Point of care genotyping assays, and algorithm for warfarin dosing

护理点基因分型测定和华法林剂量算法

基本信息

批准号：
7537596
负责人：
BERTRAND LEMIEUX
金额：
$ 15.58万
依托单位：
BIOHELIX CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-19 至 2009-03-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7537596
关键词：
Accounting Accreditation Adopted Adverse event Age Algorithms Alleles American Anticoagulants Arrhythmia Arteries Aspirin Atrial Fibrillation Bathing Biological Assay Blood Blood Circulation Blood Clot Blood Coagulation Factor Blood coagulation Brain Buffers Cells Certification Cheek structure Clinic Clinical Coagulation Process Complex Containment Cytochrome P450 DNA DNA Sequence Data Detection Development Device Designs Devices Dose Drug Formulations Drug Prescriptions Drug usage Enzymes Event Factor X Freeze Drying Gender Genes Genetic Genetic Polymorphism Genetic screening method Genotype Guanosine Monophosphate Haplotypes Healthcare Heart Heart Atrium Heating Height Hemorrhage Hepatic Hour Incubated Instruction International Label Laboratories Lateral Link Measurement Methods Monitor Multi-Institutional Clinical Trial Mutation Nanosphere Nucleic Acids Online Systems Package Insert Pathologist Patients Performance Pharmaceutical Preparations Phase Physicians Process Prohibit Prothrombin Prothrombin time assay Public Health Publishing Pump Range Reaction Reagent Regulation Risk Safety Sampling Secure Stream Stroke Swab System Technology Temperature Testing Thrombin Time Translating Travel Tube United States Food and Drug Administration Update Variant Vitamin K Waran Warfarin Water Weight Woman base caN protocol clinical application college computerized tools cost day design dosage enzyme activity experience helicase human CYP2C9 protein improved instrument instrumentation men migration molecular pathology new technology novel diagnostics novel strategies pharmacogenetic testing point of care predictive modeling prescription document prescription procedure prevent programs reduced vitamin K response tool vitamin K epoxide reductase

项目摘要

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.

描述（由申请人提供）：2007年8月16日，FDA更新了Warfarin的标签，以在“预防措施”部分中包括信息，以提醒医生，CYP2C9和VKORC1GENES中有变化的人可能需要较低的初始剂量（请参阅请参见较低的初始剂量） http://www.fda.gov/cder/drug/infopage/warfarin/default.htm）。不幸的是，FDA没有为医生提供有关如何使用此基因分型信息的具体指导。在宣布变化的华法林标签后不久，FDA清除了影响患者对华法林反应的最常见突变的基因分型测试（即纳米圈的Verigene Warfarin Test FDA 510（k）＃K070804）。不幸的是，这种新的诊断设备未包含有关如何使用遗传信息开出华法林的说明。此外，清除的华法林测试需要大量高度纯化的DNA（2 5G），并使用昂贵的仪器（这样需要高测试量来证明试剂租赁合理）。由于FDA尚未清除与Verigene Warfarin检验一起使用的核酸提取方法，只有大型诊所的实验室能够遵守美国病理学家开发的实验室认证计划分子病理学清单，因此将采用Verigene Warfarin检验。我们认为缺乏如何使用基因分型数据的信息，进行基因检测所需的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，该系统不需要大量的广泛纯化的DNA，其中包括基于Web的计算工具，该工具集成了遗传学和物理因素，以提供华法林剂量的个性化预测模型。该基因检测将使用Biohelix的专有解旋酶依赖性扩增（HDA）以及可一次性的侧向流动装置，旨在防止实验室对扩增子的污染。 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第一阶段，我们将为CYP2C9*2（又称3,608 C> T或R149C）开发3种基因分型测定法，CYP2C9*3（又称42,614 A> c或i359L），以及使用IS IS FARGES的1,639 G> AVKORC1的工程。 Biohelix。 Biohelix将生产HDA所需的散装酶。 GE Healthcare将在GMP下制造“ Isoamp Warfarin Dry试剂。在PGX实验室进行了验证。在第二阶段，我们进行了一项回顾性的多站点临床研究，旨在使用Verigene Warfarin检验作为谓词装置来验证测定法，以及PGXL Technology的算法与约500名患者进行了约500名患者，以持续的剂量给药三个月。这项研究将包括华法林剂量算法。我们相信，我们可以为华法林提供具有“适度程度的复杂性”的遗传分型测试，并链接到基于Web的计算工具，以在$ 15到20美元/20美元之间的华法林剂量的个性化预测建模。心房颤动（AF）患者的叙述，心脏的两个上腔室的不均匀和心律不齐会导致心房中的血液池，可以形成凝块。当这些凝块松动，进入血液并前往大脑以堵塞动脉时，患者可以体验中风。 AF的治疗通常包括服用阿司匹林（低风险病例）或华法林（对于更严重的病例）服用抗凝剂。与男性相比，心房颤动的女性更可能形成危险的血块（Fang等，2005），因此可能会从改善的华法林给药中受益。华法林通过抑制维生素K环氧化酶（VKOR）的抗凝作用，从而消耗了可获得还原的维生素K的池，并禁止维生素K-依赖性凝血因子的激活以及最终的凝血酶形成。 VKORC1中的遗传多态性，VKOR的一个成分影响了患者对华法林的敏感程度。患者通常患有低剂量单倍型组（A），高剂量单倍型组（B）或杂合子。肝细胞色素P450 2C9（CYP2C9）酶使华法林从血液中清除它，因此在CYP2C9基因中影响酶活性的多态性将影响血液流中的WARFARIN的稳态水平。年龄，性别，身高和体重等因素也会影响可能的华法林剂量要求的范围。毫不奇怪，在华法林治疗期间，不良药物事件（ADE）很常见。据估计，超过50％的可能从华法林中受益的患者没有服用该药物，因为他们的医生对ADE的潜力感到厌倦。最近发表的一项研究发现，将基因检测纳入华法林方案可以帮助患者避免每年85,000例严重的出血事件和17,000次中风。由于有ADE的风险，通常通过使用国际标准化评分（INR）定期确定凝血酶原时间（PT）来监测华法林治疗，以此作为标准化PT测量的一种手段。然而，华法林的抗肉眼作用，以及随之而来的INR的可解释变化，直到因子II（凝血酶原）和因子X的池在2-4天的过程中耗尽了。这会导致INR的波动，如果初始剂量被误解，可能会导致严重的AD。将所有相关遗传和物理因素整合到华法林剂量的全面，实时，个性化的预测模型中的易于使用的计算工具的开发可以将药物遗传学测试的结果转化为可操作的临床应用。但是，FDA调节要求使用基因检测验证这种算法，以便在伴随基因测试系统的包装插入包中列出。一个理想的基因测试系统用于打字华法林的剂量多态性应该易于操作，并且不需要广泛的仪器。公共卫生相关性：在此提案中，我们概述了一项计划，以开发不需要大量纯净纯化的DNA的低成本仪器基因分型系统，并且可以在医生的办公室进行。这个新颖的诊断系统将通过一种易于使用的，基于网络的剂量的计算工具来验证，该工具将所有遗传和物理因素整合到患者血液中的华法林水平，并预测当对患者进行剂量剂量的华法林目标剂量时，INR的变化。