Herein, we focused on the larvicidal effects and potential mechanisms of 5-ethenyl-2,2'-bithiophene (5 EB), a compound isolated from Echinops ritro L. on Aedes aegypti larvae. Our results show that 5 EB exhibits pronounced larvicidal activity against A. aegypti larvae, with an LC₅₀ = 0.24 mg/L, considerably lesser than that of the traditional insecticide, rotenone. Observations using fluorescence microscopy, electron microscopy, and imaging flow cytometry demonstrated that 5 EB targets the hemocytes of larvae, leading to the disruption of their intracellular membrane systems. This disruption leads to considerable damage to the cellular structure and function, leading to the death of test subjects. Note that additional investigation into the molecular mechanism of 5 EB's action was conducted using transcriptomic analysis. Both GO and KEGG enrichment analyses reported that the differentially expressed genes were predominantly associated with membranes, lysosomes, and catalytic activities. To summarize, this study provides new options for developing new, environmentally friendly, plant-based larvicides for mosquito control.

2024-08-01·Journal of Dairy Science

Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows

Article

作者: Rosa, G ; Galvão, K N ; Bicalho, R C ; Schuenemann, G M ; Figueiredo, C C ; Zas, S Rodriguez ; Chebel, R C ; Gilbert, R O ; Cunha, F ; Bisinotto, R S ; Santos, J E P ; Thatcher, W W ; Seabury, C M ; Merenda, V R ; Pinedo, P ; de Oliveira, E B ; Casaro, S

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in western and southern states of the United States. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest vaginal discharge score observed for each cow was used to allocate them into 1 of 5 possible groups (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus or lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with <50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or nonfetid reddish-brownish mucus, n = 1,643; VD 5 = fetid, watery, and reddish-brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into fever (rectal temperature ≥39.5°C; n = 334) and no fever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD 5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one AI compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. Milk production was 760 kg lower within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish-brownish vaginal discharge, which was independent of fever.

2024-05-01·Journal of Veterinary Pharmacology and Therapeutics

Metronidazole pharmacokinetics in geese (Anser anser domesticus) after intravenous and oral administrations

Article

作者: Hassoun, Georges ; Giorgi, Mario ; Fadel, Charbel ; Poapolathep, Amnart ; Bourdo, Krzysztof ; Łebkowska-Wieruszewska, Beata

AbstractMetronidazole (MTZ) is a 5‐nitroimidazole anti‐bacterial and anti‐protozoal drug. In human and companion animal medicine, MTZ remains widely used due to its effectiveness against anaerobic bacteria and protozoa. In farm animals, however, MTZ is currently prohibited in several countries due to insufficient data on nitroimidazoles. The purpose of this study was to assess its pharmacokinetics (PK) in geese after single intravenous (IV) and oral (PO) administrations. Fifteen‐month old healthy male geese (n = 8) were used. Geese were subjected to a two‐phase, single‐dose (10 mg/kg IV, 50 mg/kg PO), open, longitudinal study design with a two‐week washout period between the IV and PO phases. Blood was drawn from the left wing vein to heparinized tubes at 0, 0.085 (for IV only), 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24, and 48 h. Plasma MTZ concentrations were measured using HPLC coupled to an UV detector, and the data were pharmacokinetically analyzed using PKanalix™ software with a non‐compartmental approach. MTZ was still quantifiable and well above the LLOQ at 24 h after both routes of administration. Following IV administration, terminal elimination half‐life, volume of distribution, and total clearance were 5.47 h, 767 mL/kg, and 96 mL/h/kg, respectively. For the PO route, the bioavailability was high (85%), and the mean peak plasma concentration was 60.27 μg/mL at 1 h. When parameters were normalized for the dose, there were no statistically significant differences for any of the PK parameters between the two routes of administration. The study shows that oral administration of MTZ seems to be promising in geese, although comprehensive research on its pharmacodynamics and multiple‐dose studies are necessary before its adoption in geese can be further considered.

项与 Box 5 相关的新闻（医药）

2024-08-21

·GlobeNewswire-Health Care

Scilex Holding Company Announces the U.S. Patent and Trademark Office Will Be Issuing New ELYXYB® Patent Related to the Treatment of Acute Pain

Scilex today announced that the U.S. Patent and Trademark Office has allowed numerous claims from U.S. patent application no. 17/562,229 and will issue a new patent containing those claims related to the treatment of acute pain (the “Patent”), to Scilex in late 2024, further strengthening the Company’s intellectual property position and coverage for its acute migraine treatment drug product, ELYXYB®, a liquid, micro-encapsulation formulation of celecoxib.ELYXYBTM is a first-line treatment and the only FDA-approved, ready-to-use oral solution for the acute treatment of migraine, with or without aura, in adults.1 The U.S. oral migraine drug market size is estimated to be $1.8 billion in 2022.2ELYXYB®, a rapid onset and ready to use formulation of Celecoxib, delivers a first line non-opioid therapeutic alternative to habit-forming opioids and acetaminophen, the leading cause of acute liver failure in the U.S.3DelveInsight estimates there were approximately 100 million cases of acute pain in the U.S. and that the total acute pain market in the U.S. was approximately $3 billion in 2021.4There is strong evidence for the use of non-steroidal anti-inflammatory drugs (NSAIDs) as a first-line treatment for migraine. ELYXYB® (celecoxib oral solution) is in the same class of agents, is fast acting, and has the potential to have the lowest gastrointestinal (GI) side effects of all NSAIDs.5 PALO ALTO, Calif., Aug. 21, 2024 (GLOBE NEWSWIRE) -- Scilex Holding Company (Nasdaq: SCLX, “Scilex” or “Company”), an innovative revenue-generating company focused on acquiring, developing and commercializing non-opioid pain management products for the treatment of acute and chronic pain, today announced that the U.S. Patent and Trademark Office has allowed numerous claims from U.S. patent application no. 17/562,229 and will issue a new patent containing those claims related to the treatment of acute pain (the “Patent”), to Scilex in late 2024, further strengthening the Company’s intellectual property position and coverage for its acute migraine treatment drug product, ELYXYB®, a liquid, micro-encapsulation formulation of celecoxib. The Patent, titled “Methods of Treating Pain,” covers methods of treating acute pain. Clinicians in a recent market research study expressed their desire for fast and safe alternatives for two large pools of acute migraine patients – those who have an insufficient response to triptan therapy and those who have contraindications to triptan use. ELYXYB®’s product profile mapped with a high degree of certainty to these stated unmet needs. In clinical studies, patients treated with ELYXYB® demonstrated pain relief in as little as 15 minutes, and significant pain relief compared to placebo within 45 minutes in approximately 50% of patients.6,7 “We are very excited about the potential of ELYXYB® since launching the product in the U.S. in April 2023. This is a highly complementary commercial asset that allows us to provide physicians with another tool in their pain management armamentarium to treat migraines earlier in the patient’s journey. These new patents will provide the ability to file application in the future for acute pain,” said Jaisim Shah, Chief Executive Officer and President of Scilex. For more information on Scilex Holding Company, refer to www.scilexholding.com For more information on Semnur Pharmaceuticals, Inc., refer to www.semnurpharma.com For more information on Scilex Holding Company Sustainability Report, refer to www.scilexholding.com/investors/sustainability For more information on ZTlido® including Full Prescribing Information, refer to www.ztlido.com. For more information on ELYXYB®, including Full Prescribing Information, refer to www.elyxyb.com. For more information on Gloperba®, including Full Prescribing Information, refer to www.gloperba.com. https://www.facebook.com/scilex.pharm https://www.linkedin.com/company/scilex-holding-company/ info@scilexholding.com About Scilex Holding Company Scilex Holding Company is an innovative revenue-generating company focused on acquiring, developing and commercializing non-opioid pain management products for the treatment of acute and chronic pain. Scilex targets indications with high unmet needs and large market opportunities with non-opioid therapies for the treatment of patients with acute and chronic pain and are dedicated to advancing and improving patient outcomes. Scilex’s commercial products include: (i) ZTlido® (lidocaine topical system) 1.8%, a prescription lidocaine topical product approved by the U.S. Food and Drug Administration (the “FDA”) for the relief of neuropathic pain associated with postherpetic neuralgia, which is a form of post-shingles nerve pain; (ii) ELYXYB®, a potential first-line treatment and the only FDA-approved, ready-to-use oral solution for the acute treatment of migraine, with or without aura, in adults; and (iii) Gloperba®, the first and only liquid oral version of the anti-gout medicine colchicine indicated for the prophylaxis of painful gout flares in adults. In addition, Scilex has three product candidates: (i) SP-102 (10 mg, dexamethasone sodium phosphate viscous gel) (“SEMDEXATM” or “SP-102”), a novel, viscous gel formulation of a widely used corticosteroid for epidural injections to treat lumbosacral radicular pain, or sciatica, for which Scilex has completed a Phase 3 study and was granted Fast Track status from the FDA in 2017; (ii) SP-103 (lidocaine topical system) 5.4%, (“SP-103”), a next-generation, triple-strength formulation of ZTlido, for the treatment of chronic neck pain and for which Scilex has recently completed a Phase 2 trial in low back pain. SP-103 has been granted Fast Track status from the FDA in low back pain; and (iii) SP-104 (4.5 mg, low-dose naltrexone hydrochloride delayed-release capsules) (“SP-104”), a novel low-dose delayed-release naltrexone hydrochloride being developed for the treatment of fibromyalgia, for which Phase 1 trials were completed in the second quarter of 2022. Scilex Holding Company is headquartered in Palo Alto, California. About Semnur Pharmaceuticals, Inc. Semnur Pharmaceuticals, Inc. (“Semnur”) is a clinical-late stage specialty pharmaceutical company focused on the development and commercialization of novel non-opioid pain therapies. Semnur’s lead program, SP-102 (SEMDEXA™), is the first non-opioid novel gel formulation administered epidurally in development for patients with moderate to severe chronic radicular pain/sciatica. Semnur Pharmaceuticals, Inc. is headquartered in Palo Alto, California. About Denali Capital Acquisition Corp. Denali Capital Acquisition Corp. (“SPAC”) is a blank check company formed for the purpose of effecting a merger, share exchange, asset acquisition, share purchase, reorganization or similar business combination with one or more businesses or entities. Important Information and Where to Find It This press release relates to a proposed transaction between Semnur and the SPAC and does not contain all the information that should be considered concerning the potential business combination and is not intended to form the basis of any investment decision or any other decision in respect of the potential business combination. This press release does not constitute an offer to sell or exchange, or the solicitation of an offer to buy or exchange, any securities, nor shall there be any sale of securities in any jurisdiction in which such offer, sale or exchange would be unlawful prior to registration or qualification under the securities laws of any such jurisdiction. In connection with the transaction described herein, the SPAC will file relevant materials with the SEC, including a registration statement on Form S-4, which will include a proxy statement/prospectus. Investors and security holders of the SPAC are urged to read these materials (including any amendments or supplements thereto) and any other relevant documents in connection with the transaction that the SPAC files with the SEC when, and if, they become available because they will contain important information about the SPAC, Semnur and the proposed transaction. The preliminary proxy statement/prospectus, the definitive proxy statement/prospectus and other relevant materials in connection with the transaction (when and if they become available), and any other documents filed by the SPAC with the SEC, may be obtained free of charge at the SEC’s website (www.sec.gov). The documents filed by the SPAC with the SEC also may be obtained free of charge upon written request to: Denali Capital Acquisition Corp.437 Madison Avenue, 27th FloorNew York, NY 10022 Participants in the Solicitation The SPAC and its directors and executive officers may be deemed participants in the solicitation of proxies from the SPAC’s shareholders with respect to the proposed business combination. Information about the SPAC’s directors and executive officers and a description of their interests in the SPAC will be included in the proxy statement/prospectus for the proposed transaction and will be available at the SEC’s website (www.sec.gov). Additional information regarding the interests of such participants will be contained in the proxy statement/prospectus for the proposed transaction when available. Semnur and its directors and executive officers may also be deemed to be participants in the solicitation of proxies from the shareholders of the SPAC in connection with the proposed business combination. Information about Semnur’s directors and executive officers and information regarding their interests in the proposed transaction will be included in the proxy statement/prospectus for the proposed transaction. Non-Solicitation This press release is not a proxy statement or solicitation of a proxy, consent or authorization with respect to any securities or in respect of the potential transaction and shall not constitute an offer to sell or a solicitation of an offer to buy the securities of the SPAC, the combined company or Semnur, nor shall there be any sale of any such securities in any state or jurisdiction in which such offer, solicitation or sale would be unlawful prior to registration or qualification under the securities laws of such state or jurisdiction. No offer of securities shall be made except by means of a prospectus meeting the requirements of the Securities Act of 1933, as amended. Forward-Looking Statements This press release and any statements made for and during any presentation or meeting concerning the matters discussed in this press release contain forward-looking statements related to Scilex and its subsidiaries and the SPAC and are subject to risks and uncertainties that could cause actual results to differ materially from those projected. Forward-looking statements include statements regarding the SPAC, Scilex and its subsidiaries, including but not limited to Semnur, statements regarding the proposed business combination between Semnur and the SPAC, including the potential listing of the combined company’s common stock and warrants on Nasdaq, obtaining the approval from the SPAC’s shareholders, the expectation that the SPAC will file a registration statement on Form S-4 with the SEC, which would include a proxy statement/prospectus, the estimated or anticipated future results and benefits of the combined company following the proposed business combination, including the ability of the parties to successfully consummate the proposed business combination, the timing of the closing of the proposed business combination, future opportunities for the combined company, Semnur and the combined company’s proposed business strategies, the estimated pre-transaction equity valuation of Semnur, the estimated sales for SP-102, the Company’s outlook, goals and expectations for 2024, and the Company’s development and commercialization plans. Although each of the SPAC and Scilex and its subsidiaries believes that it has a reasonable basis for each forward-looking statement contained in this press release, each of the SPAC and Scilex and its subsidiaries caution you that these statements are based on a combination of facts and factors currently known and projections of the future, which are inherently uncertain. In addition, there will be risks and uncertainties described in the proxy statement/prospectus included in the registration statement on Form S-4 relating to the proposed transaction, which is expected to be filed by the SPAC with the SEC, and described in other documents filed by the SPAC or Scilex from time to time with the SEC. These filings may identify and address other important risks and uncertainties that could cause actual events and results to differ materially from those contained in the forward-looking statements. Neither the SPAC nor Scilex and its subsidiaries can assure you that the forward-looking statements in this communication will prove to be accurate. Risks and uncertainties that could cause actual results of Scilex and the SPAC to differ materially and adversely from those expressed in our forward-looking statements, include, but are not limited to: the inability of the parties to consummate any proposed business combination transaction for any reason, including any failure to meet applicable closing conditions; changes in the structure, timing and completion of the proposed transaction between the SPAC and Semnur; the SPAC’s ability to continue its listing on the Nasdaq Capital Market until closing of the proposed transaction; the combined company’s ability to list its securities on Nasdaq after closing of the proposed transaction; the ability of the parties to achieve the benefits of the proposed transaction, including future financial and operating results of the combined company; the ability of the parties to realize the expected synergies from the proposed transaction; risks related to the outcome of any legal proceedings that may be instituted against the parties following the announcement of the proposed business combination; risks associated with the unpredictability of trading markets; general economic, political and business conditions; the risk that the potential product candidates that Scilex develops may not progress through clinical development or receive required regulatory approvals within expected timelines or at all; risks relating to uncertainty regarding the regulatory pathway for Scilex’s product candidates; the risk that Scilex will be unable to successfully market or gain market acceptance of its product candidates; the risk that Scilex’s product candidates may not be beneficial to patients or successfully commercialized; the risk that Scilex has overestimated the size of the target patient population, their willingness to try new therapies and the willingness of physicians to prescribe these therapies; risks that the outcome of the trials and studies for SP-102, SP-103 or SP-104 may not be successful or reflect positive outcomes; risks that the prior results of the clinical and investigator-initiated trials of SP-102 (SEMDEXA™), SP-103 or SP-104 may not be replicated; regulatory and intellectual property risks; and other risks and uncertainties indicated from time to time and other risks described in Scilex’s and the SPAC’s most recent periodic reports filed with the SEC, including their Annual Reports on Form 10-K for the year ended December 31, 2023 and subsequent Quarterly Reports on Form 10-Q that the Company and the SPAC have respectively filed or may file, including the risk factors set forth in those filings. Investors are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date of this release, and Scilex and the SPAC undertakes no obligation to update any forward-looking statement in this press release except as may be required by law. Contacts: Investors and MediaScilex Holding Company 960 San Antonio RoadPalo Alto, CA 94303Office: (650) 516-4310 Email: investorrelations@scilexholding.com Website: www.scilexholding.com Investors and MediaDenali Capital Acquisition Corp. 437 Madison Avenue, 27th FloorNew York, NY 10022 Reference 1) Source: Celecoxib Oral Solution Approved for Acute Migraine March 2020. https://www.neurologylive.com/view/celecoxib-oral-solution-gets-goahead-for-acute-migraine2) Source: Evaluate Pharma data February 16, 20233) Bunchorntavakul C, Reddy K. Acetaminophen (APAP or N-Acetyl-p-Aminophenol) and Acute Liver Failure. Clin Liver Dis. 2018 May;22(2):325-346. PMID: 296050694) DelveInsight Acute Pain - Market Insight, Epidemiology And Market Forecast – 2032; Dec 2022; https://www.delveinsight.com/report-store/acute-pain-market#:~:text=The%20DelveInsight's%20acute%20pain%20market,be%20either%20acute%20or%20chronic 5) Source: Acute Migraine Headache: Treatment Strategies. https://www.aafp.org/pubs/afp/issues/2018/0215/p243.html6) Data on file. Scilex Holding Company7) Lipton RB, et al. J Pain Res 2021; 14:549-560. SEMDEXA™ (SP-102) is a trademark owned by Semnur Pharmaceuticals, Inc., a wholly-owned subsidiary of Scilex Holding Company. A proprietary name review by the FDA is planned. ZTlido® is a registered trademark owned by Scilex Pharmaceuticals Inc., a wholly-owned subsidiary of Scilex Holding Company. Gloperba® is the subject of an exclusive, transferable license to Scilex Holding Company to use the registered trademark. ELYXYB® is a registered trademark owned by Scilex Holding Company. All other trademarks are the property of their respective owners. © 2024 Scilex Holding Company All Rights Reserved.

临床1期上市批准快速通道引进/卖出临床3期

2024-05-19

·精准药物

可逆共价抑制剂（reversible inhibitor)

侃言共价抑制剂一般是指不可逆共价抑制剂；但是细分研究，可以分为不可逆共价抑制剂（ICIs）、和可逆共价抑制剂（RCIs）。这次仅聊聊可逆共价抑制剂。谈谈它的重要特征、设计规则、分析方法、以及Case Study（JAK3、BTK、EV71）、和发展展望。化合物定制1共价“喜”与“忧”共价抑制剂已经有一百多年的发展历史，相关研究有迹可循。多年以来，共价抑制让人欢喜让人忧！喜的是，共价抑制剂有非共价抑制剂（NCIs）不具有的优势：1、延长抑制剂靶向靶标蛋白的停留时间，具有抗耐药性；2、抑制传统意义上的“不可成药”靶标。包括那些具有延展的、或浅而平坦的口袋、或者根本没有口袋的靶标，以及参与蛋白质交互作用（PPIs）的大而平坦的功能作用界面。3、靶向蛋白靶标双重选择模式，赋予药物亲和力元件额外的亲和力，增强靶向效力。这是因为共价抑制一般包括两个步骤：先是如同非共价抑制的那般，抑制剂亲和性结合靶标蛋白；随后和靶标反应性氨基酸残基形成共价键，通过不可逆失活的自杀式抑制、或者通过形成共价加成物增加亲和力使抑制剂获得更强的竞争性抑制活性。共价弹头提高了抑制剂对靶标蛋白的亲和力粘性，抑制剂和靶标氨基酸残基的反应可以是永久性不可逆的，也可以是暂时性可逆的；不可逆共价抑制剂如同胶水般将亲和力元件和靶标永久性粘合在一起，而不可逆共价抑制剂好比是在抑制剂和靶标之间绑了个“活扣”，还可复原。因此，如上图，影响NCI、ICI、RCI平衡常数的因素不同。以上共价抑制的优势使其重新复兴。但是共价自身的特点，也给研究人员带来了一些担忧。从结构上看，共价抑制剂由亲和性元件、和反应性弹头构成。因此，担心它过分活力四射、脱靶，伤害了“无辜的”蛋白质靶标。ICIs还是永久性伤害；RCIs也会“伤害无辜”，但可逆性动力学致使它还会“释放无辜”。担心它的稳定性，经不起血浆或体内代谢过程中复杂环境的考验，提前“失活”，甚至反应性官能团在代谢过程中带来毒性。一些包含有活性基团的成分进入到血浆和体内，一般表现出较差的药代动力学和较低的稳定性。担心它导致免疫激活，如抑制剂与蛋白形成的加合物或者降解产物导致过敏反应等。以上的担忧曾一度限制了共价药物的发展，研究人员多是追寻着一条经验主义的黄金法则——开发可逆性药物，即非共价抑制剂。然而，即使是非共价抑制剂，也有很多因为毒性而退出了历史的舞台。也有大量的共价药物在治疗中发挥着举足轻重的作用。因此，对共价抑制不利的担忧完全可以变成研发中设计和攻克的目标。在药物开发中，研究人员致力于扩大药物的治疗效力，而降低其副作用。对于共价抑制剂来说，脱靶问题、以及药代动力学问题、和代谢稳定性问题是不可避免的，需要在药物开发过程中予以解决。2可逆共价抑制的化学结构特征和优势共价抑制剂的亲电弹头大多时候都比较明显，具有显著的结构特征，如具有环张力的β-内酰胺、α,β-不饱和羰基化合物等；还有一些隐藏在前药中，在代谢后才崭露出共价结构，如奥美拉唑等；还有一些，通过结构观察不是很明确，但在机理验证中证实为共价机制，如作为异柠檬酸裂解酶3的共价抑制剂3-硝基丙酸、作为磷酸肌醇-3激酶共价抑制剂的Wortmannin等（上图）。对于可逆共价抑制剂，反应动力学使它对“无辜的”靶标可逆的、非永久性的修饰，最后还是会释放抑制剂和“无辜者”，因此，这就减轻或者避免了脱靶效应及其毒性。符合可逆共价抑制剂的弹头近些年来也有一定的发展，如上图。靶向靶标氨基酸残基的作用机制不同，主要可以分为3类：1、靶向半胱氨酸残基类主要有α-氰基丙烯酰胺及其衍生物、氰基类、醛基类、α-氟取代丙烯磺酸酯、氟氯双取代甲基酮、巯甲基四氮嗪等。2、靶向丝氨酸/苏氨酸残基类主要有α-羰基酰胺、硼酸等。3、靶向赖氨酸残基及N末端类主要有醛基，另外有邻位羟基提供氢键稳定，或者硼酸提供氢键配位稳定亚胺结构。如下图，是近些年报道的一些发现的可逆共价抑制剂，红色标记的是可逆共价弹头。如上图，是可逆共价抑制的两步过程：亲和力结合、可逆成键。很明显，可逆共价抑制不会永久性改造蛋白靶标导致其变性等，因此一定程度上减轻了共价抑制的脱靶、和免疫激活的风险。这类弹头的反应性会更弱，在血浆和体内的稳定性相对于ICIs也会有所改善。等式中K代表平衡常数，τ代表抑制剂-靶标加合物的停留时间，研究人员的目标是获得的共价抑制剂，其Ki*尽可能地小，而τ尽可能地大。3可逆共价弹头的生化分析怎样确定抑制剂是可逆共价抑制剂呢？对机制地研究和确认，肯定不能仅靠经验从化学结构上判断。需要验证抑制剂弹头与靶标残基的可逆性反应、活性确定、保留时间测定、脱靶效应评估等。这些都需要一定的技术支持。3.1 可逆性分析可逆性的确认相对比较容易解决，一般可以通过①核磁共振（NMR）的技术方法、②质谱（MS）的方法、③生物发光共振能量转移（BRET）的方法等来确认。NMR法：对比RCI-Target加合物与未结合靶标的RCI的NMR化学信号，很容易区分两者的差别。稀释共价加合物的NMR样品浓度，如果是可逆共价结合，体系会重新平衡，降低加合物的浓度，获得更多的未结合的RCIs。此外，如果结合物或者RCIs具有明显的紫外/可见光谱信号，也可以通过这种方法确定Kd。MS法：将共价加合物注射进质谱分析仪，在MS试验的过程中，加合物会分解，抑制剂和天然蛋白会被释放，能过够在MS谱上观察到。BRET法：利用生物发光共振能量转移的竞争抑制试验，联合冲洗试验法验证可逆共价。首先蛋白靶标被共价抑制剂饱和性结合，然后引入荧光追踪试剂，用来取代共价抑制剂，如果能够竞争性取代，则共价抑制为可逆共价抑制的动力学结合。此外，共价抑制剂-蛋白加合物降解或者变性以及随后的抑制剂的复原，也进一步证明是可逆共价抑制。3.2 停留时间分析可逆共价抑制剂（RCIs）的停留时间可以通过荧光竞争法、或药代动力学/药效学（PK/PD）分析来测定。3.3 脱靶效应评估共价抑制不可避免的需要面临和解决脱靶效应的毒性问题，因此，对于共价抑制剂的脱靶效应评估至关重要，确定哪些蛋白质可以与共价抑制剂形成加合物，目前主要有三种方法报道。经典方法包括：与放射性标记的抑制剂共孵育，随后进行凝胶层析、电泳、切胶并进行Edman测序、进而鉴定出氨基酸-抑制剂加合物。现代化的方法包括质谱的方法：对目标兴趣蛋白进行分离（如色谱法、电泳法）后共价修饰分析（如果修饰位点已知，则可不进行蛋白消化；如果修饰位点未知，则需要进行蛋白消化）。化学蛋白质组学是另一种探测共价抑制剂脱靶效应的方法：通过标记可逆共价抑制剂（如亲和、荧光、或放射标记等），可以分离分析加合物（如使用亲和标记从凝胶中分离，或使用荧光或放射性标记从凝胶中分离）。这种方法已经应用于识别药物发现中的靶标化合物，也可以用于识别脱靶蛋白。需要指出的是，共价加合物的存在并不意味着与之联系的一定是不利的影响。脱靶加合物的识别可以为副作用的假设提供证据，但脱靶加合物的成分如果未知，必须进行进一步的探索，说不定会有意想不到的发现！4可逆共价抑制剂设计开发有效的可逆共价抑制剂，需要考虑一系列的问题：必须要有一个共价弹头；共价弹头必须对特定的亲核性残基有一定的选择性；共价加合物必需是可逆的；共价药物对期望靶标需要有良好的亲和结合力；一个好的可逆共价抑制剂具有一些通性，因此对于最佳可逆共价抑制剂的设计有如下一个总结性的通用流程，如上图所示。Step 1: 计算分析和虚拟建模。通过计算分析和虚拟建模确保靶标具有一个适合不可逆共价抑制剂靶向的亲核性残基（如丝氨酸、苏氨酸、赖氨酸、半胱氨酸、或者N末端氨基等）；但是计算分析和虚拟建模这一过程对于RCIs的成功开发不是必需的，尤其是对于没有靶标高质量结构数据的情况下；目前已有一些RCIs的建模理论框架，以及利用量子力学（QM）、QM/分子力学（MM）、或者QM/MM结合分子动力学方法进行建模试验。Step 2: 结合质量作用定律描述生成物和反应物的平衡状态。引入质量作用定律，通过动态平衡和停留时间评估抑制剂-靶标的结合动力学。平衡有利于共价加合物的形成；脱靶效应的逆反应的热力学有利性；如果解离常数Ka较小时，可逆性和不可逆性抑制剂没有差异；优化靶向酶和脱靶酶的停留时间等。Step 3: 抑制剂与可逆性共价抑制剂结合动力学的相似性。包括抑制剂沿着动力学描述的表征；测定组蛋白脱乙酰酶4进行生化表征后的光谱学试验方法；其他方法还有洗涤后进行标记追踪（如JAK3抑制剂）、胰蛋白酶消化后抑制剂复原等。Step 4: 抑制剂-谷胱甘肽结合物不可长期积聚。谷胱甘肽与亲电性应激有关；必需有高度的可逆性才能避免应激反应；进行竞争性谷胱甘肽消化试验以解决应激反应的问题。Step 5：最小化全局应激反应。共价抑制可能导致蛋白质错误折叠，如果是非特异性的共价抑制剂，可能会导致全局蛋白错误折叠应激反应。通过共价抑观察蛋白错误折叠应激；确保有限的脱靶效应。Step 6：对免疫细胞的影响。除了有效的靶标结合外，开发共价抑制剂的一个重要考虑因素是对免疫细胞的影响。免疫细胞对亲电应激高度敏感，可引起免疫刺激或免疫抑制作用。因此，检测抑制剂对b细胞、t细胞和巨噬细胞作用，以确定抑制剂是否引起促炎或抗炎。细胞因子的表达和分泌，对于避免免疫激活的潜在并发症很重要。5可逆共价抑制剂设计Case Study5.1 蛋白激酶类——JAK3抑制剂Janus激酶（JAK）是介导细胞信号反应的关键调控酶，特别是在涉及免疫细胞反应的细胞因子信号的分泌中。人类有四类JAK亚型，包括JAK1、JAK2、JAK3、以及TYK2，每一个都高度相似，因此开发高选择性药物非常具有挑战性。目前临床上已经开发了泛-JAK抑制剂，如FDA批准的tofacitinib (上图Comp. 5），但是其多亚型抑制效果还不清晰，特别是不同的JAK亚型在免疫健康中作用不同。研究发现JAK3拥有一个独特的半胱氨酸残基（Cys909），其余亚型在相同位置的残基为丝氨酸，因此选择性靶向的共价抑制剂的设计将非常具有潜力。利用上图中新开发的不可逆共价抑制剂1-3、已知的不可逆JAK3共价抑制剂化合物4、以及两种已知的非共价JAK3抑制剂5、6，研究人员表征和确定了新型的、具有较好活性（低nM)、和选择性(>500倍)的不可逆JAK3共价抑制。抑制剂1-3虽然具有良好的体外抑制活性，但是他们的亲电弹头在进攻JAK3之前发生了脱靶反应，导致不良反应。因此，研究人员基于化合物7的多环骨架进行可逆共价抑制剂设计，并与弹头反应性官能团饱和等价物进行对比，发现可逆共价抑制设计的抑制活性是饱和等价物的4倍以上。包含有可逆共价抑制弹头的13、14对JAK3具有良好的抑制活性（127 pM、154 pM）以及相对于其他亚型的高选择性（400倍到5800倍）。5.2 蛋白激酶类——BTK抑制剂BTK是与抗体信号和免疫细胞募集相关的免疫调节反应的关键酶，在大多数造血细胞中表达BTK，在B细胞的信号级联反应中起关键作用，是一种重要的免疫治疗靶点。BTK还有半胱氨酸残基，而类似的激酶还有丝氨酸，为设计选择性靶向的共价抑制剂提高了先天性条件。如上图，ibrutinib、acalabrutinib、zanubru-tinib、和tirabrutinib是已获批的BTK不可逆共价抑制剂，靶向Cys481残基。虽然他们对BTK具有很强的抑制作用，但是与Cys481相似的半胱氨酸的激酶也容易产生不可逆的抑制作用，造成脱靶。对此，研究人员开发了可逆共价抑制剂rilzabru-tinib。通过脱靶效应测试，其脱靶的副作用得到了很好的限制，目前已经进入了临床II/IIIq期研究。5.3 蛋白水解酶类——EV71 cPro 抑制剂蛋白水解酶（又称蛋白酶）存在于所有的生命结构域中，而蛋白水解酶抑制剂已广泛应用于抗癌、抗菌、高血压、2型糖尿病、阿尔兹海默病、非酒精性脂肪肝、和纤维化治疗中。蛋白水解酶一般根据其催化反应性残基进行分类，不同类型的作用机制不同，因此，可逆共价抑制剂可以作为选择性靶向抑制剂设计的有效工具。针对蛋白水解酶，临床上已经有不少成功的可逆共价抑制剂开发。如针对苏氨酸蛋白水解酶可逆共价抑制剂Bortezomib、Ixazomib；丝氨酸蛋白水解酶可逆共价抑制剂Boceprevir、Saxaglibtin、Telaprevir；半胱氨酸蛋白水解酶可逆共价抑制剂Nirmatrelvir等；最近一个关于半胱氨酸蛋白水解酶可逆共价抑制剂的开发的例子是肠病毒71(EV71）——用于治疗手足口病相关病原体之一。Rupintrivir是一个已知的EV71同源人源鼻病毒的不可逆共价抑制剂，共价弹头为靶向半胱氨酸残基的丙烯酸乙酯。研究人员对Rupintrivir的共价弹头进行修饰，得到共价化合物15-22，以期待获得对EV71 3C蛋白水解酶具有高效力、可逆的、选择性共价抑制剂。其中，分别选择了α-羰基-N-丙基乙酰胺、醛基、丙烯酰胺甲酯/甲胺、α-卤素取代丙烯酰胺甲酯、α-氰基取代丙烯酰胺基甲氨基叔丁基甲酸酯、α-氰基取代丙烯酰叔丁酯等作为共价弹头。研究人员利用MS检测抑制剂的可逆性，通过动力学拟合数据，检测靶标的活性。展望近年来，随着技术的发展，已经能够很好地解决共价抑制带来的脱靶效应、代谢稳定性等导致的毒性，以及药代动力学等问题，共价抑制剂再次兴起，其中一种就是开发可逆共价抑制剂（RCIs)。在RCIs的开发过程中，可以充分利用质量作用定律，其中一系列反应的平衡取决于底物的活性和浓度。其结果是，在稳态下，产物的浓度(如靶蛋白的同价加合物)将依赖于其他副反应的平衡。通过设计一种强结合抑制剂，平衡将最终导致抑制期望的目标，而非目标抑制是不利的。虽然脱靶结合会发生，但共价加合物的可逆性将限制脱靶结合的表型后果。从不可逆的共价抑制到可逆的共价抑制，显著的脱靶效应被规避，药物更安全;采用可逆性共价抑制将使有希望的药物很少因脱靶反应而产生明显的副作用。除了药物设计和酶抑制，可逆共价加合物也已被用于细胞成像、和材料科学(这里以动态共价键的名义)。RCIs未来的发展，包括：改进建模，以准确识别足够的非催化的亲核残基，以形成加合物；改进非丙烯酰胺弹头反应的反应性和可逆性的规则或指南，并开发强有力、易于执行的检测方法，以检测来自亲电应激的意外免疫反应；此外，还有新型亲电弹头的发现，特别是如果它们可以很容易地用荧光团或放射性标签进行修饰，以方便在细胞内或体内进行定位等。参考文献ACS Chem. Biol.2024, acschembio.3c00805.https://doi.org/10.1021/acschembio.3c00805.声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！长按关注本公众号粉丝群/投稿/授权/广告等请联系公众号助手觉得本文好看，请点这里↓

2023-11-10

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New asundexian Phase III study to include patients with atrial fibrillation ineligible for oral anticoagulant treatment

New study includes patients aged ≥65 years with atrial fibrillation (AF) at high risk for ischemic stroke or systemic embolism who are deemed ineligible for oral anticoagulant (OAC) treatment due to increased risk of bleeding OCEANIC-AFINA, the latest Phase III study in the OCEANIC clinical trial program, will investigate asundexian in this specific patient group OCEANIC-AFINA complements the ongoing OCEANIC-AF study, further expanding Bayer’s landmark clinical trial program with asundexian, enrolling nearly 30,000 patients in over 40 countries Asundexian is being evaluated as a potential improved treatment option in stroke prevention and could be part of an entirely new class of treatment options in thrombosis management WHIPPANY, N.J.--(BUSINESS WIRE)-- Bayer today announced it expanded its Phase III OCEANIC clinical development program for the investigational drug asundexian (BAY2433334) by initiating a third clinical study, OCEANIC-AFINA. OCEANIC AFINA is a Phase III study investigating asundexian as a potential treatment option in patients (≥65 years of age) with atrial fibrillation (AF) at high risk for stroke or systemic embolism who are deemed ineligible for oral anticoagulation (OAC) treatment due to an increased risk of bleeding. OCEANIC-AFINA complements OCEANIC-AF, an ongoing Phase III study investigating the efficacy and safety of asundexian for the prevention of stroke or systemic embolism in people with AF. Together, the results from OCEANIC-AFINA and OCEANIC-AF aim to provide robust evidence for the efficacy and safety of asundexian across a broad range of patients with AF, including those who are at high risk for both ischemic stroke or systemic embolism and major bleeding events. Studies show that 1 in 3 patients with AF are undertreated or untreated with oral anticoagulants, 1,2 leading to a substantial unmet need for an effective therapy that does not further increase the risk of bleeding during treatment. Elderly patients with AF at high risk for bleeding and patients with AF and end-stage kidney disease (ESKD) on hemodialysis are often treated with a lower dose than recommended by guidelines.3 Further, those who discontinue OACs are at increased risk of experiencing a subsequent ischemic stroke.4 Inhibiting FXIa with asundexian might be able to tackle this unmet need, as it could provide protection from thrombotic events without a corresponding increase in bleeding risk. “The promising clinical results we have seen so far indicate that asundexian could mark a new option for antithrombotic treatment for a broader range of patients”, said Dr. Christian Rommel, Member of the Executive Committee of Bayer AG’s Pharmaceutical Division and Global Head of Research and Development. “It is our vision to support patients with atrial fibrillation, including those, that up to now were deemed as not eligible for treatment with OACs. We aim to offer them a new therapy option to help physicians confidently prescribe antithrombotic treatment to protect their patients. With OCEANIC-AFINA we will evaluate asundexian in a vulnerable untreated patient population.” “For physicians, there are challenging clinical decisions we have to make when considering treatment options for atrial fibrillation patients who are at high bleeding risk, and not suitable for currently available oral anticoagulants”, said Dr. Roxana Mehran, Professor of Medicine and Director of Interventional Cardiovascular Research and Clinical Trials at Icahn School at Mount Sinai. “These patients are often overlooked and excluded from clinical trials, so it is gratifying to see Bayer’s announcement of this study.” OCEANIC-AFINA is a multicenter, international, randomized, placebo-controlled, double-blind, parallel-group, 2-arm Phase III study comparing the use of asundexian with placebo in patients (≥65 years of age) with AF at high risk for stroke or systemic embolism who are deemed ineligible to receive treatment with OACs. The primary efficacy objective of OCEANIC-AFINA is to compare the time to first occurrence of ischemic stroke or systemic embolism between AF patients treated with asundexian or placebo. The primary safety objective is to describe the time to first occurrence of ISTH (International Society on Thrombosis and Hemostasis) major bleeding in participants receiving asundexian and placebo.5 The study is expected to enroll nearly 2,000 patients with AF. Asundexian is an investigational agent and has not been approved by any health authority for use in any country, for any indication. It is currently being evaluated as a potential once-daily oral Factor XIa (FXIa) inhibitor for prevention of thromboembolic events, reducing pathological clot formation while allowing hemostasis to form clots to stop the bleeding. About the OCEANIC Program The OCEANIC clinical trial program is designed to evaluate the use of asundexian, as an oral FXIa inhibitor, in patients with atrial fibrillation (AF) at risk of stroke and in patients following acute non-cardioembolic ischemic stroke or high-risk transient ischemic attack (TIA), aiming to improve the benefit-risk pro compared to standard of care. The program started with two large multinational studies, OCEANIC-AF and OCEANIC-STROKE, and is one of the largest clinical trial programs Bayer has undertaken, expecting to enroll nearly 30,000 patients in over 40 countries. The U.S. Food and Drug Administration (FDA) has granted Fast Track Designation for asundexian as a potential treatment to prevent stroke and systemic embolism in people with atrial fibrillation (AF) as well as for the prevention of stroke in patients after an acute non-cardioembolic ischemic stroke. About OCEANIC-AF, OCEANIC-STROKE and OCEANIC-AFINA OCEANIC-AF is a multicenter, international, randomized, active comparator-controlled, double-blind, double-dummy, parallel-group, 2-arm Phase III study investigating asundexian compared to apixaban (a non-vitamin K antagonist oral anticoagulant) in patients with atrial fibrillation at risk for stroke to determine the safety and efficacy of asundexian on prevention of stroke and systemic embolism. OCEANIC-STROKE is a multicenter, international, randomized, placebo-controlled, double-blind, parallel group and event-driven Phase III study investigating the efficacy and safety of asundexian for prevention of ischemic stroke compared to placebo on top of standard-of-care antiplatelet therapy in patients after an acute non-cardioembolic ischemic stroke or high-risk transient ischemic attack (TIA) / mini-stroke. OCEANIC-AFINA is a multicenter, international, randomized, placebo-controlled, double-blind, parallel-group, 2-arm Phase III study comparing the use of asundexian with placebo in patients with atrial fibrillation (AF) (≥65 years of age) at high risk for stroke or systemic embolism who are deemed ineligible for OAC treatment. About FXIa inhibitors and Asundexian Factor XI is a protein in the blood which is converted into its active enzyme form (Factor XIa) as part of the blood coagulation cascade. FXIa inhibition specifically targets the FXIa protein involved in pathological thrombus formation, while leaving hemostasis intact. Asundexian, as an oral direct, potent inhibitor of activated coagulation factor XI (FXIa), acts selectively on the coagulation cascade, thereby offering the potential to prevent events like stroke without a corresponding increase in bleeding risk associated with the current standard of care (SoC). Asundexian is currently being evaluated as a potential treatment option in thrombosis prevention and could represent a new approach in antithrombotic treatment. Asundexian is a once-daily, oral investigational agent and has not been approved by any health authority for use in any country, for any indication. About Atrial Fibrillation AF is one of the most common sustained cardiac rhythm disorders (arrhythmias). It results from rapid, disorganized electrical signals in the upper chambers (atria) of the heart, causing them to quiver and contract quickly and irregularly. As a result, the atria do not empty completely, and blood does not flow properly, causing blood clots to form. These blood clots can break loose and travel to the brain, resulting in a stroke.6 About Bayer Bayer is a global enterprise with core competencies in the life science fields of health care and nutrition. Its products and services are designed to help people and the planet thrive by supporting efforts to master the major challenges presented by a growing and aging global population. Bayer is committed to driving sustainable development and generating a positive impact with its businesses. At the same time, the Group aims to increase its earning power and create value through innovation and growth. The Bayer brand stands for trust, reliability and quality throughout the world. In fiscal 2022, the Group employed around 101,000 people and had sales of 50.7 billion euros. R&D expenses before special items amounted to 6.2 billion euros. For more information, go to . Find more information at Follow us on Facebook: Follow us on Twitter: @BayerPharma Forward-Looking Statements This release may contain forward-looking statements based on current assumptions and forecasts made by Bayer management. Various known and unknown risks, uncertainties and other factors could lead to material differences between the actual future results, financial situation, development or performance of the company and the estimates given here. These factors include those discussed in Bayer’s public reports which are available on the Bayer website at . The company assumes no liability whatsoever to update these forward-looking statements or to conform them to future events or developments. References Steinberg BA, Gao H, Shrader P, Pieper K, Thomas L, Camm AJ, Ezekowitz MD, Fonarow GC, Gersh BJ, Goldhaber S, Haas S. International trends in clinical characteristics and oral anticoagulation treatment for patients with atrial fibrillation: results from the GARFIELD-AF, ORBIT-AF I, and ORBIT-AF II registries. American heart journal. 2017 Dec 1;194:132-40. Steinberg BA, Shrader P, Pieper K, Thomas L, Allen LA, Ansell J, Chan PS, Ezekowitz MD, Fonarow GC, Freeman JV, Gersh BJ. Frequency and Outcomes of Reduced Dose Non–Vita-min K Antagonist Anticoagulants: Results From ORBIT‐AF II (The Outcomes Registry for Better Informed Treatment of Atrial Fibrillation II). Journal of the American Heart Association. 2018 Feb 16;7(4):e007633. Al-Khatib, S. Pokorney, S. et al. (2020). Underuse of Oral Anticoagulants in Privately Insured Patients with Atrial Fibrillation: A Population Being Targeted by t. Am Heart J. (229), pp.110-117. García Rodríguez, L. et al. (2021). Discontinuation of oral anticoagulation in atrial fibrillation and risk of ischaemic stroke. Heart BMJ. (107), pp.542-548. Bayer AG. OCEANIC-AFINA Study Concept. Data on file. 2023 NHS choices. Atrial fibrillation complications. Available at: . Accessed: November 2023

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