Salivary Gland Secretion Mechanisms During Normal And Altered Functional States

正常和改变功能状态下的唾液腺分泌机制

• 批准号: 8148612
• 负责人: BRUCE J BAUM
• 金额: $ 201.25万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148612
• 关键词: Salivary Glands; Salivary Glands

Saliva maintains oral health. Building on our past studies of saliva formation and its alteration during pathology, we previously developed novel approaches to treat salivary dysfunction using principles of gene therapy, as well as strategies to use normal salivary glands as a gene transfer target site for treating systemic single protein deficiency disorders (SSPDDs). The treatment of most head and neck cancer patients includes irradiation (IR). Salivary glands in the IR field suffer irreversible damage. Much of our effort this past year focused on a clinical gene therapy trial to restore salivary function to existing IR-damaged glands. Based on previous studies with a recombinant serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (hAQP1), AdhAQP1, conducted in rats and miniature pigs, and extensive safety studies of the vector, we received an IND (BB-IND 13102) and approval to conduct a phase I clinical trial testing this vector in patients. The protocol for the trial, Open-label, dose-escalation study evaluating the safety of a single administration of an adenoviral vector encoding human aquaporin-1 to one parotid salivary gland in individuals with irradiation-induced parotid salivary hypofunction, received all required approvals and we began treating patients in 2008. We have completed the first two dose cohorts (4.8x10e7 and 2.9x10e8 vector genomes, vg, to a single parotid gland; 3 patients/dose cohort), and treated one patient in the third dose cohort (1.3x10e9 vg/gland). All patients tolerated the vector and the related study procedures well. The first patient in the third dose cohort, however, was the only patient thus far to show an inflammatory response to vector delivery, i.e., developed a mild parotitis that resolved without medical intervention. This observation may indicate that we have approached an Ad5 vector dose to the parotid at which benefits are outweighed by side effects. A practical concern with using AdhAQP1 is that a conventional Ad5 vector unlikely will direct hAQP1 expression for more than 2-4 weeks. To develop a long-term strategy for hAQP1 gene transfer, we previously began to test the use of serotype 2, adeno-associated viral (AAV2) vectors in rodents, macaques and miniature pigs. This year we completed a study in irradiated miniature pigs using the AAV2hAQP1 vector. The study goal was to see if AAV2-mediated hAQP1 gene transfer would extend the restored salivary flow in these animals beyond that seen with AdhAQP1. Sixteen weeks after IR (20 Gy) salivary flow was decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in parotid salivary flow to 35-40% of pre-IR levels after 8 weeks. Administration of either a control AAV2 vector or saline was without benefit, and led to further decreases in salivary flow. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. The findings suggest that delivery of AAV2hAQP1 to IR-damaged parotid glands may be useful in providing extended restoration of saliva output in previously irradiated patients with salivary hypofunction. We also evaluated the effect of IR on microvascular endothelial (MVE) cells in miniature pig parotid glands. Previously we showed in mice that damage to MVE cells in salivary glands occurs quite early after single dose IR. Similarly, after a single IR dose (25Gy) to parotid glands of 6 miniature pigs, local parotid gland blood flow rate decreased rapidly and remained below control levels throughout the 14 day study. Parotid microvascular density declined from 4-24 hours, and remained below control levels thereafter. The activity of both acid and neutral sphingomyelinase in parotid glands increased rapidly 4-24 hours post-IR, and then declined gradually. Also, the frequency of detecting apoptotic endothelial cell nuclei in glands followed comparable kinetics. Thus, similar to mice, miniature pigs also show marked damage to salivary gland MVE cells soon after IR. As described in many past annual reports, we have shown in multiple animal models (mice, rats, miniature pigs and rhesus macaques) that salivary glands are a potentially useful gene transfer target site for treating certain SSPDDs. This year we have extended these studies with two quite novel potential clinical applications. The first involves hypoparathyroidism. Two days after delivering an Ad5 vector encoding human parathyroid hormone, Ad.hPTH, to rat parotid glands, most secreted transgenic hPTH was detected in serum. Apparently, no vector escaped from the target tissue into the circulation, as QPCR biodistribution assays revealed vector copies only in parotid glands and not in liver or spleen. To show potential clinical applicability, Ad.hPTH (10e11 vp/gland) was administered to the parotid glands of parathyroidectomized (PTX) rats. Two days after transduction, high levels of hPTH (39.9 +/- 12.6 pg/ml) were found in serum, but no hPTH was detectable in saliva. Importantly, Ad.hPTH treatment led to normalization of serum calcium levels in the PTX rats and a significant increase in their urinary phosphorus/creatinine ratio, providing clear evidence of PTH action due to the parotid gland-produced transgenic hPTH. The second application studied involves diabetes mellitus. Glucagon-like peptide 1 (GLP-1) is a 37 amino acid peptide that is released into the circulation after a meal and acts as an incretin. Its biological half-life is quite short, 2-3 min, and it is inactivated by dipeptidyl-aminopeptidase IV (DPP IV) in the blood. An Ad5 vector encoding GLP-1 (Ad-GLP-1) was generated and initially shown able to direct biologically active GLP-1 production in vitro. The transgenic GLP-1 also was more resistant to degradation by DPP IV, due to an engineered Ala to Gly substitution at position 8. In vivo studies demonstrated that mice given the Ad-GLP-1 vector in their submandibular glands had serum levels of GLP-1 three-fold higher than those of mice transduced with a control Ad5 vector. In healthy fasted animals, serum glucose levels were similar between mice treated with Ad-GLP-1 and mice treated with a control vector. However, when challenged with a glucose tolerance test, Ad-GLP-1 treated healthy mice lowered their serum glucose levels significantly faster than the control mice. Next, we used a mouse model of drug-induced diabetes mellitus, administering alloxan, which destroys pancreatic beta cells. We found that the progression of hyperglycemia was significantly slowed in mice pre-treated with Ad-GLP-1 in their submandibular glands compared to mice pre-treated with the control vector.

唾液保持口腔健康。在我们过去对唾液形成及其在病理过程中的改变的基础上，我们先前开发了使用基因疗法原理治疗唾液功能障碍的新方法，以及使用正常唾液腺作为治疗系统性单蛋白缺乏症（SSPDDS）的基因转移靶位点的策略。 大多数头颈癌患者的治疗包括辐射（IR）。红外领域的唾液腺遭受不可逆转的损害。过去一年，我们的大部分努力都集中在一项临床基因治疗试验上，以恢复现有的IR受损腺体唾液功能。根据先前对编码人类通道1（HAQP1），ADHAQP1的重组血清型5腺病毒（AD5）载体（ADHAQP1）进行的研究，在大鼠和微型猪中进行了研究，以及对载体的广泛安全研究，我们接受了IND IND（BB-IND 13102）并批准了该阶段I临床试验训练患者。该试验的协议，开放标签的剂量降低研究研究评估了腺病毒载体的单个给药的安全性，该腺病毒载体编码人类水通道蛋白1至一个呈腮腺唾液唾液腺，辐射引起的呈辐射性唾液性唾液功能低下的人，并在2008年获得了所有需要治疗的患者。载体基因组，VG，至一个圆锥体腺体，并在第三剂量同类中治疗了一名患者（1.3x10E9 VG/腺体）。所有患者都很好地容忍了载体和相关的研究程序。然而，第三剂量队列中的第一位患者是迄今为止唯一显示出对载体递送反应的炎症反应的患者，即出现了一种轻度的耳炎，无需医疗干预而解决。该观察结果可能表明，我们已经将AD5矢量剂量剂量达到了腮腺，在腮腺上，副作用超过了益处。 使用ADHAQP1的一个实际问题是，常规的AD5载体不太可能将HAQP1表达在超过2-4周中。为了制定HAQP1基因转移的长期策略，我们先前开始测试啮齿动物，猕猴和微型猪中血清型2，与腺相关病毒（AAV2）载体的使用。今年，我们使用AAV2HAQP1载体完成了一项关于辐照微型猪的研究。研究目标是查看AAV2介导的HAQP1基因转移是否会扩大这些动物中恢复的唾液流量，而不是ADHAQP1所看到的动物。 IR（20 Gy）唾液流量后十六周减少了85-90％。 AAV2HAQP1在第17周的施用仅转导导管细胞，并导致腮腺唾液流量的剂量依赖性增加到8周后IIR水平的35-40％。对照AAV2载体或生理盐水的给药是没有益处的，并导致唾液流动进一步下降。 AAV2HAQP1递送后，在临床化学和血液学值中几乎没有观察到的变化。研究结果表明，将AAV2HAQP1递送到IR损坏的腮腺可能有助于在先前受辐照的唾液功能障碍患者中扩展唾液输出的恢复。 我们还评估了IR对微血管内皮细胞（MVE）细胞在微型猪腮腺中的影响。以前，我们在小鼠中表明，单剂量IR后很早就发生了对唾液腺中MVE细胞的损害。同样，在单个IR剂量（25GY）对6个微型猪的腮腺腺体剂量（25Gy）之后，在整个14天的研究中，局部腮腺血液流量迅速降低，并保持在控制水平以下。腮腺微血管密度从4-24小时下降，此后保持在控制水平以下。 IR后IR后4-24小时迅速增加，酸和中性鞘磷脂酶的活性迅速增加，然后逐渐下降。同样，在腺体中检测凋亡的内皮细胞核的频率遵循可比的动力学。因此，与小鼠相似，微型猪在IR后不久也显示出对唾液腺MVE细胞的明显损害。 如过去的许多年度报告中所述，我们在多种动物模型（小鼠，大鼠，微型猪和恒河猴）中表明，唾液腺是一种潜在有用的基因转移靶位点，用于治疗某些SSPDD。今年，我们通过两个非常新颖的潜在临床应用扩展了这些研究。 第一个涉及甲状腺功能减退症。在将编码人甲状旁腺激素的AD5载体AD.HPTH传递给大鼠腮腺后两天，在血清中检测到了大多数分泌的转基因HPTH。显然，由于QPCR生物分布测定法显示仅在腮腺中而不是肝脏或脾脏中，因此没有载体从目标组织逃脱到循环中。为了显示潜在的临床适用性，将AD.HPTH（10E11 VP/腺体）施用到甲状旁腺（PTX）大鼠的腮腺。转导后两天，在血清中发现了高水平的HPTH（39.9 +/- 12.6 pg/ml），但在唾液中未检测到HPTH。重要的是，AD.HPTH的治疗导致PTX大鼠血清钙水平的标准化，并显着增加其尿磷/肌酐比率，从而提供了由于腮腺产生的转基因HPTH而引起的PTH作用的明确证据。 研究的第二个应用涉及糖尿病。胰高血糖素样肽1（GLP-1）是37个氨基酸肽，在进餐后将其释放到循环中，并用作肠降低。它的生物半衰期很短2-3分钟，并且在血液中被二肽基 - 氨基肽酶IV（DPP IV）灭活。生成了编码GLP-1（AD-GLP-1）的AD5矢量，并最初显示能够在体外引导生物活性的GLP-1产生。转基因GLP-1也对DPP IV的降解更具抵抗力，因为在8位置进行了工程化的ALA替换为Gly取代。在体内研究表明，在其下颌下腺中给予AD-GLP-1载体的小鼠具有血清的GLP-1三倍高于与对照AD5 vector一起转导的小鼠的血清三倍。在健康禁食的动物中，用AD-GLP-1和用对照载体处理的小鼠治疗的小鼠之间的血清葡萄糖水平相似。但是，当通过葡萄糖耐受性测试挑战时，AD-GLP-1治疗的健康小鼠降低了其血清葡萄糖水平的速度明显快于对照小鼠。接下来，我们使用了药物诱导的糖尿病的小鼠模型，施用了甲状腺素，从而破坏了胰腺β细胞。我们发现，与用对照载体预先治疗的小鼠相比，在其下颌腺体中预先治疗的AD-GLP-1预先治疗的小鼠中，高血糖的进展显着减慢。