RNA interference (RNAi) is a naturally occurring post-transcriptional regulatory mechanism present in most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans {Fire, 1998} was awarded the Nobel Prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells, not through long dsRNAs but by means of double-stranded small interfering RNAs (siRNAs) 21 nucleotides long {Elbashir, 2001}.
The process of RNA interference is thought to be an evolutionarily-conserved cellular defence mechanism used to prevent the expression of foreign genes and is commonly shared by diverse phyla and flora, where it is called post-transcriptional gene silencing. Since the discovery of the RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise “undruggable” with traditional pharmaceutical approaches involving small molecules or proteins.
According to current knowledge, the mechanism of RNAi is initiated when long double stranded RNAs are processed by an RNase III-like protein known as Dicer. The protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) {Collins, 2005} and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3′ overhangs and a 5′ phosphate and 3′ hydroxyl group. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences {Elbashir, 2001} upon adenosine-triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity {Nykanen, 2001}. The catalytic activity of RISC, which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AGO2) {Liu, 2004; Song, 2004}. AGO2 belongs to the highly conserved Argonaute family of proteins. Argonaute proteins are ˜100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains {Cerutti, 2000}. The PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs {Song, 2004}. AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5′ end {Elbashir, 2001}. An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex {Rand, 2005}. Crystallography studies analyzing the interaction between the siRNA guide strand and the PIWI domain reveal that it is only nucleotides 2 to 8 that constitute a “seed sequence” that directs target mRNA recognition by RISC, and that a mismatch of a single nucleotide in this sequence may drastically affect silencing capability of the molecule {Doench 2004; Lewis, 2003}. Once the mRNA has been cleaved, due to the presence of unprotected RNA ends in the fragments the mRNA is further cleaved and degraded by intracellular nucleases and will no longer be translated into proteins {Orban, 2005} while RISC will be recycled for subsequent rounds {Hutvagner, 2002}. This constitutes a catalytic process leading to the selective reduction of specific mRNA molecules and the corresponding proteins. It is possible to exploit this native mechanism for gene silencing with the purpose of regulating any gene(s) of choice by directly delivering siRNA effectors into the cells or tissues, where they will activate RISC and produce a potent and specific silencing of the targeted mRNA. RNAi has been applied in biomedical research such as treatment for HIV, viral hepatitis, cardiovascular and cerebrovascular diseases, metabolic disease, neurodegenerative disorders and cancer {Angaji S A et al 2010}.
Many studies have been published describing the ideal features a siRNA should have to achieve maximum effectiveness, regarding length, structure, chemical composition, and sequence. Initial parameters for siRNA design were set out by Tuschl and co-workers in WO02/44321, although many subsequent studies, algorithms and/or improvements have been published since then. siRNA selection approaches have become more sophisticated as mechanistic details have emerged, in addition further analysis of existing and new data can provide additional insights into further refinement of these approaches {Walton S P et al 2010}. Alternatively, several recent studies reported the design and analysis of novel RNAi-triggering structures distinct from the classical 19+2 siRNA structure and which do not conform to the key features of classical siRNA in terms of overhang, length, or symmetry, discussing the flexibility of the RNAi machinery in mammalian cells {Chang C I et al 2011}.
Also, a lot of effort has been put into enhancing siRNA stability as this is perceived as one of the main obstacles for therapy based on siRNA, given the ubiquitous nature of RNAses in biological fluids. Another inherent problem of siRNA molecules is their immunogenicity, whereby siRNAs have been found to induce unspecific activation of the innate immune system. The knockdown of unintended genes (mRNAs) is a well-known side effect of siRNA-mediated gene silencing. It is caused as a result of partial complementarity between the siRNA and mRNAs other than the intended target and causes off-target effects (OTEs) from genes having sequence complementarity to either siRNA strand. One of the main strategies followed for stability enhancement and OTE reduction has been the use of modified nucleotides such as 2′-O-methyl nucleotides, 2′-amino nucleotides, or nucleotides containing 2′-0 or 4′-C methylene bridges. Also, the modification of the ribonucleotide backbone connecting adjacent nucleotides has been described, mainly by the introduction of phosphorothioate modified nucleotides. It seems that enhanced stability and/or reduction of immunogenicity are often inversely proportional to efficacy {Parrish, 2000}, and only a certain number, positions and/or combinations of modified nucleotides may result in a stable and/or non-immunogenic silencing compound. As this is an important hurdle for siRNA-based treatments, different studies have been published which describe certain modification patterns showing good results, examples of such include EP1527176, WO2008/050329, WO2008/104978 or WO2009/044392, although many more may be found in the literature {Sanghvi Y S. 2011; Deleavey et al 2012}.
Allergic diseases are characterized by an overreaction of the human immune system to a foreign protein substance (“allergen”) that is eaten, breathed into the lungs, injected or touched. Allergies have a genetic component. If only one parent has allergies of any type, chances are 1 in 3 that each child will have an allergy. If both parents have allergies, it is much more likely (7 in 10) that their children will have allergies. There are no cures for allergies; however they can be managed with proper prevention and treatment.
About 30% of people worldwide suffer from allergic symptoms and 40-80% of them have symptoms in the eyes {Key B. 2001}. Allergic diseases affecting the eyes or ocular allergies constitute a heterogenic group of diseases with a very broad spectrum of clinical manifestations. An ocular allergy usually occurs when the conjunctiva (membrane covering the eye and the lining of the eyelid) reacts to an allergen. An ocular allergy can happen independently or in conjunction with other allergy symptoms (such as rhinitis or asthma).
Basic and clinical research has provided a better understanding of the cells, mediators, and immunologic events which occur in ocular allergy. The eye, particularly the conjunctiva, has a relatively large number of mast cells. When allergens are present they can bind to immunoglobulin, IgE, in the FcϵRI receptors on the surface of these mast cells and trigger their activation and release of mediators of allergy (a process known as degranulation). Degranulation releases mast cell components, including histamine, prostaglandins, tryptase and leukotrienes, into the environment outside the mast cell. Through a variety of mechanisms these components produce the signs and symptoms of the ocular allergy. The activation of the mast cells of the allergic inflammation is frequently designated as an acute phase response or early phase of the ocular allergy. The acute phase response can progress to a late phase response characterized by recruitment of inflammatory cells to the site of the allergic inflammation, for example as an influx of eosinophils and neutrophils into the conjunctiva.
Ocular allergy represents one of the most common conditions encountered by allergist and ophthalmologists. Ocular allergy is usually associated with the following symptoms and signs: conjunctivitis, blepharitis, blepharoconjuntivitis or keratoconjunctivitis. The eye becomes red and itchy and there occurs lacrimation and slight discharge. Severe cases may also show eye burning sensation, pain and photophobia.
Allergic diseases affecting the eyes include mild forms such as seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC); and more severe manifestations such as vernal keratoconjunctivitis (VKC); atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC). The latter ones can be associated with complications such as corneal damage and may cause vision loss. SAC and PAC are commonly IgE-mast cell mediated hypersensitivity reaction to external allergens; whereas AKC and VKC are characterized by chronic inflammation involving several immune cell types. SAC and PAC allergens, with the help of antigen presenting cells (APCs), trigger a Th2-predominant immune response that induces B cells to release IgE. Activation of the allergic response usually involves infiltration and degranulation of mast cells.
SAC is the most common allergic disease in the eye, usually caused by allergens like airborne pollen, dust, and animal dander. The signs and symptoms usually occur during the spring and summer, and generally abate during the winter months. Itching, redness and swelling of the conjunctiva are the most characteristic symptoms, but also tearing, burning sensation, and photophobia. In most cases, SAC is not serious. However, it may be very disturbing to patients because it can affect their quality of life and can have significant socioeconomic impact {Kari O. and Saari K M 2010}.
PAC is the second most common allergic disease in the eye, usually caused by animals and mites. The symptoms and signs are much the same as in SAC, the difference is the specific allergens to which the patient is allergic and that PAC can occur throughout the year with exposure to perennial allergens. PAC affects all age groups but mostly young and middle-aged people of both sexes. In addition, PAC is often connected to dry eye syndrome.
SAC and PAC are the most common forms of ocular allergies. Estimates vary, but these types of allergy are said to affect at least 15-20% of the general population. SAC and PAC are often underdiagnosed and consequently undertreated. In SAC and PAC allergen induced local release of IgE prompts degranulation of mast cells in Ca2+ dependent mechanism. IgE-activated mast cells liberate preformed inflammatory mediators such as histamine and leukotriene 4 that are the first mediators of the allergic response. These mediators attract eosinophils that infiltrate the region amplifying the allergic response.
VKC is a relatively rare chronic allergic inflammation of the ocular surface that mainly affects children and young adolescents. Main symptoms are itching, redness, swelling, discharge and photophobia. The most characteristic sign is giant papillae in the upper tarsal conjunctiva.
AKC is a bilateral chronic inflammatory disease of the ocular surface and eyelid. The most characteristic sign are eczematous lesions on the eyelid which are itchy. It is not unusual for AKC patients to have cataract surgery at a young age {Kari O. and Saari K M 2010}.
GPC is an inflammatory disease characterized by papillary hypertrophy of the superior tarsal conjunctiva. GPC is caused by inert substances rather than allergens. When these irritative stimuli are removed the conjunctival papillary changes resolve. Protein deposits on the surface of the contact lens could become antigenic and stimulate the production of IgE {La Rosa M. et al 2013}.
Current treatments for ocular allergy include non-pharmacologic and pharmacologic strategies. Avoidance of the antigen is the primary behavioural modification for all types of ocular allergies. Artificial tear substitutes provide a barrier function and help to improve the first-line defence at the level of the conjunctiva mucosa. When non-pharmacologic strategies do not provide adequate symptom relief, pharmacologic treatments may be applied.
The mainstay of the management of ocular allergy involves the use of anti-allergic therapeutic agents such as antihistamine, dual-action or combination treatments and mast cell stabilizers. Topical antihistamines (such as Emedastine and Levocabastine) competitively and reversibly block histamine receptors and relieve itching and redness, but only for a short time. Antihistamines do not affect other proinflammatory mediators which remain inhibited. A limited duration of action necessitates frequent dosing and topical antihistamines may be irritating to the eye, especially with prolonged use.
Combination treatments using decongestants (such as oxymetazoline, tetrahydrozoline, and naphazonline) in combination with antihistamines act as vasoconstrictors but are known to sting or burn on instillation. Other adverse events include mydriasis and rebound hyperemia, rendering these combination treatments more suitable for short-term relief. In addition, these drugs are not recommended for use in patients with narrow-angle glaucoma. Mast cell stabilizers (such as cromoglycate, lodoxamide, nedocromil) have a mechanism of action that is unclear. They do not relieve existing symptoms and can be used only on a prophylactic basis to prevent mast cell degranulation with subsequent exposure to the allergen. They require a loading period during which they must be applied before the antigen exposure {La Rosa M. et al 2013}.
When the above mentioned anti-allergic drugs do not allow adequate control of the allergic inflammatory process, anti-inflammatory agents are used. Corticosteroids remain among the most potent pharmacologic agents used in the more severe variants of ocular allergy {La Rosa M. et al 2013}. However, steroidal drugs can have side effects that threaten the overall health of the patient. Chronic administration of corticosteroids can lead to drug-induced osteoporosis by suppressing intestinal calcium absorption and inhibiting bone formation. Other adverse side effects of chronic administration of corticosteroids include hypertension, hyperglycemia, hyperlipidemia (increased levels of triglycerides) and hypercholesterolemia (increased levels of cholesterol) because of the effects of these drugs on the body metabolic processes. It is also known that certain corticosteroids have a greater potential for elevating intraocular pressure (“IOP”) than other compounds in this class. For example, it is known that prednisolone, which is a very potent ocular anti-inflammatory agent, has a greater tendency to elevate IOP than fluorometholone, which has moderate ocular anti-inflammatory activity. It is also known that the risk of IOP elevations associated with the topical ophthalmic use of corticosteroids increases over time. In other words, the chronic (i.e., long-term) use of these agents increases the risk of significant IOP elevations. Therefore, corticosteroids may not be appropriate for the long-term treatment of ocular allergies. In addition, chronic use of corticosteroids is contraindicated due to an increased risk for the development of cataracts and glaucoma {Ono S J, and Abelson M B, 2005}.
Allergy immunotherapy is useful in reducing the response to allergens, but its role in allergic conjunctivitis has not been proven. The main objective of this treatment is to induce clinical tolerance to the specific allergen. The therapy is administered subcutaneously in progressively increasing doses to remain below the threshold of a clinical reaction. Sublingual immunotherapy (SLIT) is considered an alternative to subcutaneous allergy immunotherapy and is administered orally under the tongue, but long-term results with SLIT are not yet available. Most of the trials with this form of therapy have been for allergic rhinitis. In general, immune responses to allergen administration are not predictive of the effectiveness of the therapy and the therapy itself can produce systemic reactions, the incidence and severity of which vary dependent of the type of allergen administered {La Rosa M. et al 2013}.
In addition, the majority of newer ophthalmic anti-allergic agents have limited durations of action and twice daily dosing is required. A topical preparation with a longer duration of action would be advantageous because it may be instilled once daily. Thus, new therapies that can offer advantages in areas such as efficacy and duration of action, while offering similar safety profiles than traditional ophthalmic anti-allergic agents, are needed.
RNA interference-based therapies have been pointed out as having the potential to satisfy unmet needs in allergy treatment {Popescu F D. 2005}. It has been demonstrated that systemic administration of CD40 siRNA in mice sensitized with an allergen is capable of attenuating nasal allergic symptoms through inhibition of dendritic cell and B cell functions and generation of regulatory T cells {Suzuki M. et al 2009}. In addition, siRNA-based allergen-specific therapy for allergic rhinitis has also been developed by using CD40-silenced and allergen-pulsed dendritic cells {Suzuki M et al 2010}.
Initiation of the allergy reaction starts with the binding of the allergen to the IgE molecules in the FccRI receptors of mast cells. The activation of FccRI triggers a change in the mast cell allowing entrance of calcium form the extracellular compartment. Entrance of Ca2+ into mast cells activates phosphatidylinositol-3-kinase (PI3K). Activation of the PI3K pathway includes activation of phosphoinositide-dependent kinase (PDK1) which in turn phosphorylates downstream targets of PI3K such as PKB/Akt, SGK and PKC. These kinases are responsible for the activation of calcium channels to mobilize intracellular calcium stores and activate mast cell degranulation {Shumilina E, et al 2010}.
PDK1 is also known as PDPK1 or PDHK1. The structure of PDK1 can be divided into two domains; the kinase or catalytic domain and the Pleckstrin homology domain (PH domain). PH domain is a protein domain of approximately 120 amino acids that occurs in a wide range of proteins involved in intracellular signalling or as constituents of the cytoskeleton. The kinase domain has three ligand binding sites; the substrate binding site, the ATP binding site, and the docking site (also known as PIF pocket). PDK1 is constitutively active and it is found at the cytoplasm and the mitochondrial matrix.
EP1513947 (MEDICAL RES COUNCIL) describes a method for selecting or designing a compound for modulating the activity of PDK1.
US2009/0275570 (ASTRAZENECA AB) describes compounds possessing PDK1 inhibitory activity for the treatment of cancer.