CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Prov. Appl. 63/337,311 filed May 2, 2022, the contents of which are incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to mammalian models of eye conditions, and in particular to the use of non-human mammals which have been treated by injection of botulinum toxin into one more glands associated with tear production in the eye as models for the screening of test compounds.

BACKGROUND OF THE INVENTION

Dry eye syndrome is caused by a chronic lack of sufficient lubrication and moisture on the surface of the eye. Consequences of dry eyes range from subtle but constant eye irritation to significant inflammation and even vascularization, pigmentation and scarring of the front surface of the eye.

In addition to being called dry eye syndrome, dry eye disease, or simply "dry eye," a wide array of alternative medical terms have been used to describe dry eye. Keratitis sicca refers to dryness and inflammation of the cornea. Keratoconjunctivitis sicca refers to dry eye that affects both the cornea and the conjunctiva. Evaporative dry eye refers to incitement of ocular surface changes associated with instability of the thin tear film and rapid evaporative loss of the aqueous layer.

Dry eye syndrome is one of the most common eye conditions worldwide and a primary reason for visits to the eye doctor. In a review published in the lournal of Global Health, researchers reported that studies have shown the prevalence of dry eyes ranges from 5 percent to as high as 50 percent in different populations across the world. Risk factors for dry eye syndrome included advanced age, female sex, and computer use. Symptoms of dry eye syndrome include but are not limited to: burning sensations; itchy eyes; aching sensations; increased blinking, foreign body sensations, heavy eyes; fatigued eyes; sore eyes; dryness sensation; red eyes; photophobia; and blurred vision. An adequate and consistent layer of tears on the surface of the eye is essential to keep the eyes healthy, comfortable and seeing well. Tears bathe the eye's surface to keep it moist and wash away dust, debris and microorganisms that could damage the cornea and lead to an eye infection. A normal tear film consists of three important components: an oily (lipid) component; a watery (aqueous) component; and a mucous-like (mucin) component. Each component of the tear film serves a critical purpose. For example, tear lipids help keep the tear film from evaporating too quickly and increase lubrication, while mucin helps anchor and spread the tears across the surface of the eye.

Each tear component is produced by different glands on or near the eye. The oily component is produced by meibomian glands in the eyelids. It is noted however, that an oily component can be provided by accessory lacrimal glands such as Harderian glands in certain mammalian species such as the rabbit and in a wide array of non-mammalian vertebrate species. The watery component is produced by lacrimal glands located in a variety of locations in different species. It is located under the conjunctiva in the superolateral aspect of the globe in the dog. Accessory lacrimal glands, such as the accessory lacrimal gland of the canine third eyelid can also contribute to the aqueous element of the tear film. The Harderian gland. In species that possess one, can also contribute an aqueous component to the precorneal tear film The mucin component is produced by goblet cells in the conjunctiva that covers the white of the eye (sclera). A problem with any of these sources of tear film components can result in tear instability and dry eyes.

What is needed in the art are animal models of dry eye disease so that compounds and treatments for dry eye disease can be evaluated.

SUMMARY OF THE INVENTION

The present invention relates to mammalian models of eye conditions, and in particular to the use of non-human mammals which have been treated by injection of botulinum toxin into one more glands associated with tear production in the eye as models for the screening of test compounds and test compositions.

Accordingly, in some embodiments, the present invention provides methods of screening compounds or compositions for activity in relieving a condition of the eye comprising: injecting botulinum toxin into one or more glands associated with tear production in at least one eye of a non-human mammal so that tear production is reduced; contacting the at least one eye in which tear production is reduced with at least one test compound; and evaluating the effect of the at least one test compound or composition on the at least one eye in which tear production is reduced as compared to a control eye.

In some preferred embodiments, the condition is selected from the group consisting of dry eye disease mediated by toxic, traumatic and immune-related causes, evaporative dry eye, dry eye due to exposure keratopathy, dry eye due to facial nerve palsy, and dry eye due to neurogenic/neuroparalytic causes. In some preferred embodiments, the dry eye disease is associated with a condition selected from the group consisting keratitis sicca, keratoconjunctivitis sicca and combinations thereof. In some preferred embodiments, the condition is inflammation of the eye.

In some preferred embodiments, the test compound is administered by a route selected from the group consisting of a topical route, a subconjunctival route, a sub-Tenon’s capsule route, an iontophoresis route, an oral route, an intravenous route, a transdermal route and a transmucosal route. In some preferred embodiments, the test compound is administered in an ophthalmologically acceptable carrier.

In some preferred embodiments, the botulinum toxin is selected from the group consisting of botulinum toxin A, botulinum toxin B, and combinations thereof.

In some preferred embodiments, evaluating the effect of the at least one test compound further comprising evaluating whether the test compound relieves one or more signs of dry eye syndrome in the at least one eye in which tear production is reduced. In some preferred embodiments, the one or more signs of dry eye syndrome are selected from the group consisting of one or more of ocular discharge, rubbing at eyes, increased blink rate, squinting, uptake of fluorescein stain on the ocular surface, uptake of rose Bengal stain on the ocular surface, uptake of Lissamine green stain on the ocular surface, decreased tear film break up time (TFBUT) measured using a water soluble dye such as fluorescein, and decreased tear film break up time measured using non-invasive instruments (NITFBUT).

In some preferred embodiments, the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland, a gland of the third eyelid (nictitans gland), a harderian gland, and an accessory lacrimal gland and combinations thereof. In some preferred embodiments, at least two glands contributing to the formation of the precorneal tear film are injected with botulinum toxin.

In some preferred embodiments, the non-human animal is selected from the group consisting of a dog, a cat, a rabbit, a pig, a non-human primate and a rodent.

In some preferred embodiments, the non-human animal is a dog and the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland and a gland of the third eyelid (nictitans gland), and combinations thereof.

In some preferred embodiments, the non-human animal is a dog and the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland and a gland of the third eyelid (nictitans gland), and combinations thereof.

In some preferred embodiments, the non-human animal is a rabbit and the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland, a gland of the third eyelid (nictitans gland), a harderian gland, and combinations thereof.

In some preferred embodiments, the non-human animal is a pig and the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland, a superficial gland of the third eyelid, a deep gland of the third eyelid, and combinations thereof.

In some preferred embodiments, the non-human animal is a primate and the one or more glands associated with tear production is a lacrimal gland.

In some preferred embodiments, the non-human animal is a rodent and the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland, an accessory lacrimal gland, a harderian gland, and combinations thereof.

In some preferred embodiments, the non-human animal is a cat and the one or more glands associated with tear production are selected from the group consisting of a lacrimal gland, an accessory lacrimal gland, a gland of the third eyelid (nictitans gland), a harderian gland, and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to mammalian models of eye conditions, and in particular to the use of non-human mammals which have been treated by injection of botulinum toxin into one more glands associated with tear production in the eye as models for the screening of test compounds and compositions. The animal models are useful for screening compounds or compositions for activity in relieving disorders of the eye such as dry eye syndrome.

Conflicting results have been observed in previous attempts to create mammalian dry models. In mice, intralacrimal gland injection of botulinum toxin B (BTX-B) produced a significant decrease in aqueous tear production for one month, leading the researchers to conclude that a dry eye state had been established. Suwan-apichon et al., Botulinum Toxin B- induced Mouse Model of Keratoconjuctivitis Sicca (2006), IOVS 47(1): 133-139. However, when botulinum toxin B (BTX-B) was injected into the lacrimal gland of rabbits, tear production was reduced but no corneal pathologic factors associated with dry eye were observed.

The present invention describes novel non-human mammalian models useful for identifying compounds that are effective for relieving dry eye syndrome and associated conditions. A variety of glands located in the eye can be associated with the production of tears and proper function of the tear film. Tear production and function is not limited to the lacrimal gland. Accordingly, in some preferred embodiments, mammalian models of eye disfunction are produced by injection of botulinum toxin into one or more glands that contribute to the tear film of the eye of an animal. In some embodiments, the gland(s) of only one eye of the animal is/are injected so that the other eye may serve as a control, while in other preferred embodiments, the gland(s) of both eyes are injected so that one eye may serve as a negative control. For example, the negative control may comprise administration of the delivery vehicle without the test compound.

In some preferred embodiments, the non-human animal is a dog. In some preferred embodiments, one or both of the following glands of the eye are injected with botulinum toxin: the lacrimal gland, the gland of the third eyelid (nictitans gland). In some preferred embodiments, both the lacrimal gland and gland of the third eyelid are injected. In some preferred embodiments the lipid producing sebaceous glands associated with the lid margins (Meibomian glands) are treated, damaged or removed in such a way as to increase the severity of the model.

In some preferred embodiments, the non-human animal is a rabbit. In some preferred embodiments, one or more of the following glands of the eye are injected with botulinum toxin: the lacrimal gland, the gland of the third eyelid (nictitans gland), and the harderian gland. In some preferred embodiments, the lacrimal gland and harderian gland are injected. In some preferred embodiments, the lacrimal gland and the gland of the third eyelid are injected. In some preferred embodiments, the lacrimal gland, the harderian gland and the gland of the third eyelid are injected. In some embodiments the lipid producing sebaceous glands associated with the lid margins (Meibomian glands) are treated, damaged or removed in such a way as to exacerbate the severity of the model.

In some preferred embodiments, the non-human animal is a pig. In some preferred embodiments, one or more of the following glands of the eye are injected with botulinum toxin: the lacrimal gland, the superficial gland of the third eyelid, and the deep gland of the third eyelid. In some preferred embodiments, the lacrimal gland and superficial gland of the third eyelid are injected. In some preferred embodiments, the lacrimal gland and deep gland of the third eyelid are injected. In some preferred embodiments, the lacrimal gland, superficial gland of the third eyelid, and deep gland of the third eyelid are injected. In some embodiments the lipid producing sebaceous glands associated with the lid margins (Meibomian glands) are treated, damaged or removed in such a way as to exacerbate the severity of the model.

In some preferred embodiments, the non-human animal is a cat. In some preferred embodiments, one or both of the following glands of the eye are injected with botulinum toxin: the lacrimal gland, the gland of the third eyelid (nictitans gland). In some preferred embodiments, both the lacrimal gland and gland of the third eyelid are injected. In some embodiments the lipid producing sebaceous glands associated with the lid margins (Meibomian glands) are treated, damaged or removed in such a way as to exacerbate the severity of the model.

In some preferred embodiments, the non-human animal is a non-human primate. In some preferred embodiments, the lacrimal gland of the non-human primate is injected. In some embodiments the lipid producing sebaceous glands associated with the lid margins (Meibomian glands) are treated, damaged or removed in such a way as to exacerbate the severity of the model.

In some preferred embodiments, the non-human animal is a rodent. In some preferred embodiments, one or more of the following glands of the eye are injected with botulinum toxin: the lacrimal gland, the accessory lacrimal gland, and the harderian gland. In some preferred embodiments, the lacrimal gland and accessory lacrimal gland are injected. In some preferred embodiments, the lacrimal gland and harderian gland are injected. In some preferred embodiments, the lacrimal gland, accessory lacrimal gland, and harderian gland are injected. In some embodiments the lipid producing sebaceous glands associated with the lid margins (Meibomian glands) are treated, damaged or removed in such a way as to exacerbate the severity of the model.

In some preferred embodiments, the gland(s) identified above are injected with botulinum toxin. Botulinum toxin A (BTX-A), botulinum toxin B (BTX-B) or combinations thereof may be utilized.

BTX-A (Botox) is a purified neurotoxin complex produced from fermentation of the gram-positive spore-forming bacteria Clostridium botulinum type A. When injected into striated muscle, it produces a dose-dependent local muscle weakness by preventing the release of acetylcholine from nerve terminal at the neuromuscular junction (chemical de-innervation). The action involves a four-step process that culminates with the cleavage of the 25-kDa synaptosome-associated protein (SNAP), which is essential for the exocytosis of acetylcholine. The paralytic effect is temporary, because there is a gradual recovery of the activity of the nerve terminal over a 3 - to 6-month time-frame. The unit of measurement for BTX-A is derived from research with mice. One unit of BTX-A is the lethal dose in 50% of mice (LD50). The LD50 is 2500 to 3000 units in humans (40 units/kg).

BTX-B (Myobloc/NeuroBloc) has a similar clinical gross action as type A, but it needs to be injected in patients at an approximately 40-fold higher dose than type A to produce similar myo-relaxing effects. Such a difference in potency correlates well with the observation that type B human botulism is less severe than type A. The difference in potency of types A and B in humans is due to a point mutation in the amino-acid sequence of synaptotagmin II (BoNT/B receptor). This causes a marked reduction in the affinity of BoNT/B for synaptotagmin, as compared to its binding to rodent synaptotagmin.

In some preferred embodiments, from 0.1 to 10 units of BTX-A and/or BTX-B is introduced into the relevant gland. Amounts suitable for reduction in tear production or other activity of the relevant gland may be readily ascertained by one of skill in the art. Preferably, the injections are performed with small gauge needle, for example a 30 gauge needle. In some preferred embodiments, the animal is anesthetized prior to injection. Suitable methods for anesthetization are known in the art. Following injection, the animals are preferably monitored for cessation of tear production in the injected eye. Suitable methods for monitoring include visual inspection as well as measurement by wetting assays, for example with phenol red-impregnated cotton threads (Zone- Quick; Oasis, Glendora CA) or filter paper (often referred to as Schirmer tear test strips or tear test strips, or if of differing dimensions than is standardly employed; modified Schirmer tear test strips or modified tear test strips. See, e.g., Fernandes da Conceigao, et al., Pesq. Vet. Bras. 3 l(4):350-354 (2011), incorporated herein by reference in its entirety, for a description of a modified Schirmer test which may be employed. For example, tear production may be measured by a Schirmer test with topical anesthesia. In some embodiments, topical proparacaine ophthalmic solution 0.5% (Akom Inc., Buffalo Grove, IL) is applied to each eye. A 5 x 25-mm strip of filter paper is inserted in the lower conjunctival cul-de-sac with the end of the filter paper hanging over the edge of the lower eyelid. The degree of wetting at 5 minutes is measured in humans and typically for shorter periods in other species (60 seconds is standard in the dog and most other mammalian species). It will be noted that with even shorter periods it is possible to extrapolate to the value of a 60 second test.

In some embodiments, corneal staining may be utilized to assess for ocular surface pathological factors. For example, after topical application of 1% sodium fluorescein (Sigma- Aldrich, St. Louis, MO), the eyes are examined under cobalt blue light and photographed using a digital camera with a macro lens. Rose Bengal strips (Akom Inc.) are then applied to the inferior fornix, and the level of Rose Bengal staining was documented by an additional photograph.

The animal models in which one or more glands associated with tear production have been injected with botulinum toxin so that tear production is reduced are useful for screening and evaluating compositions for treatment of conditions associated with decreased tear production. The conditions may be due to any cause and include, but are not limited to, dry eye disease caused by toxic causes, traumatic causes and immune causes such as Sjogren’s syndrome, evaporative dry eye, such as that associated with exposure keratopathy, dry eye associated with facial nerve palsy, and dry eye associated with neurogenic/neuroparalytic causes It will further be understood that because dry eye diseases have an inflammatory component that the models described herein find use for testing compounds to treat or relieve ocular surface inflammation. In some preferred embodiments, the conditions include dry eye syndrome and associated conditions such as keratitis sicca and keratoconjunctivitis sicca. The test compounds or compositions may be delivered to the animal model via any suitable route of delivery. Suitable routes include, but are not limited to, topical administration, topically, subconjunctival administration, administration beneath the Tenon’s capsule, and administration via iontophoresis as well as oral, intravenous, transdermal and transmucosal administration.

In some preferred embodiments, the test compound or composition is applied to an injected eye. In some preferred embodiments, the test compound or composition is formulated in an ophthalmologically acceptable carrier. Components which may be included in the carrier include, without limitation, buffer components, tonicity components, preservative-components, pH adjustors, components commonly found in artificial tears, such as one or more electrolytes, and the like and mixtures thereof. In one very useful embodiment the carrier component includes at least one of the following: an effective amount of a buffer component; an effective amount of a tonicity component; an effective amount of is a preservative component; and water. These additional components preferably are ophthalmologically acceptable and can be chosen from materials which are conventionally employed in ophthalmic compositions, for example, compositions used to treat eyes afflicted with dry eye syndrome, artificial tear formulations and the like. In some preferred embodiments, the glands of both eyes of the model animal are injected with botulinum toxin. In these embodiments, the test compound, preferably in an ophthalmologically acceptable carrier, is applied to one eye of the subject while the ophthalmologically acceptable carrier without the test compound is applied to the other eye of the subject as a negative control.

In some preferred embodiments, following administration the test compound and optional negative control to the eyes of a test animal, the test animal is evaluated for efficacy the test compound or composition in relief of symptoms associated with decreased tear production. In some preferred embodiments, one or more the following signs or symptoms are evaluated: ocular discharge, rubbing at eyes, increased blink rate, squinting, uptake of fluorescein stain on the ocular surface, uptake of rose Bengal stain on the ocular surface, uptake of Lissamine green stain on the ocular surface, or decreased tear film break up time (TFBUT) measured using a water soluble dye such as fluorescein or using non-invasive instruments (NITFBUT) exemplified by but not limited to the Oculus keratography 5M or the Keeler tearscope.