糖尿病干眼症,动物造模 z-bo 2020-09-09 342

　　Background: Tear-deficient dry eye disease (TDDE) or aqueous dry eye disease (DED) refers to abnormal tear secretion related to pathophysiology and/or environmental factors. It is a multifactorial disease characterized by changes in tear film volume and eye discomfort, and may have an inflammatory component. If left untreated, severe DED can cause pain, corneal ulcers, and potential vision loss. Even milder DED can cause chronic discomfort and blurred vision. Women, old age, and autoimmune diseases (especially type 1 or type 2 diabetes) increase the risk of disease. Most treatments are over-the-counter (OTC) or prescription lubricants (artificial tears, gels, ointments), and their success depends on frequent use. Many treatments only provide temporary relief, and most do not target the underlying disease pathology. Commonly prescribed treatments for TDDE with inflammatory causes are restasis (rx) and Lifitegrost (xiidra) containing the active ingredient of cyclosporine, the latter being a lymphocyte function-related antigen-1 antagonist approved by the FDA in 2016. Both therapies are topical drugs for dry eye caused by inflammation. Restasis® has limited efficacy and requires a long period of use (for example, 4 weeks) to take effect. Compared with OTC artificial tears, gels, gel inserts and ointments, it can provide temporary relief, steroid-loaded punctal plugs, or surgical treatment of timely bites to maintain the basic level of tear retention in patients with chronic dry eye. Therefore, there is an urgent need for new topical prescription treatments. Preclinical studies report that the opioid growth factor (ogf)-ogf receptor (ogfr) regulatory pathway plays a role in reversing or improving several ocular-related complications of diabetes. Extensive studies on the re-epithelialization of corneal wounds in normal animals confirmed the overall role of OGF in cellular homeostasis. Additional studies on NTX blocking the ogf-ogfr interaction showed accelerated corneal wound healing in diabetic mice and rats. Preclinical reports indicate that topical application of NTX dissolved in Vigamox can reverse dry eye and restore tear production and eye sensitivity in animal models of type 1 and type 2 diabetes within a few hours of a single eye drop.

　　Method: NTX-001 type 1 diabetic dry eye effect study

　　Induction of animals and type 1 diabetes (T1d): Male SD rats, weighing about 160g. Male rats were selected for this experiment in order to maintain consistency with previously published studies on NTX treatment of diabetes complications. 40 mg/kg streptozotocin (ph 4.5) was injected intraperitoneally for two consecutive days to increase the blood sugar of the animal. Blood glucose levels> 250 mg/dl indicate hyperglycemia and type 1 diabetes (T1d). Non-diabetic rats were regarded as normal control group and were injected with sterile saline. Animals were used after 5 weeks of blood glucose increase. The selection criteria for T1d rats are: (i) weight loss of at least 10% compared with non-diabetic male rats, (ii) blood glucose level> 250 mg/dl, and (iii) normal rats with a baseline Schirmer value lower than 70% of the average Schirmer score.

　　Treatment: The t1d rats are randomly divided into treatment groups, twice a day (~ 08:30 and ~ 16:00), only the right eye drops (~ 0.02 ml/eye drops) (i) 20μg/ml NTX (NTX-001), (ii) Buffer/Excipient, or (iii) 5×10? 5 M NTX dissolved in Vigamox. The control group included: (i) the left eye of T1d rats was untreated; (i i) the right or left eye of T1d rats that received only excipients; and (i i i) the right or left eye of normal animals. The daily treatment lasts for 10 days and is completed within 8 weeks after the first injection of STZ.

　　Measurement of tear volume: use Schirmer strip to measure tear volume. Use a sterile single-edged blade to pre-cut the standard Schirmer tape to a length of 1 mm×17 mm, and measure it according to published procedures. Insert the strip for 1 min. Use a fine ruler with a calibration unit of millimeters to measure the wetted length of each strip to the nearest half a millimeter. After treatment (2-4 h), the Schirmer 1 test scores of T1d rats treated with NTX-001, vehicle or Vigamox® were collected. Additional measurements were performed on rats treated with NTX-001 or buffer at 96 h and 7 d. After stopping the treatment, the amount of tears in the rats was measured every day to determine when the dry eyes recovered.

　　Measurement of corneal surface sensitivity: Corneal sensitivity is measured with a cochlear cap sensory meter. Four measurements were taken for each animal and the average value was taken; the end point was the blink response. The value (g/mm2) is directly determined from the manufacturer's conversion table. The corneal surface sensitivity was measured at 48 hours and 240 hours (10 days) after treatment in T1d rats and normal animals treated with vehicle or NTX-001.

　　Intraocular pressure: At the end of the study, the intraocular pressure (IOP) of the treatment group of diabetic rats was measured. Briefly, the intraocular pressure was measured in unanaesthetized rats. The average intraocular pressure is obtained from 4 readings per eye.

　　The safety study of NTX-001 on normal rats and rabbits: rat safety study: 10 male SD rats (126-150g). The treatment program started after 7 days, including each rat receiving 4 drops (0.02 ml per drop) of the proprietary NTX-001 preparation (20μg/ml) per day, 2 drops to the right eye at 8:30, and at 16:30 2 drops of medicine. Treatment was performed twice a day for 30 days. After the animals were euthanized (>150 mg/kg sodium pentobarbital), the eyes were supported and fixed with Davidson's modified fixative. The tissue technician embeds the eyes with paraffin wax (pupil viewing direction), slices them and stains them with hematoxylin and eosin; the slides are labeled in a concealed manner and evaluated by a veterinary ophthalmologist. Check each part with an optical microscope to see if there are any adverse reactions caused by taking the test agent. Comparable anatomical structures of each section include: optic nerve, choroid, retinal pigment epithelium, neural retina, ciliary body, lens, cornea, anterior chamber and posterior segment, and extraocular structures.

　　Rabbit safety study: 10 male 2kg New Zealand white rabbits. Five rabbits took 4 drops of NTX-001 formula daily for 30 days. Right eye 08:30 2 drops (0.05 ml per drop), 16:30 2 drops. Five rabbits received 2 drops of drug in their right eyes twice a day, and the left eyes of both groups received no treatment. After 30 days, the rabbits were euthanized and injected intramuscularly with 30–40 mg/kg ketamine and 3–5 mg/kg xylazine for sedation, followed by intravenous injection of pentobarbital (>150 mg/kg) in the ear. The ocular tissues were fixed with Davidson's modified fixative containing formalin. The fixed sections were stained and observed by a pathologist.

　　Result: Clinical characteristics of T1d rats: Hyperglycemia is determined by body weight and blood glucose level. The average weight of rats when STZ was injected was 165±2 g. At the beginning of the study, normal rats weighed 386±21 g, while T1d rats weighed 324±7 g. At the beginning of eye drops, the blood glucose measurement value of T1D rats was 530±24 mg/dl, and that of normal animals was 119±6 mg/dl. The baseline Schirmer test showed that the right and left eyes of T1d rats were comparable to the measured value of 2.5 ± 0.1 mm; the right and left eyes of normal rats were comparable to the measured value of 6.7 ± 1.3 mm. The corneal sensitivity of T1d rats is 0.95 ± 0.06 g/mm2, and that of normal rats is 0.47 ± 0.05 g/mm2. At p<0.0001, all comparisons between T1d and the baseline of normal rats are significantly different. 21 T1d rats met the criteria for dry eye, 10 received ntx-001 treatment, 8 received vigamox+ntx treatment, 3 T1d rats received vehicle treatment only; 5 rats were non-diabetic rats, Normal (n). In this study, normal rats have lower tear volume, which is probably related to the low humidity in the animal facility during the experiment period from May to July; however, the tear volume of normal rats is about 2.5 times that of T1d.

　　NTX-001 can restore low tears in T1D rats: within 4 hours after a drop of NTX-001, compared with the baseline tear volume, the amount of tears in the right eye of T1d rats treated with NTX-001 or Vigamox increased; No effect on tear volume. The tear production in the right eye of T1d rats treated with ntx-001 or vigamox was different from the corresponding untreated eyes. After 96 hours of treatment (8 drops), t1d rats treated with vigamox-ntx (3.6±0.3 mm) or ntx-001 (4.3±0.4 mm) had a significant tear volume compared to t1d baseline rats (2.6±0.1 mm) Increased; the tear volume of t1d rats treated with ntx-001 was also restored relative to the excipient (2.7±0.1) treatment.

　　Corneal sensitivity recovered after NTX treatment: The baseline value of corneal sensitivity was measured with a cochlear-cap sensor. The results showed that the right and left eyes of T1D rats were comparable. The corneal surface sensitivity was measured after 4 drops of NTX-001 (48 hours after initial treatment) and 10 days later. After 4 drops of NTX-001 treatment, the sensitivity of the right eye cornea of the t1d rat returned to normal. Compared with the baseline of the T1d and T1d rats that received the excipient, the measured value was significantly different. During the 10-day treatment, the NTX-001-treated right eye still had comparable corneal surface sensitivity relative to non-diabetic normal rats. Therefore, it was necessary to significantly reduce the force measurement value to cause a T1d value relative to the baseline or receiving buffer. Response of T1d rats.

　　IOP after NTX-001 treatment: After 10 days of treatment, the IOP value of NTX-001 treated eyes (18.5 ±1.9 mmHg) is equivalent to that of normal rats (17.2 1.6 mmHg); untreated T1D The intraocular pressure of the eye was 10.9±0.7 mmHg. After 10 days, the topical administration of NTX-001 ended, and the modified Schirmer 1 test was performed on the subgroup of rats receiving NTX-001 daily to determine when the positive effect of restorative tears disappeared. Using a score of 3.0 or less indicates that the "dry eye" has recovered 24 hours after the end of treatment, and only 1 out of 8 rats has "dry eye". At 48, 72, and 96 h, in the remaining 7 rats treated with NTX-001, 50%, 20%, and 75% of the rats respectively detected a low amount of tears indicating the recovery of dry eyes.

　　Rat safety study: Rat pathology, no signs of inflammation, tumor or degenerative changes in the eyes were found. There are slight changes in three eyes. A small amount of amorphous eosinophil fragments were seen in the anterior chamber of the right eye of two rats and the left eye of one rat, and a small retinal (outer nuclear layer) wreath was seen in one eye. All of these are considered developmental injuries with no clinical consequences. During the second use every day (7-8 hours later), no signs of pain (such as screaming, crying, tearing, or vocalization) immediately after use were observed, and no redness or crusting of the eyes after treatment was observed. At the end of the 30-day study, the rats’ eyes looked clear (no cataracts) and there was no excessive tearing.

　　Rabbit safety study: In general, the optic nerve examination is insufficient. The filtering angle is visible in the normal anatomical direction. The lens is present, but sometimes fragments in the slice. Single cell types (including retinal pigment epithelium) with different structures, nucleus and cytoplasm can be seen. The pathology report showed no evidence of tumors or degenerative changes in any eyes. The conjunctiva of both eyes had mild or mild inflammatory changes, and neither received treatment. During the one-month treatment, the rabbits found no eye abnormalities, no signs of redness, swelling, irritation, excessive tearing or dryness. After eye drops or administration, the rabbit showed no signs of pain or distress. At the end of the study, all rabbits were healthy and had normal vision.

　　Conclusion: This study reports a new GLP compatible NTX external preparation, which has a significant effect on reversing dry eyes and restoring corneal surface sensitivity in diabetic rats. With the preparation for local treatment for 30 days, twice a day, gross and histopathological studies were conducted on the eyes of young rats and rabbits, and no abnormalities were found. In short, this new formulation is effective and safe in preclinical research.