Amniotic membrane and uses thereof

Abstract

The invention relates to amniotic membrane and its use in treating dry eye disease. The amniotic membrane can be applied to the subject's eye in combination with a contact lens, which may be adapted to hold the membrane by means of a recess in the lens.

Description

[0001] AMNIOTIC MEMBRANE AND USES THEREOF FIELD OF INVENTION The invention relates to amniotic membrane and its use in treating dry eye disease. BACKGROUND Dry eye disease (DED) is a multifactorial disease of the ocular surface characterised by ocular surface inflammation and damage and neurosensory abnormalities. Tear film breakup leading to localised hyperosmolarity can result in ocular surface damage either directly or through the cascade of inflammation that it initiates. DED commonly occurs in people who are otherwise healthy, but is a prominent, chronic disease leading patients to seek medical attention and treatment. DED has one of the highest annual incidences, behind refraction / accommodation and cataracts, and the annual prevalence rate that tripled between 2005 and 2012. 10% of UK and US adults (approximately 12 million individuals) are diagnosed moderate-to-severe DED. 81% of diagnosed DED is managed predominantly through artificial tears / lubricants, and prescription anti- inflammatory drugs. This accounts for 46% of the £3.5bn global DED market. Unfortunately, these options are in many cases ineffective and associated with considerable side effects meaning in the US, the annual health care treatment and overall society costs of managing DED is £3.16bn (£644 / patient) and £45.6bn (£9,305 / patient), respectively. Little progress has been made in providing DED sufferers with an effective, long-term solution. Current topical therapy options are costly, require high frequency (at least daily) and long-term application, providing only limited chronic relief. Prescription cyclosporin-based drugs are the most common therapeutic option, but efficacy is poor and protracted treatments of 12-months are required to achieve clinical benefit. There is therefore a need to have improved treatments for DED. SUMMARY In an aspect, the invention provides amniotic membrane for use in treating or preventing dry eye disease in a subject. In another aspect, the invention provides a method of treating dry eye disease in a subject, comprising administering amniotic membrane to the eye of the subject. In another aspect, there is provided the use of amniotic membrane in the manufacture of a medicament for the treatment of dry eye disease. In any aspect, the use of amniotic membrane may be in a sutureless method of treating a subject as described herein. In any aspect, the amniotic membrane may be dry amniotic membrane, such as freeze- dried amniotic membrane. In an embodiment, the amniotic membrane is to be applied to the subject’s eye in combination with a contact lens. The subject’s eye as referred to herein may refer to at least all of, or substantially all of the cornea. The amniotic membrane may be applied to the subject’s eye, and a contact lens applied over the amniotic membrane to hold it in place on the eye. The amniotic membrane may be attached or loaded to the contact lens, and the lens then applied to the subject’s eye. The amniotic membrane may be in direct contact or substantially direct contact with the subject’s eye. When in use, the amniotic membrane may be in contact with the surface of the lens and / or corneal surface. Alternatively, when in use the amniotic membrane may be held between the surface of the lens and the corneal surface, but may not be in direct contact with either the surface of the lens or corneal surface when in use. The inventors surprisingly found that the therapeutic effects of using amniotic membrane to treat dry eye can be facilitated by the presence of a contact lens holding the amniotic membrane against the eye, or substantially against the eye. This is unexpected, as the use of conventional contact lenses is considered to contribute to the pathology of dry eye disease. In an embodiment, the lens is adapted to hold the amniotic membrane. The lens may be adapted to hold the membrane by means of a recess in the lens. The recess may be on the side of the lens which is closest to the eye in use. The recessed part of the lens may form a more convex part of the lens versus the periphery of the lens. The edge of the lens may extend beyond the edge of the radius of the membrane. The surface area of the lens may exceed the surface of the membrane. The periphery of the lens may directly contact the eye, whilst the area internal to the periphery may comprise the recess. The edge of the lens therefore provides a seal which acts to retain the amniotic membrane against or substantially against the eye of the subject for the period of the treatment. This maintains the amniotic membrane in position whilst the therapeutic effects are provided. The lens may be configured to not impair the subject’s normal sight. In another aspect, the invention provides a contact lens adapted to hold amniotic membrane, for use in treating dry eye disease. The contact lens may be provided with processed amniotic membrane as described herein. The lens may be adapted to hold the amniotic membrane by means of a recess in the lens The recess may be on the side of the lens which is closest to the eye in use. The recessed part of the lens may form a more convex part of the lens versus the periphery of the lens. The edge of the lens may extend beyond the edge of the radius of the membrane. The surface area of the lens may exceed the surface of the membrane. The periphery of the lens may directly contact the eye, whilst the area internal to the periphery may comprise the recess. The edge of the lens therefore provides a seal which acts to retain the amniotic membrane against or substantially against the eye of the subject for the period of the treatment. In any aspect, the dry eye disease may be classified as moderate to severe. The amniotic membrane as described herein may comprise an aperture configured to allow the patient to see through, or substantially through, and / or to configured for the patient to retain at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90, at least 95% if their field of vision. The amniotic membrane as described herein may comprise a central or substantially central aperture. The aperture may be circular. The aperture may be about 3mm-8mm in diameter, preferably about 5mm or 6mm in diameter. This allows unimpeded vision and prevents any negative effect of the subject’s normal daily functions. Apertures outside of this range would impede the subject’s vision and / or prevent the amniotic membrane from being held securely in position between the subject’s cornea and the inner surface of the contact lens. Such an aperture would also allow for bilateral treatment of both eyes simultaneously whilst carrying on normal daily functions. Without such an aperture, a patient undergoing bilateral covering the visual axis with amniotic membrane would be prohibited from driving and other such activities. When used with a lens as described herein, amniotic membrane with an aperture as described will also be more likely to stay central and less likely to move around under the lens, which would allow the amniotic membrane to remain on the subject’s eye for longer periods. The amniotic membrane as described herein may be in the shape of a disc. The amniotic membrane may have a diameter of about 10mm or more, about 12mm or more, about 15mm or more, about 16mm or more, about 17mm or more, about 20mm or more. Application of the lens to a subject’s eye may be suture free and glue free. A contact lens as described herein may be a bandage contact lens or a soft contact lens. Such lenses and their properties are well known in the art. A contact lens as described herein may be made from any suitable material. The material may be selected from Table 1. The lens may be made from a hydrogel such as Filcon ii 2 or Samfilcon A material. The lens may be made from a silicone hydrogel such as Samofilcon A, delefilcon A, balafilcon A, comfilcon A or senofilcon A material. Preferably, the lens is made from N,N-dimethyl acrylamide (DMA) and N-vinyl-2- pyrrolidone (NVP). The lens may be highly water permeable and highly oxygen permeable, hydrophilic co-polymer soft hydrogel. The lens may have an about 60-80% water content, such as about 65-75%, about 70%, or preferably about 72% water content. In this embodiment, the DMA may make up 1% of the composition of the lens, and NVP may make up 27% of the lens; or the DMA may make up 2% of the composition of the lens, and NVP may make up 26% of the lens; or the DMA may make up 3% of the composition of the lens, and NVP may make up 25% of the lens; or the DMA may make up 4% of the composition of the lens, and NVP may make up NVP may make up 23% of the lens; or the DMA may make up 6% of the composition of the lens, and NVP may make up 22% of the lens; or the DMA may make up 7% of the composition of the lens, and NVP may make up 21% of the lens; or the DMA may make up 8% of the composition of the lens, and NVP may make up 20% of the lens; or the DMA may make up 9% of the composition of the lens, and NVP may make up 19% of the lens; or the DMA may make up 10% of the composition of the lens, and NVP may make up 18% of the lens; or the DMA may make up 11% of the composition of the lens, and NVP may make up 17% of the lens; or the DMA may make up 12% of the composition of the lens, and NVP may make up 16% of the lens; or the DMA may make up 13% of the composition of the lens, and NVP may make up 15% of the lens; or the DMA may make up 14% of the composition of the lens, and NVP may make up 14% of the lens; or the DMA may make up 15% of the composition of the lens, and NVP may make up 13% of the lens; or the DMA may make up 16% of the composition of the lens, and NVP may make up 12% of the lens; or the DMA may make up 17% of the composition of the lens, and NVP may make up 11% of the lens; or the DMA may make up 18% of the composition of the lens, and NVP may make up 10% of the lens; or the DMA may make up 19% of the composition of the lens, and NVP may make up 9% of the lens; or the DMA may make up 20% of the composition of the lens, and NVP may make up 8% of the lens; or the DMA may make up 21% of the composition of the lens, and NVP may make up 7% of the lens; or the DMA may make up 22% of the composition of the lens, and NVP may make up 6% of the lens; or the DMA may make up 23% of the composition of the lens, and NVP may make up 5% of the lens; or the DMA may make up 24% of the composition of the lens, and NVP may make up 4% of the lens; or the DMA may make up 25% of the composition of the lens, and NVP may make up 3% of the lens; or the DMA may make up 26% of the composition of the lens, and NVP may make up 2% of the lens; or the DMA may make up 27% of the composition of the lens, and NVP may make up 1% of the lens. Preferably, the lens has a Dk value of over about 20, such as over 30, such as 34. The Dk value as described herein is measure of the permeability of a material. To relate this to the oxygen performance of a contact lens, Dk is divided by t, the thickness, usually taken at the centre of that lens. This gives a measure of oxygen transmissibility that shows the amount of oxygen that can pass through a contact lens in air. The lens may be about 10-25mm in diameter, such as about 12mm, about 14mm, about 16mm, about 18mm, about 20mm, about 22mm in diameter. Preferably, the lens is between about 14 and about 18mm in diameter. The lens may have a base curve (measured in millimetres) of about 7.0-10.0, such as about 8.0-9.2, about 8.0-9.1. The lens may have a base curve of about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 8.9, about 9.0, or about 9.1. Preferably, the base curve is about 8.8. The optimal base curve prevents the amniotic membrane from moving away from the eye by allowing the lens to secure the amniotic membrane against the eye for the duration of the treatment. If the base curve is too low, the lens may cause discomfort to the patient. The base curve as described herein refers to the measurement of the back curvature of the contact lens. The base curve may be measured using a radius gauge. The lens may be afocal or powered. The power of a lens is directly correlated to the refractive ability of the lens and therefore the strength of a lens prescription a subject may have been given. The power may be positive or negative. Table 1 – exemplary lens materials

[0002] In an embodiment, the amniotic membrane is to be applied to the subject’s eye for at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, or at least 10 days. The amniotic membrane is to be kept on the subject’s eye for at least five days. Preferably, the amniotic membrane is to be kept on the subject’s eye for at least seven days. The invention has the benefit of allowing a single application of amniotic membrane to the subject’s eye to impart the therapeutic effects desired over an extended period of time. In contrast, existing therapies may require multiple applications of a therapeutic, as often as daily, resulting in lost time for the subject attending clinics, as well as increased costs. The subject may receive one or more further treatment with the amniotic membrane. The one or more further treatment may be every three months, six months, or 12 months. The amniotic membrane may be vacuum dried. The amniotic membrane may require reconstitution prior to application to a subject’s eye. The term “about” as used herein may reflect a value which is 10% above or below the value to which the term refers. DETAILED DESCRIPTION Amniotic membrane (AM) is the inner most extraembryonic membrane that surrounds the foetus in a sac of amniotic fluid, functioning as a protective barrier to ascending infection and trauma during pregnancy. Amniotic membrane is highly resistant to rupture and tearing, and this mechanical strength is attributed predominantly to a multilayered architecture and an extensive collagen type I network. This is separated from a single layer of cuboidal epithelium by a basement membrane (BM). The epithelium is metabolically active, maintaining amniotic fluid homeostasis and secreting embryonic stem cell factors. Therapeutic amniotic membrane is extensively used in ophthalmic surgery and was first applied with chorion, as a replacement for scarred conjunctival tissue. Amniotic membrane is now commonly used as a permanent graft or a temporary patch in a plethora of conjunctival and corneal procedures. These include ocular surface reconstruction and treatments for persistent epithelial defects, pterygium, bullous keratopathy and acute ocular burns. AM has been shown to act as a scaffold for cell growth, promote epithelial wound healing and to exert anti-inflammatory, anti-angiogenic, anti-fibrotic and anti- microbial effects. These mechanisms are, in part, attributed to a wide range of biological factors present in amniotic membrane, for example epidermal growth factor (EGF) and transforming growth factor (TGF)-β1. In many countries, AM is obtained from elective caesarean section deliveries and typically frozen in medium containing glycerol or dimethylsulphoxide (DMSO) while the donor is screened for a spectrum of infectious diseases. The effects of DMSO and glycerol preservatives on the structural and biochemical integrity of cryopreserved amniotic membrane (CPAM) are unclear. Following freezing amniotic membrane is considered non-viable and following thawing soluble factors presumed to be beneficial are extensively depleted from the tissue, potentially reducing its efficacy. It has been additionally shown that there are significantly lower levels of angiogenic factors in CPAM compared to fresh amniotic membrane (FrAM) but they did not assess lyophilised amniotic membrane. A number of studies have reported extensive depletion of soluble factors, presumed to be beneficial, from CPAM. While frozen preparations of amniotic membrane account for the majority of procedures, dried preparations have gained popularity as substrates for epithelial growth during ocular surface reconstruction, and to treat corneal perforations and leaks and pterygium. Moreover, as dried preparations can be kept at room temperature, they eliminate the need for a cold chain and are therefore suitable for use in developing countries and in military environments. Conventional freeze-drying requires the tissue to be frozen prior to drying, resulting in structural freeze damage and subsequent factor loss as observed with conventional cryopreservation techniques. Although rehydrated dried amniotic membrane has been reported to be thinner and more fragile than its cryopreserved counterpart, there is no significant difference in the tensile properties and collagenous structure of the two amniotic membrane preparations. Conventional freeze-drying requires the tissue to be frozen prior to drying. In amniotic membrane, this appears to result in structural freeze damage and subsequent factor loss as compared with conventional cryopreservation techniques. Therefore, in an embodiment, the amniotic membrane can be stored at room temperature with no or minimal loss to structural integrity and biochemical activity, such as retention of soluble factors. The amniotic membrane may be isolated. The amniotic membrane may be processed before being stored. Because amniotic membrane is typically less than 100 microns thick, it is possible to dry it in a freeze-dryer vacuum without the pre-freeze step, and this is herein referred to as vacuum-drying. The amniotic membrane may be collected during a natural birth or birth by caesarean section. Amniotic membranes may be vacuum-dried by spreading them flat and putting them inside a vacuum-drier. The standard method for freeze-drying includes the step of first freezing the sample (the pre-freeze step) and then putting the frozen sample in a freeze-drier. In the standard procedure it is important to pre-freeze the sample first because the freeze- drier can only remove water that has been frozen. The membrane is treated with one or more lyoprotectants before vacuum-drying. Any one or more lyoprotectant may be applied to the amniotic membrane before the vacuum-drying step. The amniotic membrane may be soaked or dipped in a solution of the lyoprotectant. The lyoprotectant may be any suitable lyoprotectant, for example a saccharide lyoprotectant, for example the lyoprotectant may be trehalose dihydrate and / or raffinose pentahydrate. The lyoprotectant may be trehalose or raffinose in combination with one or more further lyoprotectants. More than one lyoprotectant may be applied to the amniotic membrane before vacuum-drying. A combination of two or more, three or more, four or more or five or more or six or more lyoprotectants may be applied to the amniotic membrane before vacuum-drying. The amniotic membrane may be treated with an antioxidant before vacuum-drying. For example, the antioxidant is epigallocatechin (EGCG). In addition to lyoprotectants and antioxidants the amniotic membrane may be treated with other agents before vacuum-drying. For example, the amniotic membrane may be treated with glycerol / PBS, DMSO, PBS, or tertiary butyl alcohol. Prior to vacuum-drying the membrane may be washed to remove blood or debris, spread out flat and / or cut into sections. Membrane sections may be washed in a 1:10 dilution of the original saccharide lyoprotectant to remove excess residue from the surface. The amniotic membrane may be not frozen before the vacuum-drying step. The amniotic membrane should not be frozen before a vacuum-drying step is carried out. Freezing the amniotic membrane before vacuum-drying (pre-freeze step) damages the structure and / or may damage the cells in the amniotic membrane. Damage to the cells in the amniotic membrane may cause increased soluble factors to be lost when the membrane is reconstituted. Damage to the cells in the amniotic membrane may lead to soluble factors being released quickly from the amniotic membrane when it is reconstituted. It is advantageous to vacuum-dry the amniotic membrane without a pre- freeze step because this causes less damage to the amniotic membrane than when a pre-freeze step is used. The vacuum-dried amniotic membrane has similar structure to fresh amniotic membrane. The epithelial cells of the amniotic membrane may make soluble factors including growth factors. The epithelial cells may be damaged by freezing and thawing and the damaged cells may release all of the soluble factors at once. In vacuum-dried amniotic membrane the epithelial cells are not damaged by freezing and they retain soluble factors involved in cell growth and motility, for example hepatocyte growth factor (HGF); anti-angiogenesis, pigment epithelium-derived factor (PEDF); cell adhesion, intercellular adhesion molecule-1 (ICAM-1); inflammation, interleukin-8 (IL-8); cell growth and differentiation, epidermal growth factor (EGF); structural remodelling, metalloproteases 3 and 9 (MMP-3 / 9); and neuron growth and differentiation, brain- derived neurotrophic factor (BDNF), which are released more slowly and over a longer period of time from epithelial cells that are not damaged. A vacuum-dried amniotic membrane may comprise 100% of the soluble factors compared to a fresh amniotic membrane. A vacuum-dried amniotic membrane may comprise more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of the soluble factors compared to a fresh amniotic membrane. A vacuum-dried membrane may release soluble factors over a period of more than a day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 10 days, more than 12 days, more than 14 days. A vacuum-dried membrane may release soluble factors over a period of 5 to 7 days. This is advantageous over a cryo-preserved membrane, which may release all of its soluble factors in 24 hours. Vacuum-dried and reconstituted amniotic membrane comprising epithelial cells releases soluble factors over a period of at least a day, at least two days, at least three days, at least four days, at least five days, at least 6 days, at least 7 days, at least 8 days, at least 9 days or at least 10 days. This is in contrast to prior part frozen amniotic membranes, which release most of their soluble factors in the first 24 hours. It is advantageous to release growth factors over a period of several days, for example two or more days. The amniotic membrane may be treated with thermolysin before vacuum-drying. Thermolysin removes the epithelial cells from the amniotic membrane to provide a denuded amniotic membrane. The treatment of the amniotic membrane with thermolysin may be performed before the step of freeze-drying the amniotic membrane. The amniotic membrane may be treated with thermolysin by incubating processed sections of amniotic membrane with thermolysin at a suitable concentration, for example 125µg / ml, for a suitable amount of time to remove the epithelial cells, for example 9 minutes, with gentle agitation. Amniotic membrane sections may then be washed in Dulbecco’s phosphate buffered saline (DPBS) prior to drying. Further processing steps may be undertaken before or after the membrane has been vacuum dried. Further steps may include cutting the amniotic membrane to a suitable size and shape either before or after vacuum-drying; vacuum-packing the vacuum- dried amniotic membrane after drying and sterilising the amniotic membrane either before or after vacuum-drying; affixing the amniotic membrane to the side of a contact lens to be placed on the subject’s eye. Amniotic membrane may be reconstituted by adding a suitable amount of water or a suitable biocompatible solution to the membrane for a suitable time until the membrane has become rehydrated. A vacuum-dried amniotic membrane may be reconstituted by placing it directly on the eye or on a wound or operation site with or without adding liquid directly to the amniotic membrane first. Liquid naturally occurring in the eye, wound or operation site for example tears, tissue fluid, blood or liquid added to the eye, wound or operation site may reconstitute the vacuum-dried amniotic membrane while it is in contact with the surface of the eye, wound or operation site. Applying the vacuum-dried amniotic membrane directly to the eye without reconstituting is advantageous to save time and to avoid contamination of the vacuum-dried amniotic membrane while it is being reconstituted. There now follows by way of example only a detailed description of the present invention with reference to the accompanying drawings, in which; BRIEF DESCRIPTION OF THE DRAWINGS Figure 1- shows a cross sectional image of amniotic membrane held substantially against the eye of a subject, and which is held in place by a lens as described herein adapted to hold the amniotic membrane. Figure 2 - demonstrates that OSDI following BCL+ amniotic membrane treatment decreases over time from an average of 55.75 (Severe) to 18.79 (mild) for at least 6 months (p<0.001). Figure 3 - demonstrates that subjects treated with BCL + amniotic membrane as described herein see a significant reduction in limbal nasal hyperaemia (Appendix 2) at 90 days (p= 0.048, with a 95% CI of -0.69 to -0.01). Figure 4 – shows that subjects treated with BCL + amniotic membrane as described herein see a significant increase in Corneal Nerve Fibre Total Branch Density at 14 days (p =0.043, 95% CI of 1.49 to 43.59). Y axis depicts the change in CTBD in millimetres, X axis shows the days post-baseline. Figure 5 – shows an amniotic membrane with an aperture configured to retain the subject’s field of vision, by not covering the whole of the pupil. Figure 6 – shows ocular Surface Disease Index score change after one-month of treatment. Figure 7 - shows Symptom Assessment iN Dry Eye (Frequency) score change after one-month treatment. Figure 8 – shows Symptom Assessment iN Dry Eye (Severity) score change after one- month of treatment. Figure 9 – shows score change after a Dry Eye Questionnaire after one-month of treatment. Figure 10- shows Lid Wiper Epitheliopathy (LWE) Sagittal width grade change after one-month of treatment. Figure 11 - Ocular Surface Disease Index subjective comfort rating between for treatment over 6-months. Figure 12 – shows SANDE Frequency in response to treatment over 6-months. Figure 13 – shows SANDE Severity in response to treatment over 6-months. Figure 14 – shows LWE showing significant 64% (1.29 grade) reduction over 6 months -p < 0.005. Figure 15 – shows Corneal Nerve Fibre Length over 6 months. 10.6% Improvement 1.57mm / mm2 p = 0.049. Figure 16 – shows Corneal Nerve Branch Density over 6 months - 10.6% Improvement - 1.57mm / mm2 p = 0.363. Figure 17 – shows Corneal Nerve Fibre Density over 6 months - 21% improvement 4.41 mm / mm2 p = 0.030. EXAMPLES Example 1 – clinical trial demonstrating therapeutic effects of the amniotic membrane of the invention when applied in a lens adapted to hold the membrane. The inventors sought to examine changes in symptomatology and pathology following sutureless application of amniotic membrane as described herein under a specialised bandage contact lens in patients with moderate-to-severe Dry Eye Disease (DED). Table 2- clinical study parameters Study Type : Interventional (Clinical Trial) Estimated Enrolment: 42 participants Allocation: Randomized Intervention Model: Parallel Assignment Intervention Model Description: 1 month baseline followed by randomisation into treatment or control and 6 months follow-up Masking: Double (Participant, Investigator) Masking Description: Patient was not aware whether bandage contact lens has an amniotic membrane beneath in periphery or not Masked investigator will perform the clinical examinations Primary Purpose: Treatment The randomised-controlled double-masked trial (NCT04553432) enrolled 42 participants (aged 52±20 years, 31 females and 11 males) split between bilateral application of 17mm amniotic membrane disc with a central 6mm diameter window under a specialised plano 18mm BCL, or the plano BCL alone (comparator), for two consecutive 3–4-day treatments. The lens was an N,N-dimethyl acrylamide (DMA) and N-vinyl-2-pyrrolidone (NVP) copolymer soft hydrogel, was highly water permeable and highly oxygen permeable, hydrophilic, had a water content of 72%, had a base curve of 8.8, was afocal. The lens material is commercially known as Menicon 72. The lens had a recess as described herein, (see Figure 1), which held the amniotic membrane in place. A 16mm lens of this type is expected to be at least as effective. Assessed patient reported symptomatology included Ocular Surface Disease Index (OSDI), which is a patient reported scoring of dry eye symptoms, and Symptom Assessment Questionnaire iN Dry Eye (SANDE). Assessed clinical signs included Non-Invasive Breakup Time (NIBUT), Tear Meniscus Height (TMH, ocular hyperaemia, Lipid Layer Thickness (LLT), Corneal and Conjunctival Staining (CornS / ConjS), and Lid Wiper Epitheliopathy Length and Width (LWEL / LWEW - Oculus Keratograph 5M), and confocal imaging of the corneal nerve fibres and immune cells. All assessments were taken 0, 15, 30, 90 and 180 days post baseline, where baseline was before application of the amniotic membrane / lens. Treatment delivered a dramatic and significant reduction in patient-reported disease severity (OSDI) scale from an average of 55.75 (Severe) to 18.79 (mild) for at least 6 months (p<0.001, Figure 2). The frequency and severity (SANDE) of patient symptom was also significantly reduced from 67.86±21.06 to 38.46±21.13 (p= <0.001), and from 58.12±24.11 to 38.03±19.40 (p=0.002), respectively, at 30 days post treatment. There was also a significant decrease in dry-eye questionnaire results (DEQ-5) from 16.93±9.47 to 11.15±5.52 (p=0.001) at 30 days post treatment. In terms of ocular surface clinical signs, treatment delivered a 38% decrease in the width of LWE staining, from grade 2 (50-75% of the lid wiper) to grade 1 (25-50% of the lid wiper) (p=0.011) occurred in the 30-day period post treatment, and a decrease in conjunctival staining approached significance (p=0.094). The additional biological benefit of amniotic membrane was demonstrated through a significant reduction in limbal nasal hyperaemia (Figure 3) at 90 days (p= 0.048, with a 95% CI of -0.69 to -0.01), and a significant increase in Corneal Nerve Fibre Total Branch Density (CTBD) (Figure 4) at 14 days (p =0.043, 95% CI of 1.49 to 43.59), representing an increase of 22.54 mm2 compared to the lens-only group. In these models, each patient at each timepoints is compared to baseline and then the difference compared between treatment groups, and the difference represented relative to baseline, which is 0 on the y axis. Any data point above 0 is an increase compared to baseline and anything below is a decrease compared to baseline. Where an effect decreases towards baseline after towards 30 of more days, this indicates a further treatment is needed. Similarly, Corneal Nerve Fibre Length (in mm) increased over 90 days (p= 0.049, with a 95% CI of 0.03, 3.09). AM was also observed to improve the lipid thickness (p = 0.081). In summary, a 1-week treatment improved dry eye symptomatology by 60% for up to 6 months. Ocular surface signs associated friction (lid wiper damage) decreased whilst hyperaemia associated with inflammation, corneal nerve fibre length (in mm2) and branch density associated with corneal nerve health improved. Hence, the inventors show that amniotic membrane plays a significant role in improving the quality-of-life of patients with intransient moderate-severe dry eye. Table 3- quantitative of the clinical trial Measureme Enrolment 15 30 days 90 180 days p- nt days days value OSDI 55.75±19. 45.25±22.3 29.85±19. 30.62±20.2 18.79±13. <0.001 79 2 32 3 40 * DEQ-5 16.93±9.4 13.26±4. 11.15±5.5 10.45±3. 18.15±18. 0.001* 7 49 2 73 22 SANDE 67.86±21. 57.21±24.8 38.46±21. 40.10±26.1 39.95±28. <0.001 06 6 13 0 27 * Frequency SANDE 58.12±24. 53.99±26.0 38.03±19. 39.17±24.2 49.24±27. 0.002* Severity 11 2 40 7 57 VAS Overall 77.75±18. 81.54±13. 0.314 health 00 54 EQ-5D-5L 0.84±0.16 0.85±0.14 0.386 VA 0.11±0.15 0.15±0.2 0.09±0.19 0.10±0.1 0.106 0 8 TMH 0.27±0.11 0.33±0.1 0.29±0.13 0.30±0.1 0.109 6 4 Hyperaemia 1.16±0.15 1.27±0.6 1.14±0.58 1.22±0.6 0.559 - Bulbar 0 6 8 Temporal Hyperaemia 1.01±0.45 1.35±0.8 1.30±0.72 1.46±0.7 <0.001 – Bulbal 5 8 * Nasal Hyperaemia 0.83±0.38 0.95±0.5 0.79±0.45 0.85±0.4 0.099 - Limbal 4 9 Temporal Hyperaemia 0.77±0.41 0.98±0.6 0.85±0.51 0.90±0.6 0.046* -Limbal 4 1 Nasal NIKBUT 7.35±5.01 6.74±4.6 7.09±4.87 6.68±3.4 0.736 9 9 LLT 3.83±1.54 3.86±1.3 3.85±1.61 3.60±1.4 0.581 7 8 Corneal 1.33±1.18 1.48±1.2 1.20±1.25 0.90±1.0 0.032* Staining 3 5 Conjunctival 1.81±1.17 1.45±1.0 1.41±1.14 1.17±0.9 <0.001 6 9 * Staining LWE Length 1.90±1.41 2.10±1.4 1.63±1.64 0.93±1.2 0.001* 5 5 LWE Width 1.76±1.27 0.95±0.95 0.66±0.9 <0.001 1.52±0. 4 * 97 Meiborgrap 1.19±1.23 1.31±1.3 0.242 hy - UL 7 Meibograph 1.88±1.21 2.22±2.0 0.262 y - LL 4 CNFD 19.97±10. 18.93±9. 21.63±10. 23.19±9. 0.053 00 03 32 41 CNBD 25.30±20. 28.21±23.1 30.93±27. 29.50±24.5 0.444 15 6 84 6 CNFL 13.29±4.0 13.53±4. 14.43±5.0 14.84±4. 0.021* 9 55 8 30 CTBD 43.14±28. 51.96±38.0 49.52±38. 45.56±30.5 0.001* 46 4 40 1 CNFW 0.02±0.00 0.02±0.0 0.02±0.00 0.02±0.0 0.612 0 0 Langerehan 1.34±1.48 1.51±1.9 1.10±1.43 1.23±1.5 0.608 s 0 3 activated Langerhans 3.12±3.77 3.20±3.7 5.03±4.95 4.23±5.3 0.092 non- 7 6 activated CNBD: Corneal nerve branch density (branches / mm²); CNFD: Corneal nerve fibre density (fibres / mm²); CNFL: Corneal nerve fibre length (mm / mm²); CNFW: Corneal nerve fibre width; CTBD: Corneal nerve fibre total branch density; DEQ-5 – Dry Eye Questionnaire – 5; LL: Lower Lid; LLT: Lipid Layer Thickness; LWE – Lid wiper epitheliopathy; NIKBUT: non- invasive keratometric breakup time (seconds); OSDI: Ocular Surface Disease Index questionnaire; SANDE: Symptom Assessment in Dry Ey e; TMH: Tear Example 2 – inclusion of an aperture in the amniotic membrane provides numerous benefits. The normal pupil size in adults varies from 2 to 4 mm in diameter in bright light to 4 to 8 mm in the dark. The inventors tested apertures with a range of diameters and concluded that a central aperture of 5mm is the smallest possible without impacting vision in the lower lighting conditions (Figure 5). Example 3 – clinical study of bilateral sutureless application of human dehydrated amniotic membrane with a specialized bandage contact lens for moderate-to-severe dry eye disease – one month readout. Patients and Methods: In this prospective pre-post interventional study, 35 patients diagnosed with moderate-to-severe DED, with an Ocular Surface Disease Index (OSDI) score >30 on current treatment, were enrolled. Assessments were conducted at baseline (day -30), after 30 days run-in with no additional treatment (day 0 - control), and 30 days post sBCL+dAM applications (day 30 - treatment). Intervention involved two successive bilateral 4–5-day sutureless applications of dAM (17mm – 6mm central aperture) under an 18mm sBCL. Symptomatology and ocular signs were measured using OSDI, Dry Eye Questionnaire-5 (DEQ-5), Symptom Assessment iN Dry Eye (SANDE), and other ocular surface health indicators including non-invasive break-up time, corneal and conjunctival staining, and lid wiper epitheliopathy length and width (LWE). Results: While symptomatology remained stable during the 30-day no-treatment run- in, 1-month post-dAM treatment, there was a significant reduction in OSDI scores (from 55.8 to 32.3, p<0.001), DEQ-5 (from 14.6 to 10.0, p<0.001), SANDE frequency (from 65.2 to 43.6, p<0.001), and SANDE severity (from 59.8 to 41.1, p<0.001). Additionally, there was a notable decrease in the width of LWE staining, from grade 2 (50-75% of the lid wiper) to grade 1 (25-50% of the lid wiper) (p=0.011). Conclusions: A bilateral 8–10-day treatment duration with dAM applied with sBCL demonstrated a 31 to 42% improvement in symptomatology and a decrease in ocular surface signs of friction. This innovative bilateral treatment approach offers a promising treatment modality for patients with refractory moderate-to-severe DED. Participants included in the study were at least 18 years old with, at least, 1 year of longstanding positive diagnosis of DED intransigent to TFOS DEWS II stage 1 treatment, an Ocular Surface Disease Index (OSDI) score of between 25-80, the presence of at least 1 of the following signs; corneal ( ^5 punctate spots) or conjunctival ( ^9 punctate spots) staining (oxford scale), or non-invasive Keratograph breakup time (NIKBUT, ^8 seconds), and with no changes in DED therapy in the 6-weeks prior to their baseline visit. Exclusion criteria included a history of ocular herpetic keratitis, ocular surgery or IPL (Intense Pulsed Light) within 6 months prior to baseline visit, current use of glaucoma medication or other medication known to alter the tear film, use of moisture chamber googles, known sensitivity to dAM antibiotics (gentamycin, imipenem, nystatin, polymyxin B and vancomycin), active ocular surface pathologies other than DED, eyelid abnormalities or extensive ocular scarring. Eligible participants were enrolled after providing written informed consent. Study Design This prospective pre-post interventional study was designed to select refractory DED patients who exhibited stable but chronic signs and symptoms in the month leading up to the treatment. This 1-month pre-treatment period served as a control, establishing a baseline against which changes post-treatment were assessed. The study involved 5 visits: Baseline (day -30) – initial measurements to establish control data; Treatment 1 (day 0) - repeat assessment to confirm disease stability, and application of the intervention; Treatment 2 (day 4-5) - reapplication of the intervention; Treatment removal (day 8-10) - removal of the intervention to transition into post-treatment observation; and, 1-month post-treatment follow-up (day 30) - final assessment to evaluate the outcomes of the intervention. Both eyes were treated bilaterally, but to ensure consistent and standardised data comparison, only the right eye data was analysed. This approach aligns with common practices in ocular studies and ensures a reliable data set for analysis. The choice of the right eye was based on preliminary findings that suggested similar disease progression in both eyes of the target patient population. The required sample size was determined to be 33 participants. Power calculations were performed using G*Power v3.1, with OSDI as the primary outcome measure, based on detecting a minimally clinically important difference of 4.5 to 7.3, with 95% power (β = 0.05) at a two-sided statistical significance level of 5% (α = 0.05) using a pair t-test. Intervention Intervention involved all patients receiving two consecutive 4–5-day bilateral applications of dAM (17mm diameter disc with a 6mm central aperture) applied using sBCL (18mm diameter, 8.80 base radius; plano lens; Menicon 72 material). The uniqueness of this study lies in its use of a bilateral approach made possible both using a 6mm diameter central window in the dAM disc to reduce the impact on the participant’s vision, and a specialised propriety lens modified to effectively retain dAM at the ocular surface, which distinguishes it from traditional unilateral methods. The ocular surface was anesthetised with proxymetacaine hydrochloride 0.5%, to minimise discomfort during treatment application and treatment applied per the manufacturer’s recommendations. Contact lens fitting, and dAM positioning, were assessed on a digital slit lamp (CSO Phoenix, Firenze, Italy) under 16x magnification, and an assessment of dAM window-to-pupil centration was checked additionally with an anterior segment Optical Coherence Tomographer (Cirrus-HD OCT, Germany). The choice of two consecutive treatments was based on previous findings suggesting that dAM benefits typically last 3-9 days, and to avoid dAM cloudiness due to accumulation of inflammatory coagulum. The lens and any residual dAM was removed following instillation of anaesthetic. Measurements Clinical assessments were performed at baseline (day -30), on the treatment day (day 0) and 1-month post-treatment (day 30). To ensure consistency in testing conditions, all assessments were conducted by a single clinician at the same location, maintaining a mean ± SD room temperature of 21.5±1.5°C and relative humidity of 43.2±11.6%. Participants acclimatised to the room conditions for a minimum of 15 minutes before testing. The sequence of testing followed the TFOS DEWS II recommendations, adhering to an ascending order of invasiveness. The testing order involved: OSDI, Dry Eye Questionnaire 5-item (DEQ-5), and Symptoms Analysis iN Dry Eye (SANDE, frequency and severity) questionnaires, used as recommended by their respective manufacturers; Tear meniscus height (TMH), measured using infrared light and high magnification digital imaging, with an average of three measurements taken from the lower lid edge below the iris using calibrated digital callipers; NIKBUT, captured by observing the time taken for a >5% distortion in Placido disc reflections, averaged over three readings following two non-forceful blinks; lipid layer thickness (LLT), graded using tear film interferometry on the modified Guillon-Keeler grading system - 0 (non-visible / absent), 1 (open meshwork), 2 (closed meshwork), 3 (wave / flow), 4 (amorphous), or 5 (coloured fringes); Conjunctival hyperaemia, automatically and objectively assessed under high magnification on the JENVIS grading scale, to 0.1 precision; Corneal staining (CornS) and conjunctival staining (ConjS), evaluated using fluorescein (BioFluoro. Biotech Vision Care Pvt., India) observed under blue ;light with a yellow filter30 and Lissamine strips, respectively, and the number of CornS and ConjS punctate spots subjectively graded as per the modified Oxford grading scheme;31 lid wiper epitheliopathy staining (LWE) was subjectively graded relative to Korb’s grading scale. All ocular surface and tear film metrics were assessed with an Oculus Keratograph 5m (Wetzlar, Germany); Visual Acuity was assessed in all visits for safety with an Early Treatment Diabetic Retinopathy Study LogMAR chart. Statistics Data analysis was performed using IBM SPSS Statistics version 26 (New York, USA). Where normal distribution had been confirmed by the Kolmogorov-Smirnov test (p > 0.05), a repeated paired sample t-test was used. A two-tailed p-value of p<0.05 was considered significant. Results A total of 35 eligible participants (23 females, 12 males), with a mean age of 50.3±17.5 years (range: 19-77), were included in the study. This exceeded the target sample size of 33, thus ensuring adequate study power. All study participants had been diagnosed with DED which had been active for an average of 12.5±4.5 years and were refractory to previous treatments. None of the participants changed their concurrent treatment during the study. Baseline (day -30), pre- (day 0) and post- (day 30) treatment characteristics are listed in (4). The mean (± SD) duration of treatment (sBCL+dAM) remained in the eye was 4.1±1.5 days (range: 1-9) for the first application, and for 4.2±1.6 (range: 1-8) days for the second application, totalling an average of 8.1±2.7 (range: 4-16) days. Table 4. Clinical measurements pre and post (1 month) of BCL+dAM treatment. Average ± SD (p-value).*statistical significant of changes observed Baseline Treatment visit 1 month post treatment (day -30) (day 0) (day 30) DEQ-5 14.6±3.0 13.5±3.2 (p=0.210) 10.0±3.9 (p=0.045)* SANDE 65.2±21.8 70.0±19.9 (p=0.508) 43.6±22.9 (p=0.001)* frequency SANDE 59.8±19.0 62.6±20.3 (p=0.682) 41.1±22.0 (p=0.001)* severity Visual acuity 0.12±0.15 0.11±0.15 (p=0.627) 0.08±0.17 (p=0.102) (LogMAR) Bulbar 1.10±0.44 1.07±0.51 (p=0.644) 1.15±0.51 (p=0.433) hyperaemia temporal + nasal (grade) Limbal 0.80±0.34 0.76±0.48 (p=0.680) 0.77±0.37 (p=0.669) hyperaemia temporal + nasal (grade) TMH (mm) 0.26±0.09 0.25±0.08 (p=0.443) 0.25±0.11 (p=0.695) NIKBUT (s) 7.84±4.66 6.32±4.92 (p=0.125) 7.57±4.77 (p=0.777) LLT (grade) 4.14±1.14 3.74±1.29 (p=0.095) 4.00±1.26 (p=0.516) CornS (grade) 1.37±1.21 1.57±1.20 (p=0.386) 1.34±1.26 (p=0.879) ConjS (grade) 1.80±1.28 2.00±1.08 (p=0.407) 1.49±1.12 (p=0.094) LWE Width 1.74±1.34 1.31±1.23 (p=0.092) 1.09±1.07 (p=0.011)* (grade) LWE Length 1.97±1.40 2.11±1.62 (p=0.619) 1.83±1.58 (p=0.619) (grade) Abbreviations: ConjS=Conjunctival Staining, CornS=Corneal Staining, DEQ- 5=Dry Eye Questionnaire, LLT=Lipid Layer Thickness, LogMAR=Logarithm of the Minimum Angle of Resolution, LWE=Lid Wiper Epitheliopathy, NIKBUT=Non-Invasive Keratometric BreakUp Time, OSDI=Ocular Surface Disease Index, TMH=Tear Meniscus Height, SANDE=Symptoms Analysis iN Dry Eye. Dry eye symptomatology Significant improvements were observed in dry eye symptomatology 1-month post- treatment initiation. OSDI scores (6) decreased by 42% (p<0.01), whilst SANDE frequency (7) and severity scores (8) decreased by 33% and 31% (both p=0.01), respectively. DEQ-5 score (9) also showed a significant reduction of 32% (p=0.045) at the 1-month visit. Ocular surface signs The treatment positively impacted various ocular surface signs (Table 4). At the 1- month follow-up, the sagittal width of the LWE (Figure 10) significantly decreased from grade 1.7 to 1.1 (38% reduction, p=0.011) and a marked reduction in ConjS was also shown from 1.6 to 1.3 (p=0.094). A small but significant decrease in conjunctival bulbar temporal hyperaemia from 1.10 to 0.98 (p=0.031) was noted from baseline to treatment day. There were no other significant changes on bulbar conjunctival hyperaemia, ocular surface staining or tear film lipid layer thickness pattern. Safety There were no significant changes in visual acuity between visits (p=0.627 and p=0.102), suggesting that the treatment was well-tolerated without adverse effects on vision. No safety concerns were raised from the treatment. Discussion This study contributes significantly to the literature by being the first to employ dAM and using bilateral sutureless application in moderate-to-severe DED treatment. This approach not only leverages the benefits of dAM without surgical intervention, but also aligns with the trend towards less invasive treatments in ophthalmology, whilst presenting a more accessible, cost-effective, and patient-friendly option for DED management. The notable 42% (23.5 point) improvement in OSDI scores, observed in this study is comparable to, and in some cases exceeds, the outcomes from other treatments involving sutureless cryopreserved AM, such as Prokera, for DED management (Table 5). For example, in a retrospective case series by Cheng et al., immediate 46% (15.3 points) improvement in OSDI scores following the removal of Prokera. However, but the long-term benefit was not sustained, with OSDI scores reverting to baseline within an average of 4.2 ± 4.7 months. Notably, the initial severity of DED in this study, indicated by an OSDI score of 51.5, was higher than that reported by Cheng et al., where the OSDI score was 33 (Table ). This suggests that, relative to the starting severity, this study achieved a more substantial improvement in symptomology, as measured by OSDI. Table 5. Comparison of published moderate-to-severe DED study data Current Chen John57 Morkin40 McDonald McDonald study g56 58 41 Year 2023 2018 2017 2018 2018 2023 Prospectiv Retro Prospectiv Retro Retro Retro Study e pre-post e RCT 35 (70) 1 (10) 17 – 9 9 (10) 84 (97) 77 (89) Participants study (eyes) / 8 control Invention Proke Prokera Prokera Prokera Proker Sutureless ra SLIM Slim 8 SLIM a SLIM treatment SLIM / Clear 2 1-month Day 0 1 month - 1 month 1 Time point month Treatment 8.1±2.7 7.2 ± 3.4±0.7 6.4±1.1 5.4±2.8 4.9 ± duration- (4-16) 2.3 (3–5) (2-11) 1.8 days 8.5 ± (range) 2 55.8±19.0 - - - - - OSDI to Change – 32.3±19.5 points (%) -23.5 - - - - - (42.2%) 59.8±19.0 - 7.1±1.5 to 6.3±0.8 to - - to 2.2±1.1, 1.9±0.6, Pain score 41.09±22. 0, Change – 18.72 - 4.9 (69%) 4.4 - - points (%) (31%) (72.5%) Abbreviations: Ocular surface Disease Index (OSDI), Randomised Controlled Trial (RCT). Additionally, John et al. reported a reduction in patient symptomatology measured by the Visual Analog Scale (VAS) from 7.1±1.5 to 2.2±1.1, one month post-treatment with Prokera Slim, though OSDI was not a measured outcome in their study. In a retrospective case series of 10 eyes, Morkin et al. applied Prokera Slim and Prokera Clear (2 eyes) in a study on DED-related neuropathic corneal pain, reporting effective pain control in 80% of tested eyes at 6.0±2.1 months. In contrast, this study not only improved overall OSDI scores but it has also effectively reduced pain frequency and severity, as measured by the SANDE questionnaire, suggesting a more comprehensive and sustained symptom relief using bilateral dAM application. The limited improvement in signs matches that found by Craig and colleagues (2021), where symptoms improved within a month of artificial tear treatment, LWE within two months, but NIKBUT and corneal / conjunctival staining took 4 months to improve. Hence, the lack of change within these tear film homeostasis indicators would be expected. The safety profile of both dehydrated and cryopreserved AM placements has been well documented, with no significant adverse reactions. The use of sutureless sBCL as a delivery mechanism in this study potentially enhances patient comfort and treatment efficacy, in contrast to previous methods involving more rigid delivery systems, such as Prokera, associated with discomfort and shorter treatment durations. Though previously associated with reduced pain relief, recent research suggests that the duration of treatment might not correlate with treatment benefits, which is possibly due to the rapid loss of beneficial proteins in cryopreserved amnion, a limitation not present in dAM. The ability to deliver bilateral treatment with the dAM discs central 6mm aperture and sBCL amnion retention capability, is a unique aspect of this study, allowing clear visual axis maintenance and enabling patients to continue daily activities, thereby improving quality-of-life. This feature distinguishes the current study from other treatments that can potentially decrease vision from -0.22±0.06 to 0.92±0.45 (logMAR). Though Prokera Clear previously offered similar visual and clinical benefits (in 2 cases), it was not applied bilaterally. Furthermore, this bilateral treatment approach offers practical advantages in terms of potentially reducing the number of appointments or providing the option to increase the number of treatments in the current care pathway. While soft contact lenses have been used to protect the cornea, improve comfort and pain and aid epithelial recovery in many ocular surface disease states, they are not expected to deliver significant improvements in OSDI, 1-month after treatment when used alone as evidenced in cataract procedure related DED. The substantial improvements observed in this study are likely attributed to the combined use of dAM and sBCL, emphasising the efficacy of this novel treatment approach. The increasing prevalence of DED and its impact on patient quality-of-life and healthcare systems, especially post-COVID-19, further underscores the need for effective and efficient treatment strategies. The novel outpatient application of sutureless AM offers potential clinical service benefits, and may be preferable to current standard treatments like cyclosporin due to better clinical outcomes and lower societal costs. The methodology presented in this study, applicable in a primary care setting by trained eye care practitioners, such as optometrists, offers the potential for reduced treatment costs and improved patient outcomes, thereby alleviating the economic burden on public healthcare systems. Conclusion In conclusion, this is the first study to demonstrate the effectiveness of bilateral application of sutureless dAM using a specialised lens-vehicle in reducing symptomatology and improving ocular surface health in moderate-to-severe DED. This approach has the potential to transform therapeutic strategies for DED, offering an effective and patient-friendly treatment alternative. Example 4 – clinical study of bilateral sutureless application of human dehydrated amniotic membrane with a specialized bandage contact lens for moderate-to-severe dry eye disease - six moth readout. Measurements The measurements were conducted in accordance with TFOS DEWS II recommendations and in an ascending order of invasiveness. Symptomatology was assessed with OSDI, and Symptoms Analysis iN Dry Eye (SANDE), frequency and severity scales, at baseline / treatment day (day 0), 1- and 3-month post-treatment follow-up (day 30 and 90), and at final assessment (day 180). Lid wiper epitheliopathy staining (LWE), and confocal imaging of the corneal nerve fibres was assessed at baseline / treatment day (day 0), 1- and 3-month post-treatment follow-up (day 30 and 90). Participants were assessed by a single clinician, at the same location, with a mean ± SD room temperature of 21.5±1.5°C and relative humidity of 43.2±11.6% across all visits. Participants spent a minimum of 15 minutes acclimatising to the room conditions before being tested. Symptomatology questionnaires OSDI, and SANDE were used as recommended by their respective developers. Ocular surface sign assessment were captured with the Oculus Keratograph 5M (Oculus, Wetzlar, Germany). LWE staining assessment was subjectively graded and recorded relative to Korb’s grading scale. Corneal nerve branch density (branches / mm2), fibre density (fibres / mm2), fibre length (mm / mm2), fibre width and total branch density were imaged using a confocal microscope (HRTIII with Rostock Corneal Module (Heidelberg Engineering GmbH, Heidelberg, Germany) and automated software analysis CCMertrics, University of Manchester, UK). Visual Acuity was assessed in all visits for safety with an Early Treatment Diabetic Retinopathy Study LogMAR chart. Results Symptoms reduced significantly with treatment (OSDI: F=55,276, p<0.001 [Figure 1]; DEQ-5: F=4.579, p<0.001; SANDE frequency: F=19.716, p<0.001; SANDE severity: F=9.273, P<0.001). (Figures 11 to 13). Corneal staining reduced with treatment (F=3.419, p=0.018). Likewise, conjunctival staining reduced with treatment (F=10.892, p<0.001). The lid wiper staining did not reduce with treatment (F=2.279, p=0.081), but did decrease in width with treatment (F=4.358, p=0.005), Figure 14, Corneal Nerve Analysis showed an improvement in corneal nerve fibre length (Figure 15), branch density (Figure 16), and fibre density (Figure 17), as well as an increase in deactivated inflammatory cells post-treatment. Comparison of corneal nerve parameter change with treatment: Langerhans activated F=0.779, p=0.461 Langerhans deactivated F=5.355, p=0.006 The safety profile of the treatment was good, with no change in visual acuity (F=2.691, p=0.106).

Claims

CLAIMS 1. Amniotic membrane for use in treating or preventing dry eye disease in a subject.

2. The amniotic membrane for use according to claim 1, wherein the amniotic membrane is to be applied to the subject’s eye in combination with a contact lens.

3. The amniotic membrane for use according to claim 2, wherein the lens is adapted to hold the amniotic membrane.

4. The amniotic membrane for use according to claim 3, wherein the lens is adapted to hold the membrane by means of a recess in the lens, wherein the recess on the side of the lens which is closest to the eye in use.

5. The amniotic membrane for use according to claim 4, wherein the recess forms a more convex shape in of the lens versus the periphery of the lens, which is in contact with the eye in use; optionally wherein the lens has a base curve of about 8.

8.

6. The amniotic membrane for use according to any of claims 2-5, wherein the lens is a soft contact lens; 7. The amniotic membrane for use according to any of claims 1-6, wherein the amniotic membrane has been vacuum dried before being applied to the subjects eye; optionally wherein the amniotic membrane is treated with one or more lyoprotectants before vacuum-drying; and / or wherein the amniotic membrane is treated with an antioxidant before vacuum-drying.

8. The amniotic membrane for use according to any of any of claims 1-7, wherein the amniotic membrane is to be applied to the subject’s eye for at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, or at least 10 days.

9. The amniotic membrane for use according to any of any of claims 1-8, wherein the amniotic membrane is configured to retain at least 50% of a subject’s field of vision.

10. The amniotic membrane for use according to any of any of claims 1-9, wherein the amniotic membrane comprises a central or substantially central aperture.

11. The amniotic membrane for use according to claim 10, wherein the aperture is circular.

12. The amniotic membrane for use according to any of any of claims 10 or 11, wherein the aperture is about 3mm-8mm in diameter, such as about 5mm or 6mm in diameter.

13. The amniotic membrane for use according to any of any of claims 1-12, wherein the amniotic membrane is dry amniotic membrane, such as freeze-dried amniotic membrane.

14. A method of treating dry eye disease in a subject, comprising administering amniotic membrane to the eye of the subject.

15. The method according to claim 14, wherein the amniotic membrane is administered to the eye of the subject in combination with a contact lens.

16. The method according to claim 15, wherein the lens is adapted to hold the amniotic membrane.

17. The method according to claim 16, wherein the lens is adapted to hold the membrane by means of a recess in the lens, wherein the recess on the side of the lens which is closest to the eye in use.

18. The method according to claim 17, wherein the recess forms a more convex shape in of the lens versus the periphery of the lens, which is in contact with the eye in use; optionally wherein the lens has a base curve of about 8.

8.

19. The method according to any of claims 14-18, wherein the lens is a soft contact lens;20. The method according to any of claims 14-19, wherein the amniotic membrane has been vacuum dried before being applied to the subjects eye; optionally wherein the amniotic membrane is treated with one or more lyoprotectants before vacuum-drying; and / or wherein the amniotic membrane is treated with an antioxidant before vacuum- drying.

21. The method according to any of claims 14-20, wherein the amniotic membrane is applied to the subject’s eye for at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, or at least 10 days.

22. The method of any of claims 14-19, wherein the amniotic membrane is configured to retain at least 50% of a subject’s field of vision.

23. The method of any of claims 14-21, wherein the amniotic membrane comprises a central or substantially central aperture.

24. The method of any of claim 23, wherein the aperture is circular.

25. The method of any of claims 23 or 24, wherein the aperture is about 3mm-8mm in diameter, such as about 5mm or 6mm in diameter.

26. The method of any of claims 14-25, wherein the application of the amniotic membrane is sutureless.

27. The method of any of claims 14-26, wherein the amniotic membrane is dry amniotic membrane, such as freeze-dried amniotic membrane.

28. A contact lens adapted to hold amniotic membrane, for use in a method of treating dry eye disease.

29. The contact lens according to claim 28, wherein the lens further comprises vacuum dried amniotic membrane.

30. The contact lens according to claim 28 or 29, wherein the lens is adapted to hold the membrane by means of a recess in the lens, wherein the recess on the side of the lens which is closest to the eye in use.

31. The contact lens according to claim 30, wherein the recess forms a more convex shape in of the lens versus the periphery of the lens, which is in contact with the eye in use; optionally wherein the lens has a base curve of about 8.

8.

32. The contact lens according to any of claims 28-31, wherein the lens is a soft contact lens.

33. The contact lens according to any of claims 28-32, wherein the periphery of the lens directly contact the eye in use, whilst the area internal to the periphery comprises the recess.

Images