 ng/mL y es la concentración de producto requerido, para neutralizar al 50% Ia bioactividad de 5DE50 (1250 pg/mL) de rhTNFα en cultivo in vitro. La figura 2B es representativa de tres experimentos independientes realizados por triplicado. En este último caso, se utilizaron como control fragmentos F(ab')2 que neutralizan el veneno de Centruroides spp. EI control no tuvo efecto en el bioensayo, y no inhibió Ia actividad biológica de TNFα a ninguna de las concentraciones evaluadas. El ensayo de neutralización descrito se diseñó para Ia evaluación de la bioactividad del principio activo para las nuevas composiciones farmacéuticas de Ia invención. Para evitar confusiones al expresar concentración de proteína, Ia bioactividad neutralizante de F(ab')2 se expresa en Unidades / miligramo (U / mg), de acuerdo a Ia siguiente fórmula: 5x lOfi Initially, the cytolytic activity of human recombinant TNFα (rhTNFα), measured as mean effective dose (ED 50 ), was evaluated through a bioactivity test on the L929 cell line (ATCC, CCL-1 lot number 2869501), according to described methods (Hay and Cohen 1989). The ED 50 for rhTNFα was 250 pg / mL (Figure 2A) and is the concentration of rhTNFα required to kill 50% of L929 cells in in vitro culture. To evaluate the neutralizing capacity (potency) of the active ingredient, the assay of Figure 2A on the L929 cell line was modified using 5DE 50 (1250 pg / mL) of rhTNFα with different dilutions of the active ingredient [F (ab ') 2 anti -TNFα]. The potency measured as 50% Neutralizing Dose (DN 50 ) of the active substance was 507 ng / mL and is the concentration of the product required to neutralize the bioactivity of 5DE 50 (1250 pg / mL) of rhTNFα in culture in 50% vitro. Figure 2B is representative of three independent experiments performed in triplicate. In the latter case, F (ab ') 2 fragments that neutralize the venom of Centruroides spp. The control had no effect on the bioassay, and did not inhibit the biological activity of TNFα at any of the concentrations evaluated. The neutralization test described was designed for the evaluation of the bioactivity of the active ingredient for the new pharmaceutical compositions of the invention. To avoid confusion when expressing protein concentration, the neutralizing bioactivity of F (ab ') 2 is expressed in Units / milligram (U / mg), according to the following formula: 5x lO fi
= U/mg= U / mg
DE50 (ng I mL) x DN50 (ng I mL)SD 50 (ng I mL) x DN 50 (ng I mL)
Donde U son las unidades de bioactividad neutralizante de F(ab')2 anti-TNFα contenidos en un miligramo de proteína.Where U are the neutralizing bioactivity units of F (ab ') 2 anti-TNFα contained in a milligram of protein.
EJEMPLO 3EXAMPLE 3
El siguiente ejemplo es de carácter ilustrativo mas no limitativo del alcance de Ia invención. Se prepararon tres formulaciones distintas designadas como A, . B y C, conteniendo fragmentos de anticuerpos F(ab')2 anti-TNFα como principio activo. En Ia tabla de Ia figura 3 se especifica cada uno de los componentes de las tres formulaciones y los rangos de concentración adecuados se expresan en miligramos por mililitro.The following example is illustrative but not limited to the scope of the invention. Three different formulations designated as A,. B and C, containing fragments of antibodies F (ab ' ) 2 anti-TNFα as active ingredient. In the table of Figure 3, each of the components of the three formulations is specified and the appropriate concentration ranges are expressed in milligrams per milliliter.
A cada una de las formulaciones propuestas se realizaron estudios de irritabilidad ocular y determinación de Ia actividad biológica para determinar Ia compatibilidad de los excipientes con Ia proteína adicionada. La evaluación de Ia irritabilidad ocular se llevó a cabo en conejos albinos, de acuerdo al Método General de Análisis <0516> Irritabilidad Ocular (Farmacopea de los Estados Unidos Mexicanos, FEUM. 2004. 8a edición. Pags 470-471), tomando como reacción positiva Ia presencia de ulceración u opacidad de Ia cornea, inflamación del iris o inflamación de Ia conjuntiva. Después de observar a los animales a las 24, 48 y 72 horas, las formulaciones A, B, y C resultaron NO irritantes. Las formulaciones A, B y C, junto a sus respectivas matrices analíticas (excipientes sin principio activo), se evaluaron por triplicado en cuanto a Ia actividad biológica específica (potencia). Los resultados se resumen en Ia tabla de Ia figura 4. Juntos, los resultados obtenidos indican que las 3 distintas formulaciones propuestas (A, B y C) pueden servir como vehículo para el principio activo F(ab')2 anti-TNFα, debido a que resultaron NO irritantes y mantienen Ia actividad biológica del principio activo.To each of the proposed formulations, studies of ocular irritability and determination of the biological activity were performed to determine the compatibility of the excipients with the added protein. The Evaluation of the ocular irritability was conducted in albino rabbits, according to the General Method of Analysis <0516> Ocular Irritability (Pharmacopoeia of the United Mexican States, FEUM. 2004. 8 edition pp . 470-471), taking as positive reaction the presence of ulceration or opacity of the cornea, inflammation of the iris or inflammation of the conjunctiva. After observing the animals at 24, 48 and 72 hours, formulations A, B, and C were NOT irritating. The formulations A, B and C, together with their respective analytical matrices (excipients without active substance), were evaluated in triplicate in terms of the specific biological activity (potency). The results are summarized in the table of Figure 4. Together, the results obtained indicate that the 3 different proposed formulations (A, B and C) can serve as a vehicle for the active principle F (ab ') 2 anti-TNFα, due to which they were NOT irritating and maintain the biological activity of the active substance.
EJEMPLO 4EXAMPLE 4
Como prueba de identidad del principio activo se realizó un corrimiento electroforético (SDS-PAGE) de Ia formulación (tal como se describió en el Ejemplo 1), utilizando como testigo el principio activo concentrado obtenido de Ia Planta en condiciones de Buenas Prácticas de Fabricación. En las formulaciones A, B, y C el perfil de corrimiento es idéntico al del control (figura 5). EJEMPLO 5As proof of identity of the active principle, an electrophoretic shift (SDS-PAGE) of the formulation (as described in Example 1) was carried out, using as a control the concentrated active principle obtained from the Plant under conditions of Good Manufacturing Practices. In formulations A, B, and C the shifting profile is identical to that of the control (Figure 5). EXAMPLE 5
En este ejemplo, se definió Ia Formulación A como Ia formulación más adecuada, considerada como Ia formulación más completa con respecto a Ia presencia de agentes viscosantes y anti-microbianos. A partir de esta definición se formularon tres lotes distintos (denominados 211105-1, 211105-2 y 211105-3) para realizar estudios de estabilidad acelerada. A estos nuevos lotes se les evaluó Ia irritabilidad ocular, de acuerdo al Método General de Análisis <0516> Irritabilidad Ocular (FEUM. 2004. 8a edición. VoI I, Pags 470- 471). Después de observar a los animales a las 24, 48 y 72 horas, los lotes 211105-1, 211105-2 y 211105-3 (como se esperaba de los resultados anteriores de irritabilidad ocular para formulación A) resultaron NO irritantes.In this example, Formulation A was defined as the most appropriate formulation, considered as the most complete formulation with respect to the presence of viscous and anti-microbial agents. From this definition, three different batches (called 211105-1, 211105-2 and 211105-3) were formulated for accelerated stability studies. These new batches were evaluated Ia ocular irritability, according to the General Method of Analysis <0516> Ocular Irritability (FEUM. 2004. 8 edition. VoI I, pp 470- 471). After observing the animals at 24, 48 and 72 hours, lots 211105-1, 211105-2 and 211105-3 (as expected from the previous results of eye irritability for formulation A) were NOT irritating.
El protocolo de estabilidad al que se sometieron los lotes fueron 30 0C + 2 0C de temperatura con 65% + 5% de humedad relativa y 40 0C + 2 0C de temperatura con 75% + 5% de humedad relativa. Los periodos de tiempo para los análisis se establecieron a los 0, 30 60 y 90 días. Los análisis realizados incluyeron Ia evaluación de esterilidad, efectividad antimicrobiana, y más importante para nosotros fue Ia determinación de Ia concentración de proteínas y Ia determinación de Ia bioactividad neutralizante (potencia) de F(ab')2 anti- TNFα.The stability protocol to which the batches were subjected were 30 0 C + 2 0 C temperature with 65% + 5% relative humidity and 40 0 C + 2 0 C temperature with 75% + 5% relative humidity. The time periods for the analyzes were established at 0, 30, 60 and 90 days. The analyzes carried out included the evaluation of sterility, antimicrobial effectiveness, and more important for us was the determination of the protein concentration and the determination of the neutralizing bioactivity (potency) of F (ab ') 2 anti-TNFα.
EJEMPLO 6 La evaluación de Ia esterilidad de los lotes 211105-1, 211105-2 y 211105-3 se llevó a cabo por incubación en medios de cultivo en medios Tioglicolato y Dextrosa Sabouraud durante 14 días. Se determinó Ia presencia o ausencia de crecimiento de bacterias y hogos, de acuerdo a los Métodos Generales de Análisis <0381> Esterilidad. FEUM, 2004. pags 426- 434; <71> Sterility tests. USP, 2005. pags 2251-2256; <81> Antibiotics. USP, 2005. pags 2256-2263. Se utilizaron como testigos los siguientes agentes bacterianos: Staphylococus aureus, Pseudomonas aureginosa, Clostridium sporogenes, Bacillus subtillis, además de los hongos: Aspergillus niger y Candida albicans. En todos los casos las muestras evaluadas de los lotes 211105-1, 211105-2 y 211105-3 se reportaron estériles. Las bacterias y hongos que se utilizaron como testigo crecieron de manera abundante.EXAMPLE 6 The evaluation of sterility of lots 211105-1, 211105-2 and 211105-3 was carried out by incubation in culture media in Thioglycolate and Dextrose Sabouraud media for 14 days. The presence or absence of bacterial and fungal growth was determined, according to the General Methods of Analysis <0381> Sterility. FEUM, 2004. pages 426-434; <71> Sterility tests. USP, 2005. pages 2251-2256; <81> Antibiotics. USP, 2005. pages 2256-2263. The following bacterial agents were used as controls: Staphylococus aureus, Pseudomonas aureginosa, Clostridium sporogenes, Bacillus subtillis, in addition to fungi: Aspergillus niger and Candida albicans. In all cases the samples evaluated from lots 211105-1, 211105-2 and 211105-3 were reported sterile. The bacteria and fungi that were used as a control grew abundantly.
EJEMPLO 7EXAMPLE 7
Se determinó Ia efectividad antimicrobiana de las soluciones oftálmicas de Ia invención, de acuerdo a los Métodos Generales de Análisis <0305> Efectividad de preservativos antimicrobianos. FEUM, 2004. pags 401-402; <51> Antimicrobial effectiveness testing. USP, 2005. pags 2242-2243. En todos los casos, los lotes obtenidos evaluados a los tiempos señalados redujeron Ia carga bacteriana y micótica de acuerdo a las especificaciones descritas en el protocolo de estabilidad.The antimicrobial effectiveness of the ophthalmic solutions of the invention was determined, according to the General Methods of Analysis <0305> Effectiveness of antimicrobial condoms. FEUM, 2004. pages 401-402; <51> Antimicrobial effectiveness testing. USP, 2005. pages 2242-2243. In all cases, the lots obtained evaluated at indicated times reduced the bacterial and fungal load according to the specifications described in the stability protocol.
EJEMPLO 8 La concentración de proteínas y Ia bioactividad neutralizante se realizaron de acuerdo a Io descrito en los ejemplos anteriores. Un resumen de Ia concentración de proteínas y la bioactividad neutralizante recuperada de los lotes 211105-1, 211105-2 y 211105-3, a los tiempos y condiciones definidas, se resume en las tablas de las figuras 6 y 7. En todos los casos monitoreados se recuperó proteína (figura 6) de acuerdo a las especificaciones descritas en el protocolo de estabilidad, así como actividad biológica neutralizante específica contra TNFα (figura 7). La bioactividad para cada una de las condiciones en particular se evaluó contra un control que consiste del principio activo antes de formular. Por Io que si existen variaciones en Ia bioactividad, se debe comparar contra el control en cada caso particular. Todos estos datos permiten concluir que Ia formulación propuesta confiere estabilidad al principio activo a las condiciones y los tiempos descritos en el protocolo de estabilidad.EXAMPLE 8 Protein concentration and neutralizing bioactivity were performed according to what was described in the previous examples. A summary of the protein concentration and the neutralizing bioactivity recovered from lots 211105-1, 211105-2 and 211105-3, at the defined times and conditions, is summarized in the tables of Figures 6 and 7. In all cases Monitored protein was recovered (Figure 6) according to the specifications described in the stability protocol, as well as specific neutralizing biological activity against TNFα (Figure 7). The bioactivity for each of the particular conditions was evaluated against a control consisting of the active substance before formulating. Therefore, if there are variations in the bioactivity, it should be compared against the control in each particular case. All these data allow us to conclude that the proposed formulation confers stability to the active principle to the conditions and times described in the stability protocol.
EJEMPLO 9EXAMPLE 9
El siguiente ejemplo es de carácter ilustrativo mas no limitativo del alcance de Ia invención. Se evaluaron formulaciones oftálmicas cuyo vehículo contiene Ia formula A, y como principio activo contiene fragmentos F(ab')2 con capacidad neutralizante del rhVEGF recombinante humano. Para la evaluación de estas formulaciones se utilizó el bioensayo de tetrazolio/formazán para medir Ia actividad metabólica de cultivos primarios de células humanas de endotelio de cordón umbilical (HUVEC) obtenidas del Instituto Nacional de Cardiología "Ignacio Chávez", y de acuerdo a los métodos descritos, (Cory, Owen et al. 1991). La figura 8 A) muestra los resultados expresados como dosis efectiva media (DE50) de rhVEGF en ng/mL. En este ensayo, Ia DE50 se define como Ia concentración de rhVEGF en Ia cual Ia actividad es el 50% de Ia respuesta máxima. En este caso Ia figura es representativa de tres experimentos independientes y Ia DE50 fue de 1.237 ng/mL. La figura 8 B) muestra los resultados expresados como dosis neutralizante media (DN50) que se obtiene al evaluar Ia bioactividad de fragmentos F(ab')2 anti-VEGF sobre el ensayo de proliferación de células HUVEC usando 3DE50 (3.7 ng/mL). La DN50 fue de 17.34 μg/mL EJEMPLO 10The following example is illustrative but not limited to the scope of the invention. Ophthalmic formulations whose vehicle contains formula A were evaluated, and as active ingredient it contains F (ab ' ) 2 fragments with neutralizing capacity of human recombinant rhVEGF. For the evaluation of these formulations, the tetrazolium / formazan bioassay was used to measure the metabolic activity of primary cultures of human umbilical cord endothelial cells (HUVEC) obtained from the National Institute of Cardiology "Ignacio Chávez", and according to the methods described, (Cory, Owen et al. 1991). Figure 8 A) shows the results expressed as mean effective dose (SD 50 ) of rhVEGF in ng / mL. In this test, the ED 50 is defined as the concentration of rhVEGF in which the activity is 50% of the maximum response. In this case, the figure is representative of three independent experiments and the ED 50 was 1,237 ng / mL. Figure 8 B) shows the results expressed as the average neutralizing dose (DN 50 ) obtained by evaluating the bioactivity of F (ab ') 2 anti-VEGF fragments on the HUVEC cell proliferation assay using 3DE 50 (3.7 ng / mL) The DN 50 was 17.34 μg / mL EXAMPLE 10
Una indicación terapéutica de las soluciones oftálmicas de Ia invención es Ia prevención del rechazo agudo del transplante de cornea. Para evaluar esta indicación se realizó un estudio preclínico que aportó resultados sobre el eficacia de las soluciones oftálmicas inhibidoras de Ia actividad biológica de TNF-α, cuyo principio activo consiste de fragmentos F(ab')2 de anticuerpos policlonales. Dicho estudio preclínico fue prospectivo, longitudinal, aleatorizado doble ciego.A therapeutic indication of the ophthalmic solutions of the invention is the prevention of acute rejection of the cornea transplant. To evaluate this indication, a preclinical study was carried out that provided results on the efficacy of ophthalmic solutions that inhibit the biological activity of TNF-α, whose active substance consists of F (ab ') 2 fragments of polyclonal antibodies. This preclinical study was prospective, longitudinal, randomized double blind.
El objetivo del estudio fue determinar el efecto del fármaco en estudio sobre Ia frecuencia de rechazo y el tiempo de sobrevida del transplante de cornea en un modelo murino; para Io cual se evaluó Ia sobrevida de alotransplantes ortotópicos de cornea.The objective of the study was to determine the effect of the drug under study on the frequency of rejection and the survival time of corneal transplantation in a murine model; for which the survival of orthotopic corneal allografts was evaluated.
Para generar un modelo de rechazo de alto riesgo, a todos los ratones se les indujo neovascularización con tres puntos de nylon en Ia cornea. En Ia visita 1 (día 1), el investigador, de acuerdo con una tabla de aleatorización, separó dos grupos de 35 animales cada uno, con neovascularización inducida, para recibir los tratamientos posteriores.To generate a high-risk rejection model, all mice were induced neovascularization with three nylon points in the cornea. On visit 1 (day 1), the researcher, according to a randomization table, separated two groups of 35 animals each, with induced neovascularization, to receive subsequent treatments.
El tratamiento 1 es el grupo en los que se utilizó Ia solución oftálmica A ya descrita (colirio anti-TNFα), el tratamiento 2 o placebo, es el grupo a los que administró Ia solución vehículo que es dicha formulación oftálmica A pero sin principio activo. Al día 0 se evaluó Ia neovascularización y se realizó el transplante solo a aquellos animales que presentaron vascularización grado 4 o mayor. Durante las primeras 2 semanas los ratones recibieron un antibiótico como profilaxis (quinolona, una gota oftálmica 3 veces al día). Los ratones recibieron 1 gota del fármaco o del placebo (según el grupo de asignación), tres veces al día durante 8 semana. Se realizaron dos visitas semanales para Ia evaluación de opacidad corneal, Ia evaluación fue en forma enmascarada realizada por oftalmólogos, expertos en cornea, considerando el índice de concordancia.Treatment 1 is the group in which the ophthalmic solution A already described (anti-TNFα eye drops), treatment 2 or placebo was used, is the group to which the vehicle solution was administered which is said ophthalmic formulation A but without active principle . At day 0, neovascularization was evaluated and transplantation was performed only to those animals that presented grade 4 or greater vascularization. During the first 2 weeks the mice received an antibiotic as prophylaxis (quinolone, an ophthalmic drop 3 times a day). The mice received 1 drop of the drug or placebo (depending on the allocation group), three times a day for 8 weeks. Two weekly visits were made for the evaluation of corneal opacity, the evaluation was masked by ophthalmologists, cornea experts, considering the concordance index.
Criterios de inclusión: Como donadores, se utilizaron ratones machos de 8-10 semanas, cepa C57BL/6. Como receptores, se utilizaron ratones machos de 8-10 semanas, cepa BALB/C.Inclusion criteria: As donors, 8-10 week old male mice, strain C57BL / 6, were used. As receptors, male mice of 8-10 weeks, strain BALB / C, were used.
Criterio de exclusión: donadores con opacidad corneal, y receptores con neovascularización menor a 4 y complicaciones post-operatorias.Exclusion criteria: donors with corneal opacity, and recipients with neovascularization less than 4 and post-operative complications.
La evaluación de Ia opacidad de Ia cornea se basó en Ia escala descrita en Ia siguiente tabla: Grado Opacidad ObservacionesThe evaluation of the opacity of the cornea was based on the scale described in the following table: Degree Opacity Remarks
0 Sin opacidad0 No opacity
1 Mínima1 Minimum
2 Media se observan los márgenes de Ia pupila y el iris2 Average the margins of the pupil and the iris are observed
3 Moderada solo se observan los márgenes de Ia pupila3 Moderate only the margins of the pupil are observed
4 Intensa cámara anterior visible4 Intense visible anterior camera
5 Grave no se observa Ia cámara anterior5 Severe the anterior chamber is not observed
Para el análisis y Ia estadística del estudio se realizó una comparación de proporciones de rechazo evaluando el grado de opacidad corneal a través de un análisis de Xi (Chi) cuadrada considerando un P significativa de 0.003. También se efectuó un análisis de sobrevida generando curvas con el método de Kaplan-Meier para determinar Ia probabilidad acumulada de sobrevida del transplante durante el periodo de seguimiento.For the analysis and the statistics of the study, a comparison of rejection ratios was performed evaluating the degree of corneal opacity through a square Xi (Chi) analysis considering a significant P of 0.003. A survival analysis was also carried out generating curves with the Kaplan-Meier method to determine the cumulative probability of transplant survival during the follow-up period.
En el estudio de opacidad corneal en Ia visita 6 (día 18 del estudio) se encontró una diferencia significativa (p= 0.003) cuando se compararon los grados de opacidad corneal entre los dos grupos de tratamiento: 18 ratones del grupo 1 (colirio anti-TNFα) presentaron opacidad 3 en comparación con los ratones del grupo 2 (placebo) que presentaron opacidad 4 y 5 (12 y 10 ratones respectivamente).In the study of corneal opacity on visit 6 (day 18 of the study) a significant difference was found (p = 0.003) when the degrees of corneal opacity were compared between the two treatment groups: 18 mice of group 1 (anti-eye drops TNFα) presented opacity 3 compared to mice in group 2 (placebo) that presented opacity 4 and 5 (12 and 10 mice respectively).
El análisis de sobrevida se realizó mediante una prueba de Kaplan-Meier tomando en cuenta los datos de 18 visitas en 9 semanas a Io largo del estudio los datos se agruparon por grado de opacidad.The survival analysis was performed using a Kaplan-Meier test, taking into account the data of 18 visits in 9 weeks throughout the study. The data were grouped by degree of opacity.
Si se considera falla inmunológica o rechazo del transplante corneal con una opacidad grado 5, se encontró que Ia probabilidad de sobrevida con el tratamiento del grupo 1 (colirio anti-TNFα) en Ia semana 16 estudio es del 58% comparado con el 33% que presentan los ratones del grupo 2 (placebo). El grupo de tratamiento 1 tuvo una probabilidad de sobrevida del 85% hasta Ia semana 8, en contraste, el grupo de tratamiento 2, o grupo placebo, tuvo una probabilidad de sobrevida del 85% a Ia semana 4. Ver gráfico de Ia figura 9.If immunological failure or rejection of the corneal transplant with a grade 5 opacity is considered, it was found that the probability of survival with the treatment of group 1 (anti-TNFα eye drops) in week 16 study is 58% compared to 33% who the mice in group 2 (placebo) present. Treatment group 1 had a 85% chance of survival until week 8, in contrast, treatment group 2, or placebo group, had a 85% chance of survival at week 4. See graph in Figure 9 .
De acuerdo con lo anterior, se concluyó Ia eficacia del colirio anti-TNFα, y de manera generalizada se concluye Ia eficacia de los fragmentos F(ab')2 policlonales anti-TNFα, para el tratamiento preventivo del rechazo del transplante de cornea, dicho tratamiento implica Ia administración por vía tópica. REFERENCIASIn accordance with the above, the efficacy of anti-TNFα eye drops was concluded, and the efficacy of polyclonal anti-TNFα F (ab ' ) 2 fragments for the preventive treatment of cornea transplant rejection, said Treatment involves administration topically. REFERENCES
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Claims

REIVINDICACIONES: CLAIMS:
1. El uso de fragmentos de anticuerpos anti-TNFα, para fabricar un colirio o un medicamento oftálmico en solución administrable por vía tópica o, para tratar o minimizar procesos de inflamación de Ia cornea incluyendo rechazo agudo de transplante de cornea.1. The use of anti-TNFα antibody fragments, to make an eye drops or an ophthalmic medication in a solution that can be administered topically or to treat or minimize inflammation processes of the cornea including acute rejection of cornea transplantation.
2. El uso de conformidad con Ia reivindicación 1 en donde los fragmentos de anticuerpos son de tipo monoclonal, policlonal, recombinante o humanizado, de tipo Fv, Fab, F(ab')2 o scFv.2. The use according to claim 1 wherein the antibody fragments are of the monoclonal, polyclonal, recombinant or humanized type, of the Fv, Fab, F (ab ') 2 or scFv type.
3. El uso de conformidad con una de las reivindicaciones 1 ó 2, donde el medicamento ó colirio es para tratar un padecimiento ocular inflamatorio seleccionado del grupo que consiste de: queratitis, uveítis, retinitis, rechazo de transplante de cornea, blefaritis y degeneración macular.3. The use according to one of claims 1 or 2, wherein the medicament or eye drops is for treating an inflammatory eye condition selected from the group consisting of: keratitis, uveitis, retinitis, cornea transplant rejection, blepharitis and macular degeneration .
4. Una composición oftálmica caracterizada porque comprende como principio activo una cantidad farmacéuticamente efectiva dαfragmentos de anticuerpos F(ab')2 anti-TNFα.4. An ophthalmic composition characterized in that it comprises as an active ingredient a pharmaceutically effective amount of anti-TNFα F (ab ') 2 antibody fragments.
5. La composición de conformidad con Ia reivindicación 4, caracterizada porque comprende un vehículo farmacéuticamente aceptable. 5. The composition according to claim 4, characterized in that it comprises a pharmaceutically acceptable carrier.
6. La composición de conformidad con Ia reivindicación 4 que contiene como vehículo farmacéutico los siguientes componentes: de 5 a 50 mg/mL de Fosfato dibásico de sodio, de 30 a 80 mg/mL de Fosfato monobásico de sodio, 0.5 a 10 mg/mL de Cloruro de sodio, 0.05 a 10 mg/mL de Glicerina, 0.01 a 20 mg/mL de Ácido bórico, 0.05 a 10 mg/mL de Propilenglicol, todo en grado ACS disuelto en agua destilada estéril. 6. The composition according to claim 4 containing the following components as pharmaceutical vehicle: from 5 to 50 mg / mL of sodium dibasic phosphate, from 30 to 80 mg / mL of monobasic sodium phosphate, 0.5 to 10 mg / mL of sodium chloride, 0.05 to 10 mg / mL of glycerin, 0.01 to 20 mg / mL of boric acid, 0.05 to 10 mg / mL of propylene glycol, all in ACS grade dissolved in sterile distilled water.
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WO2010062857A1 (en) * 2008-11-26 2010-06-03 Allergan, Inc. Klk-13 antibody inhibitor for treating dry eye
US8491905B2 (en) 2008-11-26 2013-07-23 Allergan, Inc. KLK-13 antibody inhibitor for treating dry eye
US8821874B2 (en) 2008-11-26 2014-09-02 Allergan, Inc. KLK-13 antibody inhibitor for treating dry eye
US10538577B2 (en) 2012-10-18 2020-01-21 Inosan Biopharma S.A. Polyvalent immunotherapeutics

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