WO2007114680A1 - Préparation ophtalmique anti-inflammatoire à base de protéine - Google Patents

Préparation ophtalmique anti-inflammatoire à base de protéine 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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tnfα
ophthalmic
rejection
cornea
cytokines
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PCT/MX2007/000049
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English (en)
Spanish (es)
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/fr

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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

L'invention porte sur de nouvelles compositions ophtalmiques à usage topique et à stabilité démontrée dont le principe actif est une molécule de nature protéique apte à bloquer l'activité des cytokines ou facteurs intervenant dans des processus ophtalmiques de type inflammatoire, de type angiogénique ou de rejet aigu de transplantations. Parmi les cytokines pro-inflammatoires, le facteur de nécrose tumorale alpha (TNFα) et le facteur de croissance vasculaire endothélial (VEGF) jouent un rôle spécifique en leur qualité d'agents angiogéniques puissants, d'où une préférence pour les compositions dont le principe actif est constitué de protéines neutralisant l'activité biologique de ces deux cytokines. Les protéines actives peuvent être des anticorps monoclonaux ou polyclonaux, naturels ou recombinés ou des fragments biologiquement actifs de celles-ci qui comprennent Fv, Fab, F(ab')2 ou scFv et qui sont aptes à éviter ou minimiser le phénomène de rejet de transplantation de tissu biologique, notamment de tissus oculaires, y compris la cornée.
PCT/MX2007/000049 2006-04-03 2007-04-03 Préparation ophtalmique anti-inflammatoire à base de protéine WO2007114680A1 (fr)

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MXPA06003717A MXPA06003717A (es) 2006-04-03 2006-04-03 Composiciones oftalmicas de proteinas inhibidoras de citocinas implicadas en procesos inflamatorios o inmunologicos.
MXPA/A/2006/003717 2006-04-03

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WO2010062857A1 (fr) * 2008-11-26 2010-06-03 Allergan, Inc. Inhibiteur à base d'anticorps de klk-13 pour traiter une kératoconjonctivite sèche
US10538577B2 (en) 2012-10-18 2020-01-21 Inosan Biopharma S.A. Polyvalent immunotherapeutics

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US20040062768A1 (en) * 2001-06-05 2004-04-01 Advanced Biotherapy, Inc. Compositions and methods for treating hyperimmune response in the eye

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