TrkA; TrkB; TrkC

体外活性：Larotrectinib（以前称为 LOXO-101、ARRY-470）是一种有效的口服生物活性、高选择性、ATP 竞争性 TRK 抑制剂，IC50 处于低纳摩尔范围（2 至 20 nM），可抑制所有 TRK 家族成员在结合和细胞测定中。它的选择性比其他激酶高 100 倍，并且针对 TRKA、TRKB 和 TRKC 激酶具有 2 至 20 nM 的细胞效力。评估了 LOXO-101 对浓度为 1,000 nM 的一组非 TRK 激酶以及 Km 周围 ATP 浓度的脱靶激酶抑制作用。使用带有 TRK 融合的人源癌细胞系的 LOXO-101 临床前模型证明了融合癌蛋白和体外细胞增殖以及体内肿瘤生长的抑制作用。激酶测定：LOXO-101 是一种小分子，旨在阻断 TRK 受体家族的 ATP 结合位点，对 TRKA、TRKB 和 TRKC 激酶具有 2 至 20 nM 细胞效力。 IC50 值：2 - 20 nM 靶标：TRKA/B/C 体外：LOXO-101 是一种口服 TRK 激酶抑制剂，仅对 TRK 受体家族具有高度选择性。在化合物浓度为 1,000 nM 且 ATP 浓度接近每种酶的 Km 时，评估了 LOXO-101 对一组 226 个非 TRK 激酶的脱靶激酶抑制作用。在图中，LOXO-101 仅对一种非 TRK 激酶（TNK2 IC50，576 nM）具有超过 50% 的抑制作用。 LOXO-101 处理后的增殖测量表明，所有三种细胞系中的细胞增殖均受到剂量依赖性抑制。 CUTO-3.29 的 IC50 小于 100 nM，KM12 和 MO-91 的 IC50 小于 10 nM，这与该药物对 TRK 激酶家族的已知效力一致。细胞测定：用指定剂量的药物（ARRY-470；G，吉非替尼 1,000 nM）或 DMSO 对照处理 5 小时后，裂解表达 MPRIP-NTRK1 (RIP-TRKA) 或 EV 的 Ba/F3 细胞。细胞裂解物用于蛋白质印迹分析。

早期/持续但非晚期/急性施用 ARRY-470(LOXO-101) 可显着减轻骨癌疼痛，并显着阻止感觉神经纤维的异位萌芽和肿瘤骨中神经瘤样结构的形成，但不会对肿瘤生长或骨重塑有显着影响。它穿过血脑屏障的能力非常有限。

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使用ETV6-NTRK3的PDX模型，研究表明，与小鼠对照（脾脏重量316 vs 20mg， p<0.001）相比，使用 larorectinib (LOXO-101) （200mg/kg/day p.o o，连续6周）治疗小鼠骨髓（0 vs 75.8%人CD45/CD19骨髓母细胞，每组n=5）和脾脏的白血病浸润降低到无法检测的水平。值得注意的是，地塞米松治疗对该肿瘤有中等效果（平均为55.3%骨髓母细胞和134mg脾脏重量，n=5）。用LOXO-101治疗的小鼠在停止治疗4周后仍然存活且无白血病，这是通过Xenogen成像确定的。[3]

LOXO-101 是一种小分子，针对 TRKA、TRKB 和 TRKC 激酶具有 2 至 20 nM 的细胞效力，旨在阻断 TRK 受体家族的 ATP 结合位点。 IC50 值：2–20 nM 靶标：体外 TRKA/B/C TRK 激酶口服抑制剂 LOXO-101 仅对 TRK 受体家族具有高度选择性。针对一组 226 个非 TRK 激酶，在化合物浓度为 1,000 nM 且 ATP 浓度接近每种酶的 Km 时，测试了 LOXO-101 的脱靶激酶抑制作用。对于该组中的一种非 TRK 激酶（TNK2 IC50，576 nM），LOXO-101 表现出超过 50% 的抑制作用。当所有三种细胞系都用 LOXO-101 处理时，所产生的细胞分裂量显示出细胞分裂的剂量依赖性抑制。根据该药物对 TRK 激酶家族的已知效力，CUTO-3.29 和 KM12 和 MO-91 的 IC50 值分别小于 100 nM 和小于 10 nM。

使用指定药物剂量（ARRY-470；G，吉非替尼 1,000 nM）或 DMSO 对照处理 5 小时后，裂解表达 EV 或 MPRIP-NTRK1 (RIP-TRKA) 的 Ba/F3 细胞。对于蛋白质印迹分析，使用细胞裂解物。

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方法：在体外研究中，激酶融合物在IL3依赖性Ba/F3细胞中表达。为了生成基因工程小鼠模型，我们使用了先前报道的Etv6-NTRK3的条件敲入模型（Cancer Cell 2007;12:542-558），其中编码酪氨酸激酶结构域的NTRK3 cDNA的人类部分被插入到小鼠Etv6位点的外显子6中，位于固定转录终止序列的下游。Etv6-NTRK3蛋白的表达是由b系启动子CD19驱动的cree -recombinase完成的。体外进行磷流式细胞术分析和对 larorectinib (LOXO-101)的敏感性评估。
研究人员接下来评估了TRK抑制剂crizotinib（也抑制ALK）和一种更特异性的抑制剂 larorectinib (LOXO-101)的体外疗效。与克唑替尼（IC50 205 nM）相比， larorectinib (LOXO-101)对BaF3-ETV6-NTRK3细胞（IC5017 nM）的抑制作用强10倍，对其他激酶融合（ABL1, ABL2, CSF1R, FLT3, JAK2）在10µM范围内无影响。此外，与克唑替尼相比， larorectinib (LOXO-101)在77种人类癌细胞系的细胞毒性筛选中对TRK A、B和C具有显著的选择性。使用ETV6-NTRK3的PDX模型，我们证明了 larorectinib (LOXO-101)治疗（200mg/kg/day p.o o，持续6周）将骨髓（0 vs 75.8%人CD45/CD19骨髓母细胞，每组n=5）和脾脏中的白血病浸润减少到无法检测到的水平（脾重量316 vs 20mg， p<0.001）。值得注意的是，地塞米松治疗对该肿瘤有中等效果（平均为55.3%骨髓母细胞和134mg脾脏重量，n=5）。通过Xenogen显像确定，用 larorectinib (LOXO-101)治疗的小鼠在停止治疗后四周仍然存活且无白血病。[3]

Mice: Throughout the investigation, arthymic nude mice are employed. The mice are given a subcutaneous injection of 5x10⁵ KM12 cells into the dorsal flank region. Tumor volume is measured directly with calipers and is computed using the following formula: length × (width⁵)/2. Mice are randomly chosen to receive either diluent, 60 mg/kg/dose, or 200 mg/kg/dose of Larotrectinib (LOXO-101) after the tumor has established and reached a size of 150–200 mm⁵. For 14 days, larotrectinib (LOXO-101) is given orally via gavage once a day. Three, six, and twenty-four hours after the final dosage, tissue and blood are extracted[4].

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A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vivo.[3]

Absorption, Distribution and Excretion
The mean absolute bioavailability of larotrectinib capsules is approximately 34% (range: 32-37%). In adult patients who received larotrectinib capsules 100 mg twice daily, Cmax was achieved at about one hour after dosing and steady-state was reached within three days. The mean steady-state Cmax and AUC0-24h of larotrectinib capsules was 788 ng/mL and 4351 ng*h/mL, respectively. In healthy subjects, the AUC of the larotrectinib oral solution was similar to that of the capsules and the Cmax was 36% greater with the oral solution. As compared to a fasted state, the administration of larotrectinib in healthy subjects alongside a high-fat meal resulted in a similar AUC and a reduction in Cmax of 35%.
Following oral administration of a single 100 mg dose of radiolabeled larotrectinib in healthy subjects, 58% (5% unchanged) of the administered radioactivity was recovered in feces and 39% (20% unchanged) was recovered in urine.
Following intravenous administration to healthy subjects, the mean volume of distribution of larotrectinib at steady-state was approximately 48L.
The mean clearance CL/F of larotrectinib is 98 L/h.

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Metabolism / Metabolites
Larotrectinib is metabolized predominantly by CYP3A4. Following oral administration of a single 100 mg dose of radiolabeled larotrectinib in healthy subjects, the major circulating drug components in plasma were unchanged larotrectinib (19%) and an O-linked glucuronide (26%).

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Biological Half-Life
In healthy subjects, the half-life of larotrectinib following oral administration is 2.9 hours.

Hepatotoxicity
In early clinical trials in a total of 176 patients with various forms of solid tumors which had an NTRK gene fusion, elevations in serum aminotransferase levels occurred in 45% of patients treated with larotrectinib. Serum aminotransferase levels rose to above 5 times ULN in 6% of patients and led to early discontinuation in 2%. Serum aminotransferase elevations typically arose after 4 to 12 weeks of treatment, but usually without jaundice or alkaline phosphatase elevations. Most elevations resolved within 4 to 8 weeks and discontinuations were uncommon. Restarting larotrectinib at a reduced dose after resolution of the aminotransferase abnormalities was generally well tolerated and did not lead to recurrence of liver injury. Cases with jaundice and symptoms during larotrectinib therapy have not been reported, but the clinical experience with this kinase inhibitor has been limited and prelicensure clinical trials were carried out with careful clinical monitoring.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of larotrectinib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during larotrectinib therapy and for 1 week after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Larotrectinib is 70% bound to human plasma proteins _in vitro_ and binding is independent of drug concentration. The blood-to-plasma concentration ratio is 0.9.

[1]. LOXO-101, a pan TRK inhibitor, For The Treatment Of TRK-driven Cancers.

[2]. Infantile Fibrosarcoma With NTRK3-ETV6 Fusion Successfully Treated With the Tropomyosin-Related Kinase Inhibitor LOXO-101. Pediatr Blood Cancer. 2016 Aug;63(8):1468-70.

[3]. Genetic Modeling and Therapeutic Targeting of ETV6-NTRK3 with Loxo-101in Acute Lymphoblastic Leukemia. Blood 2016 128:278.

[4]. An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101. Cancer Discov. 2015 Oct;5(10):1049-57.

Larotrectinib is an orally administered inhibitor of tropomyosin receptor kinase (Trk), a receptor tyrosine kinase activated by neurotrophins which is mutated in a variety of cancer cell types and plays an important role in tumor cell growth and survival. Upon administration, larotrectinib binds to Trk, thereby preventing neurotrophin-Trk interaction and Trk activation, which results in both the induction of cellular apoptosis and the inhibition of cell growth in tumors that overexpress Trk. Larotrectinib was granted accelerated approval by the FDA in November 2018 for the treatment of Trk-positive solid tumors. It was notable for being the second tissue-agnostic chemotherapy ever approved by the FDA.
Larotrectinib is a Kinase Inhibitor. The mechanism of action of larotrectinib is as a Tropomyosin Receptor Kinases Inhibitor.
Larotrectinib is a selective inhibitor of neurotrophin receptor kinase (NTRK) that is used in the therapy of solid tumors harboring NTRK gene fusions. Larotrectinib is associated with a high rate of serum aminotransferase elevations during therapy but has not been linked to instances of clinically apparent liver injury with jaundice.
Larotrectinib is an orally available, tropomyosin receptor kinase (Trk) inhibitor, with potential antineoplastic activity. Upon administration, larotrectinib binds to Trk, thereby preventing neurotrophin-Trk interaction and Trk activation, which results in both the induction of cellular apoptosis and the inhibition of cell growth in tumors that overexpress Trk. Trk, a receptor tyrosine kinase activated by neurotrophins, is mutated in a variety of cancer cell types and plays an important role in tumor cell growth and survival.
See also: Larotrectinib Sulfate (has salt form).

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Drug Indication
Larotrectinib is a tyrosine kinase inhibitor that is currently indicated for the treatment of adult and pediatric patients with solid tumors that a) have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, b) are metastatic or where surgical resection is likely to result in severe morbidity, or c) have no satisfactory alternative treatments or that have progressed following treatment. These indications are approved under accelerated approval by the US FDA based on overall response rate and duration of response and continuation of support for these indications may be contingent upon the verification and description of continued clinical benefit in confirmatory trials.
FDA Label
Vitrakvi as monotherapy is indicated for the treatment of adult and paediatric patients with solid tumours that display a Neurotrophic Tyrosine Receptor Kinase (NTRK) gene fusion,who have a disease that is locally advanced, metastatic or where surgical resection is likely to result in severe morbidity, andwho have no satisfactory treatment options.
Treatment of malignant neoplasms of the central nervous system
Treatment of all conditions included in the category of malignant neoplasms (except central nervous system tumours, haematopoietic and lymphoid tissue neoplasms)

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Mechanism of Action
Tropomysoin receptor kinases (TRK) like TRKA, TRKB, and TRKC elicit activities that regulate the natural growth, differentiation, and survival of neurons when they interact with endogenous neutrotrophin ligands. TRKA, TRKB, and TRKC are themselves encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. It has been discovered that chromosomal rearrangements involving in-frame fusions of these genes with various partners, translocations in the TRK kinase domains, mutations in the TRK ligand-binding site, amplifications of NTRK, or the expression of TRK splice variants can result in constitutively-activated chimeric TRK fusion proteins that can act as oncogenic drivers that promote cell proliferation and survival in tumor cell lines. Subsequently, larotrectinib functions as an inhibitor of TRKs including TRKA, B, and C. In in vitro and in vivo tumor models, larotrectinib demonstrated anti-tumor activity in cells with constitutive activation of TRK proteins resulting from gene fusions, deletion of a protein regulatory domain, or in cells with TRK protein overexpression. Larotrectinib had minimal activity in cell lines with point mutations in the TRKA kinase domain, including the clinically identified acquired resistance mutation, G595R. Point mutations in the TRKC kinase domain with clinically identified acquired resistance to larotrectinib include G623R, G696A, and F617L.

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Infantile fibrosarcoma (IFS) is a rare pediatric cancer typically presenting in the first 2 years of life. Surgical resection is usually curative and chemotherapy is active against gross residual disease. However, when recurrences occur, therapeutic options are limited. We report a case of refractory IFS with constitutive activation of the tropomyosin-related kinase (TRK) signaling pathway from an ETS variant gene 6-neurotrophin 3 receptor gene (ETV6-NTRK3) gene fusion. The patient enrolled in a pediatric Phase 1 trial of LOXO-101, an experimental, highly selective inhibitor of TRK. The patient experienced a rapid, radiographic response, demonstrating the potential for LOXO-101 to provide benefit for IFS harboring NTRK gene fusions.[2]

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Oncogenic TRK fusions induce cancer cell proliferation and engage critical cancer-related downstream signaling pathways. These TRK fusions occur rarely, but in a diverse spectrum of tumor histologies. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein as determined by an in situ proximity ligation assay. In a phase I study of LOXO-101 (ClinicalTrials.gov no. NCT02122913), this patient's tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers. Significance: TRK fusions have been deemed putative oncogenic drivers, but their clinical significance remained unclear. A patient with a metastatic soft-tissue sarcoma with an LMNA-NTRK1 fusion had rapid and substantial tumor regression with a novel, highly selective TRK inhibitor, LOXO-101, providing the first clinical evidence of benefit from inhibiting TRK fusions.[4]

C21H22F2N6O2

428.44

428.177

C, 58.87; H, 5.18; F, 8.87; N, 19.62; O, 7.47

1223403-58-4

Larotrectinib sulfate;1223405-08-0;(R)-Larotrectinib;1223404-68-9

46188928

White to yellow solid powder

1.6±0.1 g/cm3

1.725

1.48

86

2

7

3

31

659

2

FC1C([H])=C([H])C(=C([H])C=1[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C1C([H])=C([H])N2C(=C(C([H])=N2)N([H])C(N2C([H])([H])C([H])([H])[C@@]([H])(C2([H])[H])O[H])=O)N=1)F

NYNZQNWKBKUAII-KBXCAEBGSA-N

InChI=1S/C21H22F2N6O2/c22-13-3-4-16(23)15(10-13)18-2-1-7-28(18)19-6-9-29-20(26-19)17(11-24-29)25-21(31)27-8-5-14(30)12-27/h3-4,6,9-11,14,18,30H,1-2,5,7-8,12H2,(H,25,31)/t14-,18+/m0/s1

(3S)-N-[5-[(2R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrazolo[1,5-a]pyrimidin-3-yl]-3-hydroxypyrrolidine-1-carboxamide

ARRY-470; Larotrectinib; LOXO-101; LOXO 101; ARRY-470; ARRY470; LOXO101; ARRY 470; Larotrectinib; 1223403-58-4; (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide; LOXO 101; Vitrakvi; trade name: Vitrakvi

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.84 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.84 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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