H-ras (IC50 = 1.9 nM); K-ras (IC50 = 5.2 nM); N-ras (IC50 = 2.8 nM)[1]

Lonafarnib (Sch66336) 抑制携带活化 Ki-Ras 蛋白的人类肿瘤细胞系的转化生长特性，并有效抑制全细胞中的 Ha-Ras 加工[1]。与对照治疗相比，所有含有 Lonafarnib (10 μM) 的治疗组的非法尼基化 H-Ras 含量均显着增加 (116–137%)[2]。

Lonafarnib (Sch66336) 在小鼠、大鼠和猴系统中表现出良好的口服生物利用度和药代动力学特征。 Lonafarnib 在裸鼠的多种人类肿瘤异种移植模型中表现出强大的口服疗效，包括源自结肠、肺、胰腺、前列腺和膀胱的肿瘤[1]。与媒介物治疗的对照小鼠（T/C为0.67）相比，单独的ionafarnib（口服强饲法80 mg/kg，每天一次）抑制原位U87肿瘤的能力有限。 XRT/Tem（2.5 Gy/天，持续 2 天；XRT 前 90 分钟口服 5 mg/kg）的预期结果是对肿瘤生长的中度体内抑制（T/C 为 0.42）。通过同时给予 Lonafarnib/XRT/Tem（Lonafarnib 80 mg/kg，口服管饲，每日一次，XRT 2.5 Gy）可实现最强的生长抑制作用（T/C 为 0.02），并显着优于 XRT/Tem（p<0.04）。 /天，持续 2 天，并在 XRT 前 90 分钟口服强饲 Tem 5 mg/kg）。大多数动物在 2 周后表现出肿瘤体积减小 (p<0.05)，并且效果在 4 周后持续存在 (p<0.05)[2]。

SCH 66336能有效抑制整个细胞中的Ha-Ras加工，并阻断表达活化Ki-Ras蛋白的成纤维细胞和人肿瘤细胞系的转化生长特性。许多缺乏活化ras癌基因的人类肿瘤系的锚定非依赖性生长也被SCH 66336治疗阻断。
FP敏感性是通过测量[3H]法尼烷基从[3H]法尼烷基PPi转移到三氯乙酸可沉淀的Ha-Ras CVLS来确定的。GGPT-1活性同样使用[3H]香叶基香叶基二磷酸和Ha-Ras CVLL作为底物测定[1]。

非放射性MTS细胞毒性试验[2]
按照制造商的说明，在96孔组织培养板中用5000个细胞/孔进行检测。在药物暴露后24小时照射平板，并在XRT后96小时进行检测，每天应用新鲜药物处理。为了定量，将染料直接添加到每个孔中，按照制造商的建议洗涤板，并通过光密度测定细胞存活率。采用学生T检验分析显著性。

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增殖试验[2]
用100000个细胞/孔接种12孔板。在接种后24小时开始药物治疗，每24小时更换一次培养基，共暴露96小时。在药物暴露24小时后对板进行照射。使用Z1系列库尔特计数器对来自三组重复处理的细胞进行胰蛋白酶处理，并在照射后48小时进行计数，并与第1天（药物治疗开始的那一天）计数的孔细胞数进行比较。药物治疗后的增殖与对照孔正常化，并表示为对照治疗的百分比。采用学生T检验分析显著性。

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下游路径分析[2]
每100mm3培养皿接种2.5×106个细胞，并在接种后24小时开始药物处理。在药物暴露24小时后照射板，在药物暴露48小时后（XRT后24小时）裂解细胞。用添加了蛋白酶和磷酸酶抑制剂的冰冷T-Per提取总蛋白，并使用BCA蛋白检测试剂盒进行定量。500ug总蛋白用于探测不同的人磷酸RTK人磷酸MAPK阵列。按照制造商的说明清洗和显影阵列，并将其暴露在胶片上。使用平板扫描仪扫描胶片，并使用ImageJ对点进行定量。治疗组之间的相对变化表示为对照组的百分比，通过Student的T检验评估其显著性。

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H-Ras的蛋白质印迹[2]
每100mm3培养皿接种2.5×106个细胞，并在接种后24小时开始药物处理。在药物暴露24小时后照射板，在药物暴露48小时后（XRT后24小时）裂解细胞。用添加了蛋白酶和磷酸酶抑制剂的冰冷T-Per提取总蛋白，并使用BCA蛋白检测试剂盒进行定量。样品（总蛋白20µg）在4-15%Tris-HCl SDS-PAGE标准凝胶（Biorad，Hercules，CA）上运行，并检测H-Ras和α-微管蛋白作为内载对照。将印迹暴露于胶片上，并使用平板扫描仪扫描胶片。使用ImageJ（NIH，Bethesda，MD）对条带进行定量，并使用Excel绘制图表。H-Ras被标准化为负荷对照，并表示为对照处理的百分比。使用学生T检验评估显著性。

Formulation: lonafarnib (SCH66336, Sarasar®) and Temozolomide were reconstituted in 4% DMSO in 20% (2-hydroxypropyl)-beta-cyclodextrin in PBS. Lonafarnib was given once daily at 80mg/kg with twice weekly weightings to ensure accurate dosing.

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Tumor Cell Line Xenografts[2]
Tumor cell lines were harvested in mid-logarithmic growth phase and resuspended in PBS. Homozygous NCR nude mice were anesthetized with Ketamine/Xylazine before exposure of the cranium and removal of the periosteum with a size 34 inverted cone burr. Mice were fixed in a stereotactic frame, and 5×104 cells in 10 ul of PBS were injected through a 27-gauge needle over 5 min at 2 mm lateral and posterior to the bregma and 3 mm below the dura. The incision was closed with staples. Animals were observed daily for signs of distress or development of neurologic symptoms at which time the mice were sacrificed.

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In Vivo Imaging[2]
Mice were anesthetized with Ketamine/Xylazine, injected with D-luciferin at 50 mg/kg i.p., and imaged with the Xenogen IVIS 100 Imaging System for 10–120 s, bin size 2 as previously published. To quantify bioluminescence, identical circular regions of interest were drawn to encircle the entire head of each animal, and the integrated flux of photons (photons per second) within each region of interest was determined by using the Xenogen LIVING IMAGES software package. Data were normalized to bioluminescence at the initiation of treatment for each animal. Statistical significance was assessed using the Student’s T-test.

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Glioma Neurosphere Assay[2]
Collection and use of fresh and discarded human tumor tissue was approved by the Brigham and Women’s Hospital Institutional Review Board. After frozen section diagnosis of malignant glioma by the attending neuropathologist, tumor material was grossly dissected from the tissue sample. Portions of the tumors were collected in chilled media for the studies described here and other portions were allocated for paraffin embedding for histological diagnosis and for genotyping. Expansion of tumor material and propagation was accomplished by subcutaneous implantation in Icr SCID mice (cells were never grown on plastic). When tumors reached ~1 cm, tumors were disaggregated, cells were counted and then grown in serum-free media with EGF, FGF and LIF as described previously to form tumorspheres [25, 26]. Drugs (SCH 5uM, TMZ 100uM) were added immediately after plating cells into 24 well plates and radiation given at 24hrs after plating and tumor neurospheres were counted in triplicate 10 days after plating.

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Dissolved in 20% (w/v) HPβCD; 50 mg/kg; Oral gavage

NOD/SCID mice between 6–12 weeks of age

Absorption, Distribution and Excretion
The absolute oral bioavailability of lonafarnib is unknown; in healthy subjects administration of either 75 or 100 mg of lonafarnib twice daily resulted in mean peak plasma concentrations (%CV) of 834 (32%) and 964 (32%) ng/mL, respectively. Twice daily administration of 115 mg/m2 lonafarnib in HGPS patients resulted in a median tmax of 2 hours (range 0-6), mean Cmax of 1777 ± 1083 ng/mL, mean AUC0-8hr of 9869 ± 6327 ng\*hr/mL, and a mean AUCtau of 12365 ± 9135 ng\*hr/mL. The corresponding values for a dose of 150 mg/m2 are: 4 hours (range 0-12), 2695 ± 1090 ng/mL, 16020 ± 4978 ng\*hr/mL, and 19539 ± 6434 ng\*hr/mL, respectively. Following a single oral dose of 75 mg in healthy subjects, the Cmax of lonafarnib decreased by 55% and 25%, and the AUC decreased by 29% and 21% for a high/low-fat meal compared to fasted conditions.
Up to 240 hours following oral administration of 104 mg [14C]-lonafarnib in fasted healthy subjects, approximately 62% and <1% of the initial radiolabeled dose was recovered in feces and urine, respectively. The two most prevalent metabolites were the active HM21 and HM17, which account for 14% and 15% of plasma radioactivity.
In healthy patients administered either 75 or 100 mg lonafarnib twice daily, the steady-state apparent volumes of distribution were 97.4 L and 87.8 L, respectively.

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Metabolism / Metabolites
Lonafarnib is metabolized _in vitro_ primarily by CYP3A4/5 and partially by CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1. Formation of the primary metabolites involves oxidation and subsequent dehydration in the pendant piperidine ring.

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Biological Half-Life
Lonafarnib has a mean half-life of approximately 4-6 hours following oral administration of 100 mg twice daily in healthy subjects.

Hepatotoxicity
In the small prelicensure clinical trials conducted in children with progeria, serum aminotransferase elevations occurred in 35% of lonafarnib treated subjects but were usually mild and self-limited, rising to above 3 times the upper limit of normal (ULN) in only 5%. There were no liver related serious adverse events and no patient had a concurrent elevation in serum aminotransferase and bilirubin levels. Since approval of lonafarnib, there have been no published reports of drug induced liver injury associated with its use, although clinical experience with the drug, particularly with long term therapy, has been limited.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

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Protein Binding
Lonafarnib exhibits _in vitro_ plasma protein binding of ≥99% over a concentration range of 0.5-40.0 μg/mL.

[1]. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res. 1998 Nov 1;58(21):4947-56.

[2]. Lonafarnib (SCH66336) improves the activity of temozolomide and radiation for orthotopic malignant gliomas. J Neurooncol. 2011 Aug;104(1):179-89.

[3]. Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial. Lancet Infect Dis. 2015 Oct;15(10):1167-1174.

Pharmacodynamics
Lonafarnib is a direct farnesyl transferase inhibitor that reduces the farnesylation of numerous cellular proteins, including progerin, the aberrantly truncated form of lamin A that accumulates in progeroid laminopathies such as Hutchinson-Gilford progeria syndrome. Treatment with lonafarnib has been associated with electrolyte abnormalities, myelosuppression, and increased liver enzyme levels (AST/ALT), although causation remains unclear. Also, lonafarnib is known to cause nephrotoxicity in rats and rod-dependent low-light vision decline in monkeys at plasma levels similar to those achieved under recommended dosing guidelines in humans; patients taking lonafarnib should undergo regular monitoring for both renal and ophthalmological function. In addition, based on observations from animal studies with rats, monkeys, and rabbits with plasma drug concentrations approximately equal to those attained in humans, lonafarnib may cause both male and female fertility impairment and embryo-fetal toxicity.

C27H31BR2CLN4O2

638.82

636.05

C, 50.76; H, 4.89; Br, 25.02; Cl, 5.55; N, 8.77; O, 5.01

193275-84-2

(Rac)-Lonafarnib;193275-86-4

148195

White to off-white solid powder

1.5±0.1 g/cm3

710.4±70.0 °C at 760 mmHg

214.5-215.9° (monohydrate); mp 222-223°

383.5±35.7 °C

0.0±2.3 mmHg at 25°C

1.630

5.03

79.53

1

3

3

36

790

1

C1CN(CCC1CC(=O)N2CCC(CC2)[C@@H]3C4=C(CCC5=C3N=CC(=C5)Br)C=C(C=C4Br)Cl)C(=O)N

DHMTURDWPRKSOA-RUZDIDTESA-N

InChI=1S/C27H31Br2ClN4O2/c28-20-12-19-2-1-18-13-21(30)14-22(29)24(18)25(26(19)32-15-20)17-5-9-33(10-6-17)23(35)11-16-3-7-34(8-4-16)27(31)36/h12-17,25H,1-11H2,(H2,31,36)/t25-/m1/s1

4-[2-[4-[(2R)-6,15-dibromo-13-chloro-4-azatricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14-hexaen-2-yl]piperidin-1-yl]-2-oxoethyl]piperidine-1-carboxamide

Lonafarnib; SCH66336; Sarasar; Sch 66336; Sch66336; Sch-66336; Zokinvy; lonafarnibum; Trade name: Sarasar; SCH 66336; SCH-66336;

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 (3.91 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 (3.91 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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配方 3 中的溶解度: ≥ 2.5 mg/mL (3.91 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 900 μL 玉米油中并混合均匀。

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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));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。