Pharmacology Of Ocular Complications

糖尿病是一个日益严重的公共卫生问题，与糖尿病相关的神经病变、肾病、微血管病变、视网膜病变和白内障等长期并发症导致视力丧失、生活质量下降、肢体和运动功能丧失以及死亡率增加。眼部糖尿病并发症包括白内障、角膜病变和视网膜病变。在工业化国家，视网膜病变导致的失明是成年人失明的主要原因，糖尿病患者比非糖尿病患者更常接受白内障手术。过去30年进行的研究已经在细胞内山梨醇水平的过量积累与糖尿病并发症的发生之间建立了明确的联系。山梨醇是一种由葡萄糖通过醛糖还原酶（AR）形成的糖醇。我们发现哺乳动物组织中含有一种内禀醛糖还原酶抑制剂（IARI）。IARI的发现意义重大，因为它代表了一类新的无毒（或毒性显著降低）抑制剂。研究表明，IARI是一种热稳定的化合物，对醛糖还原酶的抑制作用明显在微摩尔到纳摩尔范围内。它的分子量似乎小于1000，证据表明它是一个小多肽。部分纯化的IARI体外晶状体培养研究表明，该化合物可以跨膜抑制晶状体内山梨醇的产生。初步的大鼠研究也表明这种IARI在体内是活跃的。将含有牛晶状体IARI的部分纯化提取物腹腔注射半乳糖喂养24小时的大鼠，半乳糖醇的形成在注射0.3 mL提取物的3只大鼠中被抑制了94%，在注射0.1 mL提取物的大鼠中被抑制了54%。估计不到0.1%的材料代表紧密结合抑制剂，注射水平低于6微克/千克。醛糖还原酶引发糖尿病并发症的病理生理机制也在研究中。由于缺乏犬多元醇途径酶的序列信息用于基础分子生物学研究，因此对犬醛糖还原酶、醛还原酶和山梨醇脱氢酶进行了克隆和测序。此外，为了开发抑制醛糖还原酶的新方法，设计和筛选了一些抑制人和大鼠AR基因在细胞培养中的表达的反义寡聚物。磁共振成像研究也正在进行，以确定醛糖还原酶是否会引发人类糖性白内障。使用非侵入性的磁化转移对比（MTC）增强磁共振成像（MRI）工具，我们获得了半乳糖喂食狗眼睛的高对比度图像，表明在白内障形成过程中，这些狗的晶体中发生了与醛糖还原酶相关的渗透变化。这项技术现在正应用于临床环境，使用年龄从28岁到72岁的正常志愿者。初步研究表明，MTC-MRI所获得的高对比度图像是临床研究白内障病变的良好工具。磁共振研究也被应用于非侵入性研究眼部代谢。由于药物的药代动力学性质的变化和血眼屏障降解的变化，通常很难确定适当的眼内药物水平，如醛糖还原酶抑制剂，以充分抑制眼组织中醛糖还原酶的活性。利用局部磁共振波谱（MRS-SLOOP），我们开发了目前的方法，通过非侵入性测量眼内多元醇途径活性，在玻璃体内注射3-氟-3-脱氧-d -葡萄糖（3FDG）后，确定晶状体中醛糖还原酶活性的充分抑制。对新西兰大白兔进行麻醉下局部MRS，通过测定眼内3FDG生成的3-氟-3-脱氧-d -山梨醇（3FS）和3-氟-3-脱氧-d -果糖（3FF）代谢物水平来评估多元醇通路活性。MRS能够跟踪3FDG从玻璃体到眼睛前段的损失，特别是进入晶状体和水晶状体。MRS观察到3FDG的主要代谢是醛糖还原酶在晶状体中催化形成3FS。3FS的产生在时间上呈线性，并随着口服醛糖还原酶抑制剂而降低。因此，局部MRS似乎是一种潜在的强大技术，可以通过眼睛和其他组织的代谢途径无创地研究通量。

Diabetes is a rapidly increasing public health problem and the long-term complications such as neuropathy, nephropathy, microangiopathy, retinopathy and cataract associated with diabetes result in loss of vision, decreased quality of life, loss of limbs and motor function and increased mortality. Ocular diabetic complications include cataract, keratopathy and retinopathy. Blindness due to retinopathy is the leading cause of blindness in of adults industrialized countries, and diabetics undergo cataract surgery more often than non-diabetics. Studies conducted over the last 30 years have established a clear link between the excess accumulation of intracellular sorbitol levels and the onset of diabetic complications. Sorbitol is a sugar alcohol formed from glucose by the enzyme aldose reductase (AR). We have discovered that mammalian tissues contain an intrinsic aldose reductase inhibitor (IARI). The discovery of an IARI is significant because this represents a new class of nontoxic (or significantly less toxic) inhibitor. Studies indicate that the IARI is a heat-stable compound which inhibits aldose reductase in apparently the pico- to nanomolar range. Its molecular weight appears to be less than 1,000 and evidence suggests that it is a small polypeptide. In vitro lens culture studies with partially purified IARI indicate that this compound can cross membranes to inhibit the intralenticular production of sorbitol. Preliminary rat studies also indicate that this IARI is active in vivo. When the partially purified extract containing the IARI from bovine lenses was injected intraperitoneally into 24 hr galactose-fed rats, galactitol formation was inhibited by 94 % in the three rats receiving ca. 0.3 mL injection of extract and 54% in the rat receiving ca. 0.1 mL injection. Estimating that less than 0.1% of the material represents the tight-binding inhibitor, the injected level was under 6 microg/kg. Studies are also being conducted on the pathophysiological mechanism of how aldose reductase initiates diabetic complications. Since no sequence information on the enzymes of the polyol pathway in the dog is available for basic molecular biological studies, canine aldose reductase, aldehyde reductase and sorbitol dehydrogenase have been cloned and sequenced. In addition, to develop new methods of inhibition of aldose reductase, a number of antisense oligomers to suppress human and rat AR gene expression in cell cultures have been designed and screened. Magnetic resonance imaging studies are also being conducted to determine if aldose reductase initiates human sugar cataracts. Using the noninvasive tool of magnetization transfer contrast (MTC) enhanced magnetic resonance imaging (MRI), we have obtained high contrast images of the eyes of galactose-fed dogs that indicate that osmotic changes linked to aldose reductase occur in the lenses of these dogs during cataract formation. This technique is now being applied to a clinical setting using normal volunteers ranging from 28 to 72 years in age. Preliminary studies indicate that the high contrast images obtained by MTC-MRI are a good tool for clinically investigating cataractous changes. Magnetic resonance studies are also being applied to non-invasively investigate metabolism in the eye. Due to variations in the pharmacokinetic properties of drugs and variations in the degradation of the blood-ocular barrier, it is often difficult to determine the proper intraocular levels of drugs such as aldose reductase inhibitor required for adequate inhibition of aldose reductase activity in ocular tissues. Utilizing localized magnetic resonance spectroscopy (MRS-SLOOP) we have developed the present method for determining adequate inhibition of aldose reductase activity in the lens by noninvasively measuring polyol pathway activity in the eye Following intravitreal injection of 3-fluoro-3-deoxy-D-glucose (3FDG). to New Zealand White rabbits, under anesthesia localized MRS was used to assess polyol pathway activity by determining the levels of 3-fluoro-3-deoxy-D-sorbitol (3FS) and 3-fluoro-3-deoxy-D-fructose (3FF) metabolite formation from 3FDG in the eye. MRS was able to follow the loss of 3FDG from the vitreous into the anterior segment of the eye and particularly into the lens and aqueous. The primary metabolism of 3FDG observed by MRS was the formation of 3FS in the lens that is catalyzed by aldose reductase. Production of 3FS was linear in time and decreased with the oral administration of an aldose reductase inhibitor. Therefore, it appears that localized MRS is a potentially powerful technique for non-invasively investigating flux through metabolic pathways in the eye and other tissues.