Treatment of eye disorders characterized by an elevated intraocular pressure by siRNAs

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DESCRIPTION

SiRNA treatment of eye disorders characterized by elevated intraocular pressure

Field of the invention

The present invention relates to methods and compositions for the treatment of eye disorders; in particular, but not exclusively, to the treatment of glaucoma. In preferred embodiments, the invention relates to the use of RNAi technology to down-regulate the expression of aqueous formation genes and aqueous secretion genes. Also provided are methods and compositions for treating eye disorders.

Background of the invention

RNAi as a tool to downregulate gene expression

Homologous recombination gene targeting is generally used to determine gene function in mammals, but it is a costly and time-consuming process. Alternatively, the functions of many genes can be determined after inhibition of mRNA with ribozyme or antisense technologies. While these technologies have been successful in some situations, their universal application has been difficult. The advent of siRNA-directed knockdown has triggered a revolution in somatic cell genetics, enabling rapid and economical analysis of gene function in mammals.

Establishing a convenient and reliable method to silence gene expression at the mRNA level has been a recurring theme in molecular biology for the past 15 years. In the effort to generate cells or organisms with loss of function, several molecules have been examined, including for example antisense sequences, ribozymes and chimeric oligonucleotides, but the design of these molecules has been based on trial and error, depending on the properties of the target gene.

On the other hand, the desired effects have been difficult to predict and often only weak suppression has been achieved (Braasch & Corey, 2002).

Following the discovery of the phenomenon in plants in the early 1990s, in 1998, Andy Fire and Craig Mello demonstrated for the first time, with the worm Caenorhabditis elegans, that dsRNA (double-stranded RNA) could specifically and selectively inhibit gene expression. in a highly efficient way (Fire et al., 1998). In their experiment, the sequence of the first strand (the so-called sense RNA) matches that of the corresponding region of the target messenger RNA (mRNA). The second strand (antisense RNA) is complementary to its mRNA. The dsRNA obtained was found to be considerably more efficient (by several orders of magnitude) than the corresponding single-stranded RNA molecules (in particular, antisense RNA). Fire et al., 1998 named the RNAi phenomenon to refer to RNA interference. This powerful gene silencing mechanism has been shown to work in various species, especially among phylogenetic phylogeny.

RNAi begins when an enzyme called DICER encounters dsRNA and cuts it into pieces called small interfering RNAs, or siRNA. This protein belongs to the RNase III nuclease family. A protein complex assembles these RNA residues and uses their code as a guide to search for and destroy any RNA in the cell with a mating sequence such as target mRNA (for review see Bosher & Labouesse, 2000).

The RNAi phenomenon (Akashi et al., 2001) could be summarized as follows:

• Stage 1: dsRNA recognition and exploration process.

• Stage 2: cleavage of dsRNAs through RNase III activity and production of siRNA.

• Stage 3: association of siRNAs and associated factors in RISC complexes.

• Stage 4: recognition of complementary target mRNA.

• Step 5: cleavage of the target mRNA in the center of the complementary region of the siRNA.

• Stage 6: degradation of target mRNA and recycling of the RISC complex.

In trying to apply the RNAi phenomenon as a technology for gene silencing, it was soon realized that mammalian cells have evolved various protective phenomena against viral infections that could impede the use of this approach. In fact, the presence of extremely low levels of viral dsRNA triggers an interferon response resulting in global non-specific suppression of translation which, in turn, triggers apoptosis (Williams, 1997, Gil & Esteban, 2000).

In 2000, a first attempt with dsRNA resulted in the specific inhibition of 3 genes (MmGFP under the control of elongation factor 1a, E-cadherin, and c-mos) in the mouse oocyte and early embryo. Translation arrest and therefore a PKR response was not observed as the embryos continued to develop (Wianny & Zernicka-Goetz, 2000). One year later, research at Ribopharma AG (Kulmbach, Germany) demonstrated by

ES 2 632 913 T3 first time RNAi functionality in mammalian cells. Using short dsRNAs (20-24 base pairs) called SIRPLEX ™ - they specifically turned off genes even in human cells without initiating the acute phase response. Other similar experiments carried out by other teams of researchers (Elbashir et al., 2001; Caplen et al, 2001) continued to confirm these results.

One year later, Paddison et al. (Paddison et al, 2002) tried to use small RNA folded into hairpin structures to inhibit the function of specific genes. This work was inspired by previous studies in which it was shown that some genes in Caenorhabditis e / egans naturally regulate other genes through RNAi encoding RNA of hairpinned structure. Tested in a variety of normal and tumor human and mouse cell lines, short hairpinned RNAs (shRNAs) are capable of silencing genes with the same efficiency as their siRNA counterparts. Furthermore, shRNAs show better reassociation kinetics in vivo than equivalent double-stranded ones. It should be noted that these authors generated transgenic cell lines obtained by genetic engineering to synthesize hcRNA that have a long-lasting suppression effect throughout cell divisions (Eurogenetec). Recently, another group of small RNAs (also in the 21-25 nt range) have been shown to mediate down-regulation of gene expression. These RNAs, known as small temporally regulated RNAs (tRNA), have been described in Caenorhabditis elegans where they regulate the timing of gene expression during development. It should be noted that tRNAs and siRNAs, despite their obvious similarities, proceed through different modes of action (for a review see Banerjee & Slack, 2002). In contrast to siRNA, 22 nt long tRNAs down-regulate target mRNA expression after initiation of translation without affecting mRNA integrity. Recent studies indicate that the two tRNAs first described in nematodes are members of a vast family of hundreds of additional micro-RNAs (miRNAs) that exist in metazoans (Grosshans & Slack, 2002).

Scientists have initially used RNAi in various systems, including Caenorhabditis elegans, Drosophila, trypanosomes, and various other invertebrates. Also, applying this approach, some teams have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines specifically in HeLa cells - demonstrating that RNAi is a method with a broad spectrum of application for gene silencing in vitro. Based on these results, RNAi has quickly become a well-recognized tool for validating (identifying and assigning) gene functions. RNA interference with the use of short dsRNA oligonucleotides will further decipher the function of genes that are only partially sequenced. Therefore, RNAi will become unavoidable in studies such as:

• Inhibition of gene expression at the post-transcriptional level in eukaryotic cells. In this context, RNAi is a simple tool to rapidly assess gene function and reveal null phenotypes.

• Development of RNAi technology for use in post-implantation embryos.

• The predominant economic importance of RNA interference is established with its application as a therapeutic principle. Thus, RNAi can produce RNA-based drugs to treat human diseases.

Glaucoma

Glaucoma is one of the main causes of blindness. Approximately 15% of the cases of blindness in the world derive from glaucoma. The most common type, primary open-angle glaucoma, has a prevalence of 1/200 among the general population over 40 years of age.

Glaucoma has been defined simply as the process of destruction of ocular tissue caused by a sustained elevation of intraocular pressure (IOP) above normal physiological limits.

It is becoming increasingly clear that many forms of glaucoma have a genetic component, and much of the current research is aimed at identifying chromosomal regions and genes that contribute to glaucoma. The etiology of GAA is likely to be multifactorial, as a result of a combination of mutations in more than one gene and environmental factors that have not yet been identified. Regarding juvenile and adult-onset GAA, several loci have been identified. However, only one gene is known, namely the myocillin / TIGR (trabecular meshwork inducible glucocorticoid response) gene at the GLC1A locus on chromosome 1q21-q31. More than thirty mutations of this gene have been identified in ethnically diverse populations around the world. Studies have shown that it is responsible for only about 5% of GAA in total (see reviews in Wirtz & Samples, 2003, and Khaw et al, 2004a).

Pathogenesis

Most glaucomas are characterized by an elevated IOP, although the level of elevation can vary. In glaucomas in which this elevation is initially low (ie, open-angle glaucoma, melanocytic glaucoma) and in some secondary glaucomas, damage to the ganglion cells of the retina and the optic nerve progresses slowly. In angle-closure glaucoma, the sudden elevation of the IOP usually causes blindness of the eye, mainly and undoubtedly as a consequence of the cessation of axoplasmic flow at the level of the lamina cribrosa.

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In human studies, it has been widely accepted that tissue ischemia plays a role in the initiation or progression of optic disc damage that occurs in glaucoma. Retinal ganglion cell degeneration may consist of necrosis, but it may be apoptosis triggered by elevated IOP, and the corresponding roles of nitric oxide and glutamate are believed to be relevant during disease progression. (For a recent review on the subject see Osborne et al, 2003).

Treatment

Although the etiology related to the glaucoma complex is diverse, the absolute determinant for selecting therapy is the amount of primary and / or induced pressure change within the iridocorneal angle.

Current therapies include medications or surgery that aim to reduce pressure, although the pathophysiological mechanisms through which elevated IOP leads to neuronal damage in glaucoma are unknown.

Medically suppressing elevated IOP can be attempted using four types of drugs: aqueous suppressants (including carbonic anhydrase inhibitors, beta-adrenergic blocking agents, or alpha 2-adrenoceptor agonists), miotics (i.e. parasympathomimetics -cholinergics -, or anticholinesterase inhibitors); enhancers of uveoscleral secretion; and hyperosmotic agents (which produce an osmotic pressure gradient across the blood / water barrier within the ciliary epithelium). All four are used in the treatment of glaucoma, the first three generally as emergency treatment and in long-term control, while the hyperosmotic agents are enormously valuable as emergency and preoperative treatment. A fifth category of drugs is beginning to appear, neuroprotective agents, which may be added in a relevant way as another medical therapy. In fact, since it has been observed that the levels of NOS and glutamate are elevated in glaucoma and that they are related to the necrosis or apoptosis of the ganglion cells of the retina, the possibility of neuroprotective therapies and even neuro-regeneration has increased. Therefore, NOS inhibitors that excite amino acid antagonists, glutamate receptor antagonists, apoptosis inhibitors, and calcium channel blockers are all potential candidates in the development of future glaucoma therapies. Calcium channel blockers may reduce the effect of impaired microcirculation at the optic nerve head, while possibly increasing the ease of secretion at the trabecular cell level.

Reviews on different eye disorders and their treatments are provided in the references, particularly in Bunce (2005), Costagliola (1995, 2000), Cullinane (2002), Sakaguchi (2002), Shah (2000), and Wang (2005) .

The therapies available to us today still fall short, and the practical difficulties associated with assessing ease of secretion, precision monitoring of therapy, and complexity of surgical techniques combine to confuse prognosis. The fundamental factor in all glaucomas is the degeneration of the retinal glanglion cells, so neuroprotection through effective ocular hypotension is an essential requirement of any therapy we use (for a recent review on the subject, see Khaw et to 2004b).

WO2004 / 042024 describes the use of siRNA to down-regulate the expression of HIF-1 alpha which in turn down-regulates VEGF expression. Control of VEGF expression can be used to inhibit angiogenesis, in particular, in diseases such as diabetic retinopathy, age-related macular degeneration and certain cancers.

Brief summary of the invention

The present invention provides the use of siRNA in the preparation of a medicament for the treatment of an ocular condition characterized by an increase in intraocular pressure (IOP), wherein said medicament is formulated for topical administration on the surface of the cornea and down regulates the expression of a target gene in the eye selected from the group consisting of carbonic anhydrases II, IV and XII; adrenergic receptors: beta I and 2 and alpha 1A, 1B and 1D; acetylcholinesterase; cyclooxygenases 1 and 2; ATPases; alpha1, alpha2, alpha3, beta1, beta2; leukocyte endothelium adhesion molecule I (ELAM-1); angiotensin II, Angiotensin II conversion enzymes (ACE I and ACE II)), Angiotensin II Receptors (ATR1 and ATR2) and renin; Cochlin.

Isolated siRNA molecules are also provided for use in the treatment of an ocular condition characterized by an increase in intraocular pressure (IOP) in the patient, the siRNA being complementary to a nucleotide sequence selected from SEQ ID 1 to SEQ ID 1829. , wherein said siRNA is for topical administration on the corneal surface; and pharmaceutical compositions comprising siRNA.

Other features of the invention are described in the appended claims.

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In the present disclosure the inventors describe a method for treating ocular conditions characterized by altered IOP in animals, including humans. In particular, eye conditions can include glaucoma, uveitis, and inflammation. The method is based on the down-regulation of the expression of genes related to watery formation and watery secretion in the eye. Downregulation can be accomplished through the use of double-stranded nucleic acid fractions, termed csNAs or small interfering ANs that are targeted for interference with the mRNA expression of various candidate genes. SiRNAs are siRNAs, although modified nucleic acids or similar chemically synthesized entities are also included within the scope of the invention.

Preferred embodiments of the disclosure relate to a topical application of cNA. Embodiments of the disclosure also provide pharmaceutical compositions for use in treating eye conditions. The invention can be used within the fields of local ocular treatments, of related target genes in the pathogenesis of glaucoma, as well as the use of chemically synthesized entities to treat animal diseases (including humans).

In addition to the treatment of glaucoma, the method of the present invention is also suitable for the treatment of other diseases of the anterior chamber of the eye. In particular, the method can be applied for the treatment of diseases characterized by an alteration in the formation or aqueous secretion in the eye. Examples of conditions that can be treated include local conditions, such as infections or inflammations, and general conditions, such as uveitis or expression of systemic diseases. Also, certain embodiments of the invention provide treatment for diabetic retinopathy.

Detailed description of the invention

Target genes

In the present disclosure, the inventors define a list of target genes, the expression levels of which can alter IOP. Said genes can be classified into the groups of genes related to aqueous formation or the group of genes related to aqueous secretion. A list of our target genes is presented below.

Carbonic ahidrases II, IV and XII

Adrenergic receptors: beta1 and 2 and alpha 1A, 1B and 1D

Acetylcholinesterase

Cyclooxygenases 1 and 2

ATPases: alpha1, alpha2, alpha3, beta1, beta2

Leukocyte endothelium adhesion molecule I (ELAM-1)

Angiotensin System: Angiotensin II, Angiotensin II Conversion Enzymes (ACE I and ACE II), Angiotensin II Receptors (ATR1 and ATR2) and Renin • Cochlin

Design by ANic

Although the mechanisms for RNAi remain unknown, the steps required to generate specific dsRNA oligonucleotides are clear. Double stranded dsRNA strands that are 21-26 nucleotides in length have been shown to work most efficiently to produce RNA interference. Selection of the right homologous region within the gene is also important. Factors such as the distance from the start codon, the G / C content and the location of the adenosine dimers are important when considering the generation of dsRNA for RNAi. However, a consequence of this is that different sequences may need to be tested to determine the most efficient RNAi and may be costly.

In 1999, Tuschl et al., Deciphered the silencing effect of siRNA demonstrating that its efficacy is a function of the length of the bicaternary, the length of the 3 'end of the protrusion, and the sequence in these protrusions. Based on this work, Eurogentec recommends that the target mRNA region and thus the sequence of the piRNA double stranded should be selected using the following guidelines:

Since RNAi depends on the establishment of complex protein interactions, it is clear that the mRNA target should be devoid of unrelated binding factors. In this context, both the 5 'and 3' untranslated regions (UTR) and the regions near the start codon should be avoided as they may be richer in regulatory protein binding sites. The sequence of siRNA is therefore selected as follows:

• In the mRNA sequence, a region located 50 to 100 nt upstream of the AUG start codon or upstream of the stop codon is selected.

• In this region, the following regions are searched: AA (N19), CA (N19).

• The G / C percentage is calculated for each identified sequence. Ideally, the G / C content is 50% but can

ES 2 632 913 T3 be less than 70% or more than 30%.

• Preferably, sequences containing the following repeats are avoided: AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT.

• An accessibility prediction is made according to the secondary structure of the mRNA as well.

• A BLAST analysis (ie NCBI EST database) is also performed with the nucleotide sequence that best fits the above criteria to ensure that only one gene is to be inactivated.

To maximize the interpretation of the result, the following precautions should be taken when using siRNA:

• Always examine sense and antisense single chains in separate experiments.

• Test a messy siRNA double stranded. This should have the same nucleotide composition as the siRNA obtained but will lack significant sequence homology with any other gene (including the one obtained).

• If possible, silence the same gene with two independent piRNA double strands to control the specificity of the silencing process.

Practically, each of the selected genes is entered as a nucleotide sequence in a prediction program that takes into account all the variables that have been described for the design of optimal oligonucleotides. The program scans any mRNA nucleotide sequence for regions that can be targeted via siRNA. The result of this analysis is a score of possible siRNA oligonucleotides. Higher scores are used to design double-stranded RNA oligonucleotides (typically 21 bp in length, although other lengths are also possible) that are typically obtained by chemical synthesis.

In addition to siRNA, modified nucleotides can also be used. The inventors envision examining various chemical modifications that are well known in the art. These modifications are intended to increase the stability or availability of the ANic.

Examples of suitable modifications are described in the publications listed below in the references, each being incorporated herein by reference.

Studies show that replacing the 3'-terminal protruding nucleotide segments of a 21 mer dsRNA having two 3 'protruding nucleotides with deoxyribunucleotides does not have an adverse effect on RNAi activity. Replacement of up to four nucleotides at each end of siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete replacement with deoxyribonucleotides results in lack of RNAi activity (Elbashir 2001). On the other hand, Elbashir et al. They also report that substitution of siRNA with 2'-O-methyl nucleotides completely eliminates RNAi activity.

Affinity-modified nucleosides can be used, as described in WO2005 / 044976. In said publication, oligonucleotides are described that comprise nucleosides modified to have a greater or lesser affinity for their complementary nucleotide in the target mRNA and / or the complementary ANic chain.

Alternative modified oligonucleotides chemically modified to provide better resistance to degradation or better absorption are described in GB2406568. Examples of such modifications include phosphorothioate internucleotide linkages, 2'-O-methyl ribonucleotides, 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, universal base nucleotides, 5-C-methyl nucleotides, and incorporation of an inverted deoixiabasic remainder.

Modified oligonucleotides are described in WO2004 / 029212 to enhance the stability of siRNA or to increase targeting efficiency. Modifications include chemical crosslinking between two complementary strands of a siRNA and chemical modification of a 3 'end of a strand of a siRNA. Preferred modifications are internal modifications, for example sugar modifications, nucleobase modifications, and / or backbone modifications. 2'-fluoro modified ribonucleotides and 2'-deoxy ribonucleotides are described.

Modified oligonucleotides that can be used are further mentioned in WO2005 / 040537.

In addition to employing modified ANdc and ANdc, the present invention may utilize short bracketed AN (hc AN); The two chains of the cNA molecule can be connected by a linker region, which can be a nucleotide linker or a non-nucleotide linker.

In addition to icNA that is perfectly complementary to the target region, redundant siNA sequences can be used to target homologous target regions. Document WO2005 / 045037 describes the design of ANic molecules to direct said homologous sequences, for example incorporating non-canonical base pairs,

ES 2 632 913 T3 eg mismatches and / or wobbly base pairs, which may provide additional target sequences. In cases where mismatches are identified, non-canonical base pairs (eg, mismatches and / or wobbly bases) can be used to generate siNA molecules that target more than one gene sequence. In a non-exhaustive example, non-canonical base pairs such as UU and CC base pairs are used to generateicAN molecules that are capable of targeting sequences for different targets that share sequence homology. Thus, an advantage of using the siNA of the invention is that a single siNA can be designed to include a nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between homologous genes. In this approach, a single cNA can be used to inhibit the expression of more than one gene rather than using more than one cNA molecule to target different genes.

The siNA molecules of the invention are double-stranded. A cNA molecule of the invention may comprise blunt ends, ie ends that do not include any protruding nucleotides. In one embodiment, a siNA molecule of the invention may comprise one or more blunt ends. In preferred embodiments, the ANic molecules have 3 'overhangs. The siNA molecules of the invention may comprise 3 'overhang double-stranded nucleic acid molecules with n nucleotides (5> n> 1). Elbashir (2001) demonstrates that 21 nucleotide piRNA double strands are most active when they contain 3'terminal protruding dinucleotides.

Candidate oligonucleotides are further filtered for interspecies sequence conservation to facilitate the transition from animal studies to human clinical studies. In preferred embodiments of the invention, conserved oligonucleotides are used; this allows a single oligonucleotide sequence to be used both in animal models and in human clinical trials.

In Figure 1, the GenBank accession numbers corresponding to the selected human target genes are shown. In some of these genes, alternative splicing produces a family of transcripts that differ in exon content. The present invention allows individual targeting of each form of transcription.

Selected oligonucleotide sequences against which RNAi is targeted are shown in Figure 2. The displayed sequences are DNA sequences targeted by ANic. Therefore, the invention would make use of double-stranded ANs with sequences complementary to the indicated DNA sequences.

The sequences shown in Figure 2 are not exhaustive. In fact, the target DNA need not necessarily be preceded by AA or CA. Also, the target DNA could be made up of sequences included in Figure 2 flanked by any continuous sequence.

Animal and in vitro studies

Obtaining double-stranded siRNA

Preferably, the RNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. Substitution of one or both strands of a siRNA double stranded by 2'-deoxy or 2'-O-methyl oligoribonucleotides eliminated silencing in fly extract (Elbashir et al. 2001). In mammalian cells, however, it seems possible to replace the sense siRNA with a 2'-O-methyl oligoribonucleotide (Ge et al. 2003).

It is especially convenient to obtain the siRNAs from commercial RNA oligo synthesis providers, who sell RNA synthesis products of different qualities and prices. In general, 21 nt RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi.

Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow, United Kingdom), Qiagen (Germany), Ambion (United States) and Invitrogen (Scotland). The above commissioned RNA synthesis companies are entitled to provide siRNA with a license for target validation. In particular, the inventors' suppliers of siRNA are Ambion, Dharmacon and Invitrogen, companies that offer a traditional custom chemical synthesis service for siRNA and supply the siRNA with HPLC purification and supplied in dry form along with free water. RNase. A central website-based resource for RNAi and RNApi methodologies, along with links for additional RNAi-related products and services can be found on the websites of the listed providers.

When working with single-stranded RNA molecules, a hybridization step is necessary. It is crucial that all handling steps are carried out under sterile conditions, without RNase. To hybridize the RNAs, the oligos must first be counted by UV absorption at 260 nanometers (nm). The following protocol is then applied based on Elbashir et al. (2001) for hybridization:

ES 2 632 913 T3 • Aliquots are separated and each RNA oligo diluted to a concentration of 50 µΜ.

• 30 µl of each RNA oligo solution and 15 µl of 5X Hybridization Buffer are combined. The final buffer concentration is: 100 mM potassium acetate; 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate. The final volume is 75 μ !.

• The solution is incubated for 1 minute at 90 ° C, the tube is centrifuged for 15 seconds, 1 is allowed to stand for 1 hour at 37 ° C, then a room temperature is used. The solution can be stored frozen at -20 ° C and freeze-thaw up to 5 times. The final concentration of double stranded siRNA is typically 20 µΜ.

Alternatively, it can be purchased from already hybridized dsRNA suppliers.

Chemically modified nucleic acids can also be used. For example, in WO03 / 070744, a description of the types of modification that can be used is given. Special attention should be paid to pages 11 to 21 of this publication. Other possible modifications are those that have been described. Those skilled in the art will be aware of other types of chemical modification that can be incorporated into RNA molecules.

In vitro system

To check the specificity of the siRNA interference, different cell cultures expressing the target genes were used. The cells used for these experiments were: rabbit unpigmented ciliary epithelium NPE cells, human ciliary epithelium OMDC cells and human embryonic kidney HEK293 cells. Cells were incubated with the corresponding siRNA double strands and analysis of the downregulation of target gene expression was carried out. For the silenced siRNA binding to specific phenotypes in cultured cells, it is necessary to demonstrate the reduction of the targeted protein or at least demonstrate the reduction of the targeted mRNA.

The mRNA levels of the target gene can be quantified by real-time PCR (RT-PCR). Also, protein levels can be determined in various ways known in the art, such as Western blot analysis with antibodies specific for different targets that allow direct monitoring of targeted protein reduction.

DsRNA double-stranded transfection

The following are several examples of techniques well known in the art: a single transfection of siRNA double strands can be performed using a cationic lipid, such as RNAiFect transfection reagent (Qiagen) and Lipofectamine 2000 reagent (Invitrogen) and the silencing 24 determined, 28 and 72 hours after transfection.

A typical transfection protocol can be applied as follows: For one well of a 6-well plate, transfection is carried out using 100 nM final concentration of siRNA. Following the RNAiFect protocol, they are seeded the day before transfection, 2-4 x 10⁵ cells per well in 3 ml of suitable growth medium containing DMEM, 10% serum, antibiotics and glutamine, and the cells are incubated under normal growth conditions (37 ° C and 5% CO2). On the day of transfection, cells should be 30-50% confluent. 15 ul of 20uM dsRNApi (corresponding to 100 nM final concentration) is diluted in 85 ul of ECR ​​buffer, to give a final volume of 100 ul, and mixed with vortex. For complex formation, 19 ul of RNAiFect transfection reagent is added to the diluted siRNA and mixed by pipetting or vortexing. After incubation of the samples for 10-15 minutes at room temperature to allow the formation of transfection complexes, the complexes are added dropwise to the cells with 1.9 ml of new low antibiotic growth medium. After vortexing the plates to ensure uniform distribution of the transfection complexes, the cells are incubated under their normal growth conditions. The next day, the complexes are removed and new, complete growth medium is added. To monitor gene silencing, cells are harvested 24, 48, and 72 hours after transfection. The Lipofectamine 2000 reagent protocol is quite similar. The day before transfection, sow 2-4 x 10⁵ cells per well in 3 ml of an appropriate growth medium containing DMEM, 10% serum, antibiotics and glutamine and the cells are incubated under normal growth conditions (37 ° C and 5% CO2). On the day of transfection, cells should be 30-50% confluent. 12.5 ul of 20uM dsRNApi (corresponding to 100 nM final concentration) are diluted in 250 ul of DMEM to give a final volume of 262.5 ul and mixed. Likewise, 6ul of Lipofectamine 2000 are diluted in 250ul of DMEM and mixed. After a 5 minute incubation at room temperature, the diluted oligomer and diluted Lipofectamine are combined to allow complex formation for a 20 minute incubation at room temperature. The complexes are then added dropwise to the cells with 2 ml of fresh low-antibiotic culture medium and mixed gently by rocking the plate back and forth to ensure even distribution of the transfection complexes. Cells are incubated under their normal growth conditions and the next day, complexes are removed and fresh, complete growth medium is added. To monitor gene silencing, cells were harvested 24, 48, and 72 hours after transfection.

ES 2 632 913 T3

The efficiency of transfection may depend on the cell type, but also on the passage number and the confluence of the cells. The timing and manner in which the ppi-liposome complexes are formed (eg inversion vs. vortex) are also crucial. Low transfection efficiencies are the most common cause of failed silencing. Good transfection is not a trivial matter and requires careful examination with each new cell used. Transfection efficiency can be determined by transfection of reporter genes, for example, a CMV-activated EGFP expression plasmid (eg from Clontech) or a BGal expression plasmid, and then evaluated by phase contrast microscopy and / or fluorescence the next day. Double-stranded siRNA Assay

Depending on the abundance and lifetime (or turnover) of the targeted protein, a silenced phenotype may be apparent after 1 to 3 days or even later. In cases where no phenotype is seen, protein depletion can be observed by immunofluorescence or Western blotting.

After the transfections, the total RNA fractions extracted from the cells were pre-treated with DNase I and used for reverse transcription using a random primer. PCR is amplified with a specific primer pair covering at least one exon-exon junction to control pre-mRNA amplification. RT / PCR of an undirected mRNA is also required as a control. Efficient mRNA suppression despite the yet undetectable reduction of the target protein may indicate that there may be a large pool of stable protein in the cell. Alternatively, real-time PCR amplification can be used to more accurately examine the decrease or disappearance of mRNA. Real-time reverse transcriptase (RT) PCR quantifies the initial amount of matrix in a very specific, sensitive and reproducible way. Real-time PCR tracks the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescent emission in each cycle, it is possible to track the PCR reaction during the exponential phase, in which the first significant increase in the amount of PCR product is correlated with the initial amount of the target matrix. .

To verify the interference pattern of the differently expressed genes identified in the cell cultures, qRT-PCR was carried out according to the manufacturer's protocol. For quantitative RT-PCR (qRTPCR), approximately 250 ng of total RNA was used for reverse transcription followed by PCR amplification with primers specific for each gene in the reaction mix containing Master SYBR Green I. Basic PCR conditions comprised an initial step of 30 minutes at 91 ° C, followed by 40 cycles of 5 s at 95 ° C, 10 s at 62 ° C, and 15 s at 72 ° C. Specific primer sequences corresponding to each target gene were used. Quantification of b-actin mRNA was used as a control for data normalization. Relative gene expression comparisons work best when the gene expression of the chosen endogenous / internal control is more abundant and remains constant, in proportion to total RNA, between samples. By using an invariant endogenous control as an active reference, the quantitation of an mRNA target can be normalized for differences in the amount of total RNA added to each reaction.

Pharmaceutical formulations

The present disclosure may comprise the administration of one or more species of siNA molecules simultaneously. Such species can be selected to target one or more target genes.

The siNA molecules of the invention and formulations or compositions thereof are administered topically (eg, locally) to the eye, as is generally known in the art. For example, a cNA molecule can comprise a delivery vehicle, including liposomes, for administration to a patient. Carriers and diluents and their salts may be present in pharmaceutically acceptable formulations. Nucleic acid molecules can be delivered to cells by various methods known to those skilled in the art, including, but not limited to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, poly (lactic-glycolic acid) cyclodextrins (PLGA) and PLCA microspheres, biodegradable nanocapsules and bioadhesive microspheres or through proteinaceous vectors. In another embodiment, the nucleic acid molecules of the disclosure can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or derivatives of polyethyleneimine-polyethylene glycol-tri -Nacetylgalactosamine (PEI-PEG-triGAL).

A cNA molecule of the disclosure may complex with membrane disrupting agents and / or a cationic lipid or an auxiliary lipid molecule.

Delivery systems that can be used with the disclosure include for example aqueous and non-aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and may contain excipients such as solubilizers, permeation enhancers (eg fatty acids, fatty acid esters, fatty alcohols and amino acids) and hydrophilic polymers (eg. polycarbophil and polyvinyl pyrrolidone). In one embodiment, the pharmaceutical carrier

Acceptable ES 2,632,913 T3 is liposome or a transdermal enhancer.

A pharmaceutical formulation according to the disclosure is presented in a form suitable for administration, eg systemic or local administration, to a cell or subject, including for example a human. Suitable forms will depend in part on use or route of entry, such as oral, transdermal, or injection. Other factors are known in the art and include considerations such as toxicity and ways that prevent the composition or composition from exerting its effect.

The present disclosure also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Carriers or diluents acceptable for therapeutic use are well known in the pharmaceutical art. For example, preservatives, stabilizers, colorants and flavoring agents can be provided. These include sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters. In addition, antioxidants and suspending agents can be used.

The pharmaceutically effective dose is that required to prevent, inhibit the appearance or treat (alleviate a symptom to some degree, preferably all symptoms) of a pathology. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the particular mammal being considered, the concurrent medication and other factors that people will be able to recognize. specialized in medical technology.

Generally, an amount between 0.1 mg / kg and 100 mg / kg of body weight per day of active principle is administered.

The formulations of the disclosure can be administered in unit dose formulations containing conventional non-toxic pharmaceutically acceptable excipients, adjuvants and / or carriers. The formulations may be presented in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs. Compositions intended for oral use can be prepared according to a method known in the art for the manufacture of pharmaceutical compositions and said compositions can contain one or more sweetening agents, flavoring agents, coloring agents or preserving agents to provide elegant and pleasant preparations. to the palate. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.

These excipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as cornstarch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid, or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases, such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide a sustained action over a longer period of time. For example, a retarding material such as glyceryl monostearate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules, in which The active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil.

The aqueous suspensions contain the active principles mixed with excipients suitable for the manufacture of the aqueous suspensions. Said excipients are suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum acacia; the wetting or dispersing agents can be naturally occurring phosphatides, such as lecithin or condensation products of an alkylene oxide with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with aliphatic alcohols of long chain, for example heptadecaethylenexykethanol, o condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene monooleate sorbitan. Aqueous suspensions may also contain one or more preservatives, such as, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil, such as liquid paraffin. Oil suspensions may contain a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide

ES 2 632 913 T3 palatable oral preparations. Said compositions can be preserved by adding an antioxidant, such as ascorbic acid.

Dispersible powders and granules suitable for the preparation of an aqueous suspension by adding water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents or suspending agents include those mentioned above. Also, additional excipients such as sweetening, flavoring and coloring agents may be present.

The pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oily phase can consist of a vegetable oil or a mineral oil, or mixtures of them. Suitable emulsifying agents include natural gums, such as acacia gum or tragacanth gum, natural phosphatides, such as soybeans, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, such as monooleate of sorbitan, and condensation products of said partial esters with ethylene oxide, such as, for example, polyoxyethylene monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations may also contain a demulsifier, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of an injectable aqueous or oleaginous suspension.

Said suspension can be formulated according to the known art using the dispersing or wetting agents and the suitable suspending agents mentioned above.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, such as a 1,3-butanediol solution. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, and isotonic sodium chloride solution. Also, sterile non-volatile oils are conventionally employed as solvents or suspending media. For this purpose, any mild non-volatile oil can be used, including synthetic mono- or diglycerides. Also, fatty acids such as oleic acid have a place in the preparation of injections.

The nucleic acid molecules can also be administered in the form of suppositories, eg, for rectal drug administration. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures, but liquid at rectal temperatures and therefore melts in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

The nucleic acid molecules can be administered parenterally in a sterile environment. The drug, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. Advantageously, adjuvants can be dissolved in the vehicle, such as anesthetics, preservatives and buffering agents.

It should be understood that the specific dose level for any particular patient will depend on a number of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, timing of administration, route of administration, rate of excretion, drug combination, and severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can be added to forage or water to be fed to the animal. It may be desirable to formulate the animal feed or water compositions so that the animal consumes a therapeutically appropriate amount of the composition in combination with its diet. It may also be convenient to present the composition as a premix to be added to forage or water for drinking.

The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to enhance the overall therapeutic effect. The use of multiple compounds to treat an indication can increase beneficial effects while reducing the presence of side effects.

Alternatively, certain icNA molecules can be expressed within eukaryotic promoter cells. Recombinant vectors capable of expressing icNA molecules and persisting in target cells can be delivered. Alternatively, vectors that provide transient expression of nucleic acid molecules can be used. The administration of said vectors can be repeated as many times as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of the vector-expressing icNA molecule can be systemic, such as by intravenous or intramuscular administration, by administration to the target cells explanted from the patient followed by reintroduction into the patient, or by other means that allow introduction into the patient. the desired target cell.

ES 2 632 913 T3

Animal studies

The New Zealand rabbit is the gold standard in experimental platforms designed to study IOP. It is easy to handle and has large eyes, similar in size to the human organ. On the other hand, the current equipment to measure IOP is not suitable for use in animals with small eyes, such as mice or rats. Finally, rabbits have an IOP (around 23 mm Hg) that can be reduced to 40% of its value using local commercial hypotensive medication. Therefore, although it is possible to generate rabbit glaucoma models (for example, surgical episclerotic vein block or artificial occlusion of the trabecular meshwork), normotensive rabbits were used, since, in the hands of the inventors, the pharmacological decrease IOP can be easily and reproducibly measured.

Experimental protocol

Normotensive New Zealand white rabbits (male, 2-3 kg) were used. The animals were kept in individual cages with free access to food and water. They were subjected to 12-hour light / dark cycles artificially to avoid uncontrolled circadian oscillations in IOP. The handling and treatment of the animals was carried out in accordance with the Directive of the Council of the European Communities (86/609 / EEC) and the statement of the Association for Research in Vision and Ophthalmology on the Use of Animals in Research in Ophthalmology and Vision.

Typically, drugs were delivered by instillation of a small volume (typically 40 µl) onto the corneal surface. Contralateral eyes were treated with vehicle only and could be used in each experiment as controls unless a sympathetic phenomenon occurred with the other eye. Multiple experiments on the same animal should be avoided.

IOP measurements were made using a contact tonometer (TONOPEN XL, Mentor, Norwell, Massachusetts). The TonoPen tonometer is very convenient because of its reliability and its small size. Measurements were made with this instrument delicately by applying the tonometer sensor to the corneal surface. This device has been shown to be the ideal tonometer for measuring intraocular pressures within the range of 3 to 30 mm Hg in rabbits (Abrams et al., 1996). All measurements fall within this range: mean baseline intraocular pressure was 17.0 ± 0.39 mm Hg (n = 100). Since IOP changes from night to day, all experiments were performed at the same time to allow for more stable IOP and to allow an objective comparison with vehicle treatment. To avoid suffering to the animals, rabbits were topically anesthetized (oxybuprocaine / tetracaine, 0.4% / 1%, in a saline solution (1/4 v: v). The solution (10 pl) was applied in the cornea before taking each intraocular pressure measurement. SiRNA or saline was applied topically to the cornea in 40 µl volumes.

The standard protocol for the application of siRNA in rabbits was as follows. Doses of siRNA in saline solution (0.9% w / v) were applied to one eye up to a final volume of 40ul, every day for four consecutive days. The opposite eye was taken as a control and 40 µl of sterile saline solution (0.9% w / v) was instilled in it at the same time points. IOP was measured before each application and after 2 h, 4 h and 6 h from the moment of instillation for 10 days. The maximum responses were observed between the second and the third day. To compare the effect of siRNA with other hypotensive compounds, Xalatan (Iatanoprost 0.005%) and Trustop (Dorzolamide 2%) were tested and IOP was measured under the same conditions.

Results

Example 1 In vitro tests

To determine the inhibition of different glaucoma-related targets and using RNAi technology, the first stage consisted of conducting experiments in cell cultures. For each target, several siRNAs were designated using specific software according to the rules described above. Those with the best characteristics were selected for testing. The siRNAs were applied to cell cultures, such as NPE, OMDC, and HEK293. The effect of siRNA on the target gene was analyzed by real-time PCR and semi-quantitative PCR according to normal protocols. The transcriptional levels of the gene target were normalized using actin as the housekeeping gene. Representative results of real-time PCR experiments for some of the target genes described above are shown in Table 1, below. The values ​​represent the mean of the percentage of interference of siRNA on each gene expression once normalized with the control cells and their standard deviations. Compared with control cells, the level of the different transcripts at both the 24 hr and 48 hr time points was significantly reduced after treatment with siRNA. Some of the different siRNAs that were tested and their different efficiencies in targeting gene interference are listed in the Table. The siRNAs used in the Table correspond to the human siRNA targets listed in Figure 2 as follows.

AC2: siRNA1: rabbit sequence homologous to SEQ. ID. 73 human siRNA2: identical rabbit sequence to SEQ. ID. 54 human

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SiRNA3: identical rabbit sequence to SEQ. ID. 66 human PTGS1 ARNpi1: rabbit sequence homologous to SEQ. ID. 353 human siRNA2: rabbit sequence homologous to SEQ. ID. 369 human PTGS2 siRNA1: identical rabbit sequence to SEQ. ID. 426 human siRNA2: rabbit sequence homologous to SEQ. ID. 421 human siRNA3: rabbit sequence homologous to SEQ. ID. 477 human

Table I: Treatment with siRNA reduces the levels of target gene transcripts. RNA was prepared from cells treated with different siRNA for 24 h and 48 h. Samples were analyzed by real-time PCR using specific primers. The values ​​show the average of the expression levels of different transcripts normalized for actin in relation to the cellular control.

<td></td><td></td><td colspan="2">% level of gene transcription</td>

<td>Diana</td><td></td><td>24 h</td><td>48 h</td>

<td>AC2</td><td>ARNpi1</td><td> 76,25 ± 12,60</td><td> 84,57 ± 14,70</td>

<td></td><td>ARNpi2</td><td> 37,97 ± 9,78</td><td> 61,45 ± 9,62</td>

<td></td><td>ARNpi3</td><td> 35,30 ± 9,73</td><td> 51,14 ± 16,49</td>

<td></td><td></td><td></td><td></td>

<td>PTGS1</td><td>ARNpi1</td><td> 42,25 ± 13,76</td><td> 42,68 ± 17,00</td>

<td></td><td>ARNpi2</td><td> 34,98 ± 14,33</td><td> 26,30 ± 10,91</td>

<td></td><td></td><td></td><td></td>

<td>PTGS2</td><td>ARNpi1</td><td> 68,68 ± 12,48</td><td> 70,17 ± 19,21</td>

<td></td><td>ARNpi2</td><td> 81,00 ± 13,54</td><td> 66,85 ± 18,67</td>

<td></td><td>ARNpi3</td><td> 75,45 ± 14,71</td><td> 61,83 ± 16,96</td>

In Figure 3 some representative semi-quantitative genes for some of the targets described above are shown. The decrease in gene expression for each target gene depends on the efficiency of siRNA silencing. For each target, the most efficient siRNA obtained by the in vitro studies was administered to the animal model. RNA was prepared from cells treated with different siRNA. Samples were analyzed by semi-quantitative PCR using specific primers. The figure shows a representative semi-quantitative gel for the expression of Receptor 2 beta adrenergic (A) and another for the expression of acetylcholinesterase (B). M: Marker MW; C: Control cells; TC: Transfection control; 1: siRNA1; 2: siRNA2; 3: siRNA3; NC: Negative control. The expression levels for each target depend on the efficacy of siRNA silencing. The siRNAs used in the figure correspond to the human targets given in Figure 2 as follows:

Panel A (Receptor 2 beta adrenergic)

1: rabbit sequence homologous to SEQ. ID. 122 human 2: identical rabbit sequence to SEQ. ID. 125 human 3: rabbit sequence homologous to SEQ. ID. 139 human

Panel B (Acetylcholinesterase)

1: rabbit sequence homologous to SEQ. ID. 162 human 2: rabbit sequence homologous to SEQ. ID. 177 human

Example 2 In vivo tests

Prior to the therapeutic application of siRNA, in vivo assays were validated to determine appropriate siRNA delivery.

The selected siRNAs were applied through in vitro tests in the animal model, following the protocol described above. To avoid the effect of IOP fluctuations as a consequence of circadian cycles, all applications were performed at the same time. To determine the effect of siRNA, intraocular pressures (IOP) were measured as mentioned above.

Given that glaucoma pathology presents an increase in intraocular pressure, the objective was to obtain a decrease in levels after the application of siRNA.

Most of the results for the different targets showed a significant decrease in IOP levels in comparison with the controls and with the commercial drugs (Latanoprost and Dorzolamide) and the animals treated with vehicle only (negative control) did not present any significant change with with respect to the baseline IOP value. The data are summarized in Table II in which the values ​​represent the mean of the percentage

ES 2 632 913 T3 maximum of IOP reduction after treatment with siRNA once normalized and its standard deviations. The decrease in IOP was statistically significant for all treated targets. These results indicate that siRNA and commercial drugs act in a similar way, reducing IOP levels by around 20%, although siRNA has a more sustained effect. No side effects were observed in the animals throughout the experimental protocols. The siRNA used in these experiments corresponded to the human targets given in Figure 2 as follows:

AC2: rabbit sequence homologous to SEQ. ID. 73 human

AC4: identical rabbit sequence to SEQ. ID. 5 human

AC12: identical rabbit sequence to SEQ. ID. 522 human

ADRB1: identical rabbit sequence to SEQ. ID. 105 human

ADRB2: rabbit sequence homologous to SEQ. ID. 139 human

ADRA1A: rabbit sequence homologous to SEQ. ID. 546 human

ADRA1B: rabbit sequence homologous to SEQ. ID. 619 human

ACHE: rabbit sequence homologous to SEQ. ID. 189 human

PTGS1: rabbit sequence homologous to SEQ. ID. 322 human

PTGS2: identical rabbit sequence to SEQ. ID. 426 human

SELE: rabbit sequence homologous to SEQ. ID. 262 human

ACE1: rabbit sequence homologous to SEQ. ID. 866 human

AGTR1: rabbit sequence homologous to SEQ. ID. 705 human

AGTR2: identical rabbit sequence to SEQ. ID. 774 human

ATP1A1: identical rabbit sequence to SEQ. ID. 1399 human

ATP1B2: identical rabbit sequence to SEQ. ID. 1820 human

Table II: Effect of siRNA on IOP reduction in normotensive New Zealand rabbits. The values ​​represent the mean of the IOP reduction percentage over the control (contralateral eye only with vehicle) and its standard deviation (SEM).

<td>Diana</td><td>IOP reduction (% of control)</td>

<td>AC2</td><td> 24,84 ± 3,41</td>

<td>AC4</td><td> 14,47 ± 5,00</td>

<td>AC12</td><td> 24,30 ± 1,29</td>

<td>ADRB1</td><td> 28,04 ± 2,98</td>

<td>ADRB2</td><td> 21,18 ± 1,88</td>

<td>ADRA1A</td><td> 9,51 ± 1,04</td>

<td>ADRA1B</td><td> 17,48 ± 1,30</td>

<td>ACHE</td><td> 25,25 ± 2,70</td>

<td>PTGS1</td><td> 14,62 ± 1,93</td>

<td>PTGS2</td><td> 23,78 ± 2,27</td>

<td>SELE</td><td> 21,80 ± 1,74</td>

<td>ACE1</td><td> 17,51 ± 1,28</td>

<td>AGTR1</td><td> 9,72 ± 1,35</td>

<td>AGTR2</td><td> 11,22 ± 1,53</td>

<td>ATP1A1</td><td> 18,13 ± 1,39</td>

<td>ATP1B2</td><td> 16,32 ± 0,91</td>

<td>Latanoprost</td><td> 25,46 ± 5,24</td>

<td>Dorzolamide</td><td> 16,41 ± 2,38</td>

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