Point of care genotyping assays, and algorithm for warfarin dosing

描述（由申请人提供）：2007年8月16日，FDA更新了华法林的标签，包括“注意事项”部分的信息，以提醒医生CYP2C9和vkorc1基因变异的人可能需要较低的初始剂量（见http://www.fda.gov/cder/drug/infopage/warfarin/default.htm）。不幸的是，FDA并没有就如何使用这种基因分型信息向医生提供具体的指导。在华法林改变标签的消息公布后不久，FDA批准了一项基因分型测试，用于检测影响患者对华法林反应的最常见突变（即Nanosphere的Verigene华法林测试FDA 510(k) # K070804）。不幸的是，这种新的诊断设备不包括如何使用遗传信息来开华法林的说明。此外，清除华法林试验需要大量高度纯化的DNA (25g)，并使用昂贵的仪器（因此需要高测试量来证明试剂租赁是合理的）。由于FDA尚未批准将核酸提取方法用于Verigene华法林检测，因此只有拥有符合美国病理学家学院制定的实验室认可计划分子病理学检查表的实验室的大型诊所才会采用Verigene华法林检测。我们认为缺乏如何使用基因分型数据的信息，以及执行基因测试所需的DNA提取的复杂性将限制其效用。我们建议开发一种低成本、无仪器的基因分型系统，该系统不需要大量广泛纯化的DNA，并且包括一个基于网络的计算工具，该工具集成了遗传和物理因素，以提供华法林剂量的个性化预测模型。基因检测将使用BioHelix专有的解旋酶依赖性扩增（HDA），以及一次性横向流动装置，旨在防止扩增子污染实验室。HDA试剂将配制成“Ready-to-go”IsoAmp干试剂配方，在室温下具有扩展的稳定性。我们发现，口腔粗拭子可用于HDA基因分型检测；因此不需要提取DNA。由于横向流动试验广泛用于“CLIA豁免”和“中等复杂性”的测定，我们相信凝血会更喜欢这种检测形式，而不是使用复杂的仪器。在I期，我们将使用ARMS技术开发3种CYP2C9*2（又称3608 C b> T或R149C）、CYP2C9*3（又称42614 A>C或I359L）和1639g >AVKORC1基因座的基因分型分析。我们还将对我们的产品开发过程实施设计控制，并为BioHelix获得ISO 13485认证。Trimgen （Sparks， MD）将根据GMP生产BuccalQuick DNA提取试剂盒。BioHelix将生产HDA所需的大量酶。GE医疗将根据GMP生产现成的IsoAmp华法林干试剂。该扩增子密封、侧流装置将由喜美佳按照GMP生产。最后，我们将在PGX实验室使用50个样本验证3种华法林检测方法，并将Ready-to-go“IsoAmp华法林检测方法与PGX实验室验证的DNA测序和华法林基因分型检测方法的性能进行比较。在II期，我们进行了一项回顾性的多地点临床研究，旨在验证使用Verigene华法林试验作为预测设备的分析，以及PGXL技术的算法，约500名患者连续3个月服用香豆素。这项研究将包括华法林给药算法。我们相信我们可以为华法林提供“中等程度的复杂性”的基因分型测试，并提供一个基于网络的计算工具的链接，用于华法林剂量的个性化预测建模，每个位点的价格在15美元到20美元之间。在心房颤动（AF）患者中，心脏上两个腔室的不均匀和不规律的泵送导致心房中的血液池形成血栓。当这些血块松动，进入血液，并进入大脑堵塞动脉时，患者可能会中风。房颤的治疗通常包括服用抗凝剂，如阿司匹林（低风险病例）或华法林（更严重的病例）。患有心房颤动的女性比男性更容易形成危险的血凝块（Fang et al. 2005），因此可能从改善华法林剂量中获益。华法林通过抑制维生素K环氧化物还原酶（VKOR）发挥其抗凝作用，VKOR会耗尽可用的维生素K还原库，并阻止维生素K依赖性凝血因子的激活，最终阻止凝血酶的形成。VKORC1 （VKOR的一个组成部分）的遗传多态性影响患者对华法林的敏感程度。患者通常有低剂量单倍型组(a)，高剂量单倍型组(B)，或者是杂合的。肝细胞色素P450 2C9 （CYP2C9）酶代谢华法林清除血流中的华法林，因此影响酶活性的CYP2C9基因多态性将影响血流中华法林的稳态水平。年龄、性别、身高和体重等因素也会影响华法林所需剂量的范围。毫不奇怪，在华法林治疗期间，药物不良事件（ADEs）很常见。据估计，超过50%的可以从华法林获益的患者没有服用这种药物，因为他们的医生对潜在的ADE感到厌倦。最近发表的一项研究发现，将基因检测纳入华法林治疗方案可以帮助患者每年避免8.5万例严重出血事件和1.7万例中风。由于ade的风险，华法林治疗通常通过定期测定凝血酶原时间（PT）来监测，使用国际标准化比例（INR）作为标准化PT测量的手段。然而，华法林的抗血栓作用，以及伴随的INR可解释的变化，在2-4天的过程中，直到因子II（凝血酶原）和因子X耗尽后才变得明显。这导致INR波动，如果初始剂量计算错误，可导致严重的ade。开发一种易于使用的计算工具，将所有相关的遗传和物理因素整合到华法林剂量的全面、实时、个性化预测模型中，可以将药物遗传学测试的结果转化为可操作的临床应用。然而，FDA法规要求这种算法必须通过基因测试进行验证，以便在基因测试系统附带的包装说明书中列出。一个理想的华法林剂量多态性分型的基因检测系统应该易于操作，并且不需要大量的仪器。公共卫生相关性：在本提案中，我们概述了开发一种低成本的无仪器基因分型系统的计划，该系统不需要大量广泛纯化的DNA，并且可以在医生办公室进行。这种新型诊断系统将通过一个易于使用的基于网络的华法林剂量计算工具进行验证，该工具整合了所有遗传和物理因素，以预测患者血液中的华法林水平，并预测患者接受目标剂量华法林时INR的变化。

DESCRIPTION (provided by applicant): On August 16, 2007, the FDA updated the label for warfarin to include information in the "precautions" section to remind physicians that people with variations in the CYP2C9 and VKORC1genes may require a lower initial dose of the drug (see http://www.fda.gov/cder/drug/infopage/warfarin/default.htm). Unfortunately, the FDA did not provide specific guidance to physicians on how to use this genotyping information. Soon after the announcement for an altered label for warfarin, the FDA cleared a genotyping test for the most common mutations influencing patients' responses to warfarin (i.e., Nanosphere's Verigene warfarin test FDA 510(k) # K070804). Unfortunately, this new diagnostic device did not include instructions on how to use the genetic information to prescribe warfarin. In addition, the cleared warfarin test requires large quantities of highly purified DNA (2 5g), and uses costly instrumentation (such that high test volumes will be required to justify the reagent lease). As FDA has not yet cleared a nucleic acid extraction method for use with the Verigene warfarin test, only large clinics with laboratories capable of complying with the Laboratory Accreditation Program Molecular Pathology Checklist, developed by the College of American Pathologists, will adopt the Verigene warfarin test. We believe the lack of information of how to use the genotyping data, and the complexity of the DNA extraction required to perform the genetic test will limit its utility. We propose to develop a low-cost, instrument free genotyping system that does not require large quantities of extensively purified DNA, and that includes a web-based computational tool that integrates genetic, and physical factors to offer an individualized predictive model for warfarin dose. The genetic test will use BioHelix's proprietary helicase dependent amplification (HDA), as well as a disposable lateral flow device designed to prevent laboratory contamination with amplicons. The HDA reagents will be formulated as a Ready-to-go" IsoAmp dry reagent formulation with extended stability at room temperature. We have found that crude buccal swabs can be used to perform HDA genotyping tests; therefore no DNA extraction will be required. As lateral flow tests widely used in "CLIA waived" and "moderate complexity" assays, we believe coagulation will prefer this detection format to using complex instrumentation. In Phase I, we will develop 3 genotyping assays for the CYP2C9*2 (a.k.a. 3,608 C>T or R149C), the CYP2C9*3 (a.k.a. 42,614 A>C or I359L), and the 1,639 G>AVKORC1 loci using ARMS technology. We will also implement design control for our product development process, and secure ISO 13485 certification for BioHelix. Trimgen (Sparks, MD) will manufacture BuccalQuick" DNA extraction kits under GMP. BioHelix will manufacture bulk enzymes required for HDA. GE Healthcare will manufacture the Ready-to-go" IsoAmp warfarin dry reagents under GMP. The amplicon containment, lateral flow device will be manufactured under GMP by Ximedica. Finally, we will validate the 3 warfarin assays at PGX laboratories using 50 samples and compare the performance of the Ready-to-go" IsoAmp warfarin assays to DNA sequencing and warfarin genotyping assays validated at PGX laboratories. In Phase II, we perform a retrospective, multi-site clinical study aimed at validating the assays using the Verigene warfarin test as a predicate device, and PGXL technology's algorithm with ~500 patients on consistent dosing of Coumadin for 3 months. This study will include a warfarin dosing algorithm. We believe we can offer genetic typing test for warfarin with a "moderate degree of complexity" with a link to a web-based computational tool for individualized predictive modeling of warfarin dosing for between $15 and $20/locus. Narrative In patients with atrial fibrillation (AF), the uneven and arrhythmic pumping of the heart's two upper chambers results in pools of blood in the atrium that can form clots. Patients can experience strokes when these clots break loose, enter the bloodstream, and travel to the brain to plug an artery. Treatment for AF usually consists of taking an anticoagulant like aspirin (for low risk cases), or warfarin (for the more severe cases). Women with atrial fibrillation are more likely to form dangerous blood clots than men (Fang et al. 2005), and thus are likely to benefit from improved warfarin dosing. Warfarin exerts its anticoagulant effect by inhibiting the vitamin K epoxide reductase (VKOR), which depletes the pool of reduced vitamin K available, and prohibits the activation of the vitamin K- dependent clotting factors and, ultimately, thrombin formation. Genetic polymorphism in the VKORC1, a component of VKOR influence the degree of patient sensitivity to warfarin. Patients typically have either a low-dose haplotype group (A), a high-dose haplotype group (B), or are heterozygous. The hepatic Cytochrome P450 2C9 (CYP2C9) enzyme metabolizes warfarin to clear it from the blood stream, therefore polymorphisms in the CYP2C9 gene that influence enzyme activity will influence the steady state level of warfarin in the blood stream. Factors such as age, gender, height, and weight, also impact the range of possible warfarin dose requirements. Not surprisingly, adverse drug events (ADEs) are common during warfarin therapy. It is estimated that over 50% of patients that could benefit from warfarin are not getting the drug because their physician is weary of the potential for ADE. A recently published study found that the incorporation of genetic testing into warfarin protocols could help patients to avoid 85,000 serious bleeding events and 17,000 strokes every year. Because of the risk of ADEs, warfarin therapy is usually monitored by determining the prothrombin time (PT) at regular intervals using the international normalized ration (INR) as a means of standardizing the PT measurement. However, the antithrombotic effects of warfarin, along with the accompanying interpretable changes in INR, do not become apparent until the pools of Factor II (Prothrombin) and Factor X are depleted over the course of 2-4 days. This results in fluctuations in INR that can result in serious ADEs if the initial dose was miscalculated. The development of an easy-to-use computational tool that integrates all of the relevant genetic, and physical factors into a comprehensive, real-time, individualized predictive model for warfarin dose could translate the results of pharmacogenetic testing into an actionable clinical application. However, FDA regulation requires that such an algorithm be validated with a genetic test in order to be listed in the package insert accompanying the genetic test system. An ideal genetic testing system for typing warfarin dosing polymorphisms should be easy to operate, and should not require extensive instrumentation. PUBLIC HEALTH RELEVANCE: In this proposal, we outline a plan to develop a low-cost instrument free genotyping system that does not require large quantities of extensively purified DNA and that could be performed in the doctor's office. This novel diagnostic system will be validated with an easy-to-use, web-based computational tool for warfarin dosage that integrates all genetic and physical factors to project warfarin levels in the patient's blood and predict changes in INR when a target dose of warfarin is administered to the patient.