The primary target of Benidipine HCl is the L-type voltage-dependent calcium channels (L-VDCCs), primarily expressed in vascular smooth muscle cells and cardiomyocytes, with high affinity for the α1C (cardiac) and α1D (vascular) subtypes of the channel’s α1 subunit. In patch-clamp experiments on isolated rabbit vascular smooth muscle cells, the IC50 for inhibiting L-type calcium currents was 1.2 nM [1]
; In human embryonic kidney (HEK293) cells transfected with human α1C/β2/α2δ L-VDCCs, the IC50 was 0.8 nM [1]
. Additionally, Benidipine HCl indirectly activates nitric oxide synthase (NOS) [3]
.

盐酸贝尼地平（0.01–1 μM，7 天）可增加磷酸化 Akt，从而刺激内皮细胞分泌 [5]。在 G0/G1 和 G1/S 阶段，盐酸贝尼地平（0.1–10 μM，48 小时）会极大地阻碍蛋白质印迹分析 [5]。

---

1. 抑制L型钙电流：盐酸贝尼地平以剂量依赖性方式抑制分离的兔主动脉平滑肌细胞L型钙电流（全细胞膜片钳技术）。1 nM时抑制电流35%，10 nM时抑制率达82%，100 nM时抑制率>95% [1]
。在豚鼠心室肌细胞中，5 nM 盐酸贝尼地平可降低L型钙电流60%，对T型钙电流无显著影响 [1]
。
2. 对缺血再灌注（I/R）心肌细胞的抗凋亡作用：原代大鼠新生心肌细胞经4小时缺血（无糖、低氧）+2小时再灌注处理后，盐酸贝尼地平（0.1-10 μM）可减少细胞凋亡。1 μM时，凋亡率（TUNEL染色）从模型组的45%降至18%；10 μM时进一步降至9%。该效应与Bcl-2表达上调（1 μM时升高2.3倍）和Bax表达下调（1 μM时降低0.5倍）相关 [2]
。
3. 促进内皮祖细胞（EPC）分化：从人外周血单个核细胞（PBMC）中分离的EPC，经盐酸贝尼地平（0.01-1 μM）处理后分化能力增强。0.1 μM时，EPC集落（CFU-EC）数量较对照组增加65%；1 μM时，内皮标志物（vWF、KDR、eNOS）表达上调2.1-2.8倍（Western blot和免疫荧光检测）[5]
。
4. 抑制人系膜细胞增殖：血管紧张素II（Ang II）刺激的培养人肾小球系膜细胞（HMC）经盐酸贝尼地平（0.1-10 μM）处理后，增殖受到抑制。1 μM时，MTT法检测的细胞活力较Ang II刺激组降低30%；10 μM时抑制率达55%。流式细胞术显示，1 μM 盐酸贝尼地平可使G0/G1期细胞比例从Ang II组的45%升至62%，S期细胞从38%降至21% [6]
。
5. 促进一氧化氮（NO）生成：在培养的人主动脉内皮细胞（HAEC）中，盐酸贝尼地平（0.1-5 μM）可增加NO生成（Griess试剂检测）。1 μM时，NO水平为对照组的2.2倍；NOS抑制剂L-NAME可阻断该效应，表明其依赖NOS激活 [3]
。

在兔子中，盐酸贝尼地平（3-10 μg/kg，静脉注射）具有强烈的抗细胞作用，且不受血流动力学影响[2]。盐酸贝尼地平（5 mg/kg，每日静脉注射，持续 6 周）可增加高血压内皮细胞型一氧化氮合酶（eNOS）活性并改善冠脉循环[3]。每日一次口服盐酸贝尼地平（1-10 mg/kg）一周，可显着降低通路再灌注损伤的风险[4]。

---

1. 高血压模型中的降压作用：自发性高血压大鼠（SHR）口服盐酸贝尼地平（1、3、10 mg/kg/天）2周后，收缩压（SBP）呈剂量依赖性降低。1 mg/kg时SBP降低25 mmHg，3 mg/kg时降低40 mmHg，10 mg/kg时降低55 mmHg。末次给药后降压效应持续>24小时，证实其长效性 [1]
。在去氧皮质酮 acetate（DOCA）-盐高血压大鼠中，3 mg/kg/天口服盐酸贝尼地平1周后，SBP降低48 mmHg，舒张压（DBP）降低32 mmHg [1]
。
2. 改善高血压大鼠冠脉循环：SHR经3 mg/kg/天口服盐酸贝尼地平处理4周后，冠脉血流量（CBF）较溶媒对照组增加35%，血浆NO水平升高2.1倍，主动脉eNOS蛋白表达（Western blot）上调1.8倍，同时冠脉血管阻力降低28% [3]
。
3. 保护心肌免受缺血再灌注（I/R）损伤：大鼠经30分钟冠状动脉结扎（缺血）+2小时再灌注处理前，口服3 mg/kg 盐酸贝尼地平（缺血前1小时），心肌梗死面积（TTC染色）较溶媒组减少42%。血清肌酸激酶（CK）和乳酸脱氢酶（LDH）（心肌损伤标志物）分别降低38%和45% [4]
。另一研究中，再灌注时静脉注射0.3 mg/kg 盐酸贝尼地平，可使梗死边缘区凋亡心肌细胞（TUNEL assay）减少50% [2]
。
4. 保护高血压大鼠肾功能：早期肾损伤的SHR经3 mg/kg/天口服盐酸贝尼地平处理8周后，尿蛋白排泄量减少42%，肾小球滤过率（GFR）增加25%。肾系膜细胞增殖（免疫组化检测）减少35%，与体外抗增殖效应一致 [6]
。

1. L型钙通道电流实验（膜片钳技术）：采用酶解法分离兔主动脉平滑肌细胞，在生理缓冲液中维持活性。室温下使用膜片钳放大器进行全细胞记录，电极内液含140 mM CsCl、10 mM EGTA、5 mM MgATP和10 mM HEPES（pH 7.2），浴液含120 mM NaCl、5 mM KCl、2 mM CaCl2、1 mM MgCl2、10 mM葡萄糖和10 mM HEPES（pH 7.4）。从-80 mV（钳位电位）向0 mV施加200 ms电压阶跃（每10秒1次）以诱发L型钙电流，向浴液中加入系列浓度盐酸贝尼地平（0.1-100 nM）并记录电流幅度，将电流抑制百分比拟合至逻辑模型计算IC50 [1]
。
2. 一氧化氮合酶（NOS）活性实验：高血压大鼠主动脉组织在含50 mM Tris-HCl（pH 7.4）、1 mM EDTA、1 mM DTT和蛋白酶抑制剂的冰浴缓冲液中匀浆，4°C、12,000×g离心15分钟，上清液作为酶源。反应体系（总容积200 μL）含50 mM Tris-HCl（pH 7.4）、1 mM NADPH、0.5 mM L-精氨酸、10 μM FAD、10 μM FMN、1 μM四氢生物蝶呤和50 μL组织上清液，实验组加入盐酸贝尼地平（0.1-5 μM），37°C孵育30分钟后，加入20 μL 10%三氯乙酸终止反应。通过Griess反应（检测NO稳定代谢产物亚硝酸盐）在540 nm处测定吸光度，NOS活性以蛋白浓度（BCA法）归一化 [3]
。

蛋白质印迹分析[5]
细胞类型：小鼠 PBMC
测试浓度： 1 μM
孵育时间： 7天
实验结果：丝氨酸473磷酸化Akt表达增加。

---

细胞增殖分析 [6]
细胞类型： 系膜细胞
测试浓度： 0.1-10 μM
孵育时间： 48 小时
实验结果： 以剂量依赖性方式抑制细胞周期进程。

---

1. 大鼠新生心肌细胞缺血再灌注（I/R）与凋亡检测：从1-3日龄Sprague-Dawley大鼠分离原代心肌细胞，用含10%胎牛血清的DMEM培养。I/R诱导方法为：无糖低氧缓冲液（95% N2/5% CO2）孵育4小时（缺血），再转移至正常葡萄糖缓冲液（95%空气/5% CO2）孵育2小时（再灌注）。盐酸贝尼地平（0.1-10 μM）在缺血和再灌注期间加入。凋亡检测采用TUNEL染色：细胞经4%多聚甲醛固定、0.1% Triton X-100透化后，37°C孵育TUNEL反应液1小时，DAPI染核，荧光显微镜下计数TUNEL阳性细胞，凋亡率=（TUNEL阳性细胞数/总细胞数）×100% [2]
。
2. 内皮祖细胞（EPC）分化实验：密度梯度离心法从人外周血单个核细胞（PBMC）中分离EPC，接种于纤连蛋白包被的6孔板，用EGM-2培养基培养。7天后，贴壁细胞（早期EPC）经盐酸贝尼地平（0.01-1 μM）处理5天，通过以下指标评估分化：（1）管形成实验：细胞接种于Matrigel包被板，6小时后测量管长；（2）免疫荧光：抗vWF（内皮标志物）和抗CD31抗体染色，计数阳性细胞；（3）Western blot：分析细胞裂解物中eNOS和KDR的表达 [5]
。
3. 人系膜细胞（HMC）增殖与细胞周期实验：HMC用含10%胎牛血清的RPMI 1640培养基培养，血清饥饿24小时使细胞同步于G0期，再用100 nM Ang II刺激增殖，同时加入盐酸贝尼地平（0.1-10 μM）。48小时后，MTT法检测增殖：加入20 μL MTT（5 mg/mL）孵育4小时，DMSO溶解甲瓒结晶，570 nm处测吸光度。细胞周期分析：收集细胞，70%乙醇固定，PI（50 μg/mL）和RNase A（100 μg/mL）染色，流式细胞术检测，计算G0/G1、S、G2/M期细胞比例 [6]
。

Animal/Disease Models: MI/R rabbit model [2]
Doses: 3-10 μg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results:caused a significant decrease in HR (heart rate), MABP (mean arterial blood pressure) and PRI (pressure rate index) ), the concentration is 10 µg/kg. At 3 µg/kg, apoptosis-positive cells were diminished to 7.4%.

---

Animal/Disease Models: Renovascular hypertension rat (RHR) model [3]
Doses: 5 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Reduce blood pressure and coronary vascular resistance index. Nitrite production and eNOS mRNA expression as well as resting coronary flow and capillary density were increased.

---

Animal/Disease Models: Rat heart model [4]
Doses: 1-10 mg/kg
Route of Administration: Oral
Experimental Results: Increased LVDP and post-ischemic recovery of LV dP/dt max (LVDP: 87.5±10.1 vs 64.6±11.9%; LV dP /dt max: 97.8±10.4 vs 70.2±15.7%), 3 mg/kg.

---

1. Spontaneously Hypertensive Rat (SHR) antihypertensive study: Male SHRs (12 weeks old, n=6 per group) were randomized into vehicle control (0.5% methylcellulose) and Benidipine HCl groups (1, 3, 10 mg/kg/day). Benidipine HCl was suspended in 0.5% methylcellulose and administered orally via gavage once daily for 2 weeks. Systolic and diastolic blood pressure were measured using a tail-cuff plethysmograph before treatment and every 3 days during treatment. At the end of the study, blood samples were collected for plasma drug concentration analysis, and aortic tissues were harvested for eNOS expression analysis [1]
.
2. Rat myocardial ischemia-reperfusion (I/R) model: Male Sprague-Dawley rats (250-300 g, n=5 per group) were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal). The left anterior descending coronary artery (LAD) was ligated with a 6-0 silk suture for 30 minutes (ischemia), followed by suture release for 2 hours (reperfusion). For pretreatment groups, Benidipine HCl (3 mg/kg) was suspended in 0.5% methylcellulose and administered orally 1 hour before ischemia. For post-reperfusion treatment groups, Benidipine HCl (0.3 mg/kg) was dissolved in normal saline and injected intravenously immediately after reperfusion. At the end of reperfusion, the heart was excised, and infarct size was measured by TTC staining. Serum was collected for CK and LDH analysis [4, 2]
.
3. Hypertensive rat coronary circulation study: Male DOCA-salt hypertensive rats (n=6 per group) were treated with Benidipine HCl (3 mg/kg/day, oral) or vehicle for 4 weeks. Benidipine HCl was formulated in 0.5% methylcellulose. Coronary blood flow (CBF) was measured using a Doppler flow probe placed around the left circumflex coronary artery under anesthesia. Plasma NO levels were determined by Griess reagent, and aortic tissues were collected for NOS activity and eNOS protein analysis [3]
.
4. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4 per group) were fasted for 12 hours before administration. Two groups were established: intravenous (IV) and oral (PO). For IV administration, Benidipine HCl was dissolved in 10% ethanol + 90% normal saline and injected via the tail vein at 1 mg/kg. For PO administration, Benidipine HCl was suspended in 0.5% methylcellulose and administered orally at 5 mg/kg. Blood samples (0.3 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-administration. Plasma was separated by centrifugation (3,000×g for 10 minutes at 4°C), and Benidipine HCl concentration was measured by HPLC-UV. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of Benidipine HCl was 18% after oral administration at 5 mg/kg (vs. IV 1 mg/kg) [1]
; In beagle dogs, oral administration of 2 mg/kg resulted in an F value of 25% [1]
; In healthy human volunteers, oral administration of 4 mg Benidipine HCl (tablet formulation) showed an absolute bioavailability of ~10% (due to first-pass metabolism) [1]
.
2. Plasma pharmacokinetic parameters: In humans, after oral administration of 4 mg Benidipine HCl, the maximum plasma concentration (Cmax) was 5.2 ng/mL, the area under the plasma concentration-time curve (AUC₀₋₂₄) was 28.6 ng·h/mL, and the terminal half-life (t₁/₂) was 14.5 hours [1]
. In rats, IV administration (1 mg/kg) yielded a Cmax of 85 ng/mL, AUC₀₋∞ of 62 ng·h/mL, and t₁/₂ of 2.8 hours; oral administration (5 mg/kg) resulted in a Cmax of 12 ng/mL, AUC₀₋∞ of 58 ng·h/mL, and t₁/₂ of 3.2 hours [1]
.
3. Tissue distribution: In rats, 1 hour after IV administration of 1 mg/kg Benidipine HCl, the highest tissue concentrations were found in the liver (1200 ng/g) and kidneys (850 ng/g), followed by the aorta (150 ng/g) and heart (120 ng/g). The brain concentration was low (15 ng/g), indicating poor blood-brain barrier penetration [1]
. The volume of distribution (Vd) in rats was 8.5 L/kg, suggesting extensive tissue binding [1]
.
4. Metabolism: Benidipine HCl is primarily metabolized in the liver via cytochrome P450 (CYP) enzymes. In human liver microsomes, CYP3A4 is the major metabolic enzyme (accounting for 75% of total metabolism), followed by CYP2D6 (15%). The main metabolites are dihydropyridine ring-hydroxylated derivatives and pyridine ring-oxidized derivatives, which are inactive [1]
.
5. Excretion: In rats, after IV administration of 1 mg/kg Benidipine HCl, 72% of the dose was excreted in feces (mostly as metabolites) within 72 hours, and 15% was excreted in urine (only metabolites, no parent drug detected) [1]
. In humans, fecal excretion accounts for ~60% of the dose, and urinary excretion for ~25% (both as metabolites) [1]
.

1. Acute toxicity: In Sprague-Dawley rats, the oral median lethal dose (LD50) of Benidipine HCl was >2000 mg/kg; the intravenous LD50 was 50 mg/kg [1]
. In mice, oral LD50 was >1500 mg/kg [1]
. At lethal doses, animals showed hypotension, respiratory depression, and hypothermia [1]
.
2. Chronic toxicity: In rats treated with Benidipine HCl (10, 30, 100 mg/kg/day, oral) for 6 months, no significant toxicity was observed at 10 mg/kg. At 30 mg/kg, mild myocardial hypertrophy was noted (no functional impairment). At 100 mg/kg, severe hypotension (SBP <80 mmHg), renal tubular degeneration, and hepatic steatosis were detected. The no-observed-adverse-effect level (NOAEL) was 10 mg/kg [1]
. In dogs treated with 5, 15, 50 mg/kg/day for 6 months, NOAEL was 15 mg/kg (50 mg/kg caused gastrointestinal disturbances and weight loss) [1]
.
3. Plasma protein binding: In human plasma, the protein binding rate of Benidipine HCl was >99% (determined by equilibrium dialysis) [1]
; In rat and dog plasma, binding rates were 98.5% and 99.2%, respectively [1]
.
4. Drug-drug interactions: Benidipine HCl co-administration with CYP3A4 inhibitors (e.g., ketoconazole) increased human plasma AUC by 3.2-fold, due to reduced metabolism [1]
. Co-administration with other antihypertensive drugs (e.g., ACE inhibitors, β-blockers) enhanced hypotensive effects (additive effect), requiring dose adjustment [1]
. No significant interactions with CYP2C9, CYP2C19, or CYP2E1 substrates were observed [1]
.
5. Reproductive and developmental toxicity: In pregnant rats, oral Benidipine HCl (10, 30, 100 mg/kg/day) during gestation had no teratogenic effects at 30 mg/kg; 100 mg/kg caused fetal growth retardation (reduced birth weight) [1]
.

[1]. Pharmacological, pharmacokinetic, and clinical properties of benidipine hydrochloride, a novel, long-acting calcium channel blocker. J Pharmacol Sci. 2006 Apr;100(4):243-61.

[2]. Anti-apoptotic effect of benidipine, a long-lasting vasodilating calcium antagonist, in ischaemic/reperfused myocardial cells. Br J Pharmacol. 2001 Feb;132(4):869-78.

[3]. Benidipine stimulates nitric oxide synthase and improves coronary circulation in hypertensive rats. Am J Hypertens. 1999 May;12(5):483-91.

[4]. Orally administered benidipine and manidipine prevent ischemia-reperfusion injury in the rat heart. Circ J. 2004 Mar;68(3):241-6.

[5]. Benidipine, a dihydropyridine-Ca2+ channel blocker, increases the endothelial differentiation of endothelial progenitor cells in vitro. Hypertens Res. 2006 Dec;29(12):1047-54.

[6]. Broad antiproliferative effects of benidipine on cultured human mesangial cells in cell cycle phases. Am J Nephrol. 2002 Sep-Dec;22(5-6):581-6.

1. Chemical class and mechanism of action: Benidipine HCl is a long-acting dihydropyridine-type calcium channel blocker (DHP-CCB). Its mechanism involves: (1) blocking L-type calcium channels in vascular smooth muscle cells, reducing calcium influx and vasodilation (main antihypertensive effect); (2) activating endothelial NOS, increasing NO production to enhance vasodilation and protect vascular endothelium; (3) inhibiting myocardial cell apoptosis and mesangial cell proliferation, exerting organ protection (heart, kidney) [1, 2, 3, 6]
.
2. Clinical indications and dosage: Benidipine HCl is clinically approved for the treatment of essential hypertension and chronic stable angina pectoris. The recommended oral dose for hypertension is 4-8 mg once daily (maximum 12 mg/day); for angina, 4 mg twice daily [1]
. Its long-acting property (t₁/₂ ~14 hours in humans) allows once-daily administration, improving patient compliance [1]
.
3. Advantage over other DHP-CCBs: Compared to short-acting DHP-CCBs (e.g., nifedipine), Benidipine HCl has a longer duration of action and less reflex tachycardia (due to weaker cardiac calcium channel blocking effect relative to vascular effects). Compared to other long-acting DHPs (e.g., amlodipine), it shows stronger endothelial protection and renal protective effects (reducing mesangial proliferation) [1, 3, 6]
.
4. Preclinical organ protection evidence: Beyond antihypertension, Benidipine HCl demonstrates: (1) myocardial protection against I/R injury (reducing infarct size, inhibiting apoptosis); (2) renal protection (reducing proteinuria, preserving GFR); (3) coronary circulation improvement (increasing CBF via NO activation) [2, 3, 4, 6]
.

C28H31N3O6.HCL

542.02

541.197

91599-74-5

Benidipine;105979-17-7

656667

Light yellow to yellow solid powder

1.3±0.1 g/cm3

625.2±55.0 °C at 760 mmHg

199-201ºC

331.9±31.5 °C

0.0±1.8 mmHg at 25°C

1.622

4.92

124.28

2

8

8

38

933

2

CC1=C([C@H](C(=C(N1)C)C(=O)O[C@@H]2CCCN(C2)CC3=CC=CC=C3)C4=CC(=CC=C4)[N+](=O)[O-])C(=O)OC.Cl

KILKDKRQBYMKQX-MIPPOABVSA-N

InChI=1S/C28H31N3O6.ClH/c1-18-24(27(32)36-3)26(21-11-7-12-22(15-21)31(34)35)25(19(2)29-18)28(33)37-23-13-8-14-30(17-23)16-20-9-5-4-6-10-20;/h4-7,9-12,15,23,26,29H,8,13-14,16-17H2,1-3H3;1H/t23-,26-;/m1./s1

5-O-[(3R)-1-benzylpiperidin-3-yl] 3-O-methyl (4R)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: ≥ 2.08 mg/mL (3.84 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 20.8 mg/mL澄清DMSO储备液加入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。

---

配方 2 中的溶解度: 2.08 mg/mL (3.84 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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。

---

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。