GHS/growth hormone secretagogue (Ki =7 nM for hGHS-R1a; EC50 = 3 nM in rat pituicyte )

通过在HEK293细胞中使用[125I]-ghrelin作为放射性配体过表达克隆的人GHS-R1a（hGHS-R1a）的竞争性结合试验测量结合亲和力Capromorelin的效力比GHS-R1a的内源性配体ghrelin低17倍（见表1）。Capromorelin的结合活性显示出立体特异性，吡唑啉酮哌啶杂环的3aR异构体优于3aS异构体。PP基团上N-甲基基团的去除导致活性损失两倍。N-乙基PP衍生物5b在测定中与胃饥饿素等效。[1]

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尽管表现出30倍的结合亲和力，但Capromorelin和类似物5b和5c以相似的效力刺激GH释放（见表1）。Capromorelin（5a）的3aS非对映异构体显示出明显较弱的活性，EC50值大于1μM。有趣的是，化合物3的3aR-和3aS-非对映异构体（PP二肽的结构前体）都能有效刺激GH分泌（EC50s<25 nM），尽管3aS-非对映异构体稍受青睐。9除5a外，GHS的结合亲和力并不能预测细胞测定中的活性，这可能是因为全细胞测定中蛋白质结合的潜在混杂效应。

在静脉注射后，在麻醉的大鼠模型中测量了PP二肽类似物的体内GH活性Capromorelin和化合物5b和5c在单次1mg/kg剂量后刺激GH分泌，尽管Capromerelin和5c的平均GH峰高明显高于5b的平均GH峰值高度（数据未显示）Capromorelin和5c在模型中显示出类似的剂量-反应关系，ED50值小于0.05 mg/kg iv。N-乙基PP衍生物5b的体内活性较弱归因于其亲脂性增加，这可能减少了血浆室中能够与GHS-R1a相互作用的未结合药物的量[1]
在狗模型中检查了Capromorelin的口服活性，发现该模型可以预测人类的GHS活性。在单次口服1 mg/kg剂量后，Capromorelin迅速增加了血浆GH水平，最大峰高为73 ng/mL。在低至0.05 mg/kg的剂量下观察到明显的GHS活性。当以1 mg/kg口服给药5天后，Capromolelin在研究的第一天和最后一天刺激了GH分泌；然而，第5天的给药后GH反应有所减弱。

结合分析[1]
用质粒pcDNA3.1neo中稳定转染人GHS-R1a受体cDNA的HEK293细胞（ATCC）制备膜。用预浸在0.3%聚乙烯亚胺中的GF/C过滤器以96孔格式进行竞争性放射性配体结合分析。在室温下使用50 pM[125I]-ghrelin和每孔1μg膜在50 mM HEPES、pH 7.4、10 mM MgCl2、0.2%牛血清白蛋白和以下蛋白酶抑制剂中进行1小时的两次检测：100μg/mL杆菌肽、100μg/mL苯甲脒、5μg/mL抑肽酶、5μg/mL亮肽。收集膜并用含有50mM HEPES、pH 7.4和10mM MgCl2的冰冷洗涤缓冲液洗涤三次。使用Prism by Graphpad™测定IC50和Ki值。[125I]-ghrelin在表达人GHS受体的膜上的Kd计算为0.2 nM。在未转染的HEK293细胞膜中未观察到可检测的结合（数据未显示）。

体外功能活性（大鼠垂体细胞培养中GH的释放）[1]
通过酶促分离6周龄雄性Wistar大鼠的垂体前腺建立原代垂体细胞培养物。将细胞悬浮在Dulbecco改良的Eagle培养基（DMEM，4.5 g/L葡萄糖，补充1 mM丙酮酸钠，1%MEM非必需氨基酸，10%热灭活马血清，2.5%胎牛血清加抗生素）中，以每孔1×105个细胞的速度接种在24孔组织培养板上，并在37°C下在加湿的5%CO2/95%空气培养箱中培养。电镀后3-4天进行激素释放。将细胞培养物漂洗两次，然后在37°C下在释放培养基（DMEM，含25 mM HEPES缓冲液，pH 7.4和5 mg/mL牛血清白蛋白）中平衡30分钟。将该培养基吸出，并用含有测试试剂的预热释放培养基替换。在37°C下孵育15分钟后，取出培养基并测定GH。结果以四个孔的平均值±SEM表示。除非另有说明，否则采用非配对学生的双侧t检验对各组进行比较。数据被报告为产生半最大响应（EC50）所需的浓度。MK-0677用作对照板的参考标准。

Pharmacokinetics of Capromorelin in rats[1]
Eight adult female Sprague–Dawley rats were prepared for use by surgical implantation of a cannula in the femoral vein while under methoxyflurane anesthesia the day before study initiation. Study rats were fasted overnight prior to dosing, allowed free access to water and housed in standard polycarbonate rodent cages. The rodents were allowed access to food 4 h post-dose. The dose (intravenous and oral) was prepared as a 0.5 mg/mL base equivalent (0.5 mgA/kg) solution of Capromorelin in deionized water. Four Sprague–Dawley rats (weighing 0.280–0.305 kg) received an iv dose of 1 mgA/kg of Capromorelin. The dose was administered via the femoral vein catheter followed by a 1 mL infusion of normal saline to rinse the catheter. Blood samples (0.5 mL) were taken from the femoral vein catheter pre-dose, 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose and transferred into heparinized microtainers. The blood volume withdrawn was replaced with normal saline. Plasma was harvested from the centrifuged microtainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes. Four Sprague–Dawley rats (weighing 0.275–0.290 kg) received an oral dose of 1 mgA/kg Capromorelin. The dose was administered via an 18-gauge, 3 in., curved gavage needle. Blood samples (0.5 mL) were taken from the femoral vein catheter pre-dose, 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose and transferred into heparinized microtainers. The blood volume withdrawn was replaced with normal saline. Plasma was harvested from the centrifuged microtainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes.

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Pharmacokinetics of Capromorelin in dogs[1]
Four adult beagle dogs (2 males and 2 females weighing 8.9–12.9 kg) were fasted overnight prior to dosing but were allowed free access to water. The dogs were allowed access to food 4 h post-dose. The iv dose was prepared as a 10 mgA/mL solution of Capromorelin in deionized water and filter sterilized. The oral dose was prepared as a 2 mgA/mL solution of Capromorelin in deionized water. Four beagle dogs (weighing 8.9–12.9 kg) received an oral dose of 1 mgA/kg of Capromorelin. The dose was administered via an oral gavage tube. Blood samples (2 mL) were taken from the jugular vein pre-dose, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose. Blood samples were collected directly into heparinized vacutainers. Plasma was harvested from the centrifuged vacutainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes. Following a 2 day wash-out period, the same four beagle dogs received an iv dose of 1 mgA/kg of Capromorelin. The dose was administered via the cephalic vein. Blood samples (2 mL) were taken from the jugular vein pre-dose, 0.083, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6 and 8 h post-dose. Blood samples were collected directly into heparinized vacutainers. Plasma was harvested from the centrifuged vacutainers and stored frozen at −70 °C in 500 μL polypropylene microcentrifuge tubes.

Pharmacokinetic properties of two PP dipeptide GHSs, Capromorelin and 5c, were measured in female Sprague–Dawley rats. For Capromorelin, plasma clearance (CL), volume of distribution (Vd) and plasma elimination half-life (t1/2) following a 1 mgA/kg iv dose were 34±5 mL/min/kg, 1.7 L/kg and 0.79±0.21 h respectively. Capromorelin was rapidly absorbed after a 1 mgA/kg oral (po) dose, reaching maximum systemic concentrations (Cmax) of 329±158 ng/mL in 0.25 h. The unbound plasma free fraction was high (Fu=27%), possibly explaining the robust GHS activity in the in vivo anesthesized rat model. Oral bioavailability was 65%, far greater than the reported bioavailabilities of other peptidyl or peptidomimetic GHSs in the literature. Compound 5c was detected as a circulating metabolite, though exposure generally represented less than 10% of parent drug. Pharmacokinetic values for 5c were similar to those for Capromorelin. Following a 1 mgA/kg iv dose, systemic clearance was high and volume of distribution was moderate, resulting in a short half-life (CL=56.7 mL/min/kg; Vd=2.6 L/kg; t1/2=0.83 h). Oral bioavailability was low, most likely due to the combination of incomplete intestinal absorption and high CL (F=12%).[1]
As the only PP GHS with superior bioavailability and good absorption properties in a rat model, Capromorelin was selected for pharmacokinetic evaluation in the dog. After a 1 mgA/kg iv dose, the plasma clearance of the compound was 19±5 mL/min/kg, the volume of distribution was 2.0±0.4 L/kg and the elimination half-life was 1.3 h. Mean values for Cmax and Tmax following a 1 mgA/kg po dose were 180±66 ng/mL and 1 h. The plasma free fraction of drug was 51% and the bioavailability was 44%. Compound 5c was also identified as a circulating plasma metabolite.[1]
Pharmacokinetic characterization of Capromorelin in rats and dogs revealed short plasma elimination half-lives. In dogs, the short half-life was attributed to a moderate volume of distribution and a moderate-to-high clearance due in part to de-methylation of the PP ring and oxidation of both the benzyl group in the (d)-O-Bn-Ser moiety and a methyl group in the Aib moiety (data not shown). The compound did not undergo proteolytic degradation in plasma. The des-methyl metabolite 5c exhibited a similarly short half-life, but because of its lower rat bioavailability, it did not offer any advantages over Capromorelin. A short half-life was considered pharmacologically desirable for stimulating GH secretion (rather than increasing IGF-1 levels) and preventing attenuation of the post-dose GH response during repeat administration.[1]

[1]. Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs). Discovery of capromorelin. Bioorg Med Chem. 2003 Feb 20;11(4):581-90.

Capromorelin is under investigation in clinical trial NCT00527046 (Effects Of An Oral Growth Hormone Secretagogue In Older Functionally Limited Adults).
See also: Capromorelin Tartrate (has salt form).

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Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituicyte EC(50)=3 nM) and the des-methyl analogue 5c (hGHS-R1a K(i)=17 nM, rat pituicyte EC(50)=3 nM), increased plasma GH levels in an anesthesized rat model, with ED(50) values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-duration GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal frailty in elderly adults.[1]

C28H35N5O4

505.6086

505.268

193273-66-4

193273-66-4; 193273-69-7 (tartrate)

216208

Typically exists as solid at room temperature

1.3±0.1 g/cm3

1.621

4.07

117

2

6

9

37

878

2

O=C1[C@@]2(C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])C(C([H])([H])C([H])([H])N(C([C@@]([H])(C([H])([H])OC([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])N([H])C(C(C([H])([H])[H])(C([H])([H])[H])N([H])[H])=O)=O)C2([H])[H])=NN1C([H])([H])[H]

KVLLHLWBPNCVNR-SKCUWOTOSA-N

InChI=1S/C28H35N5O4/c1-27(2,29)25(35)30-22(18-37-17-21-12-8-5-9-13-21)24(34)33-15-14-23-28(19-33,26(36)32(3)31-23)16-20-10-6-4-7-11-20/h4-13,22H,14-19,29H2,1-3H3,(H,30,35)/t22-,28-/m1/s1

N-[(2R)-1-[(3aR)-3a-benzyl-2-methyl-3-oxo-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl]-1-oxo-3-phenylmethoxypropan-2-yl]-2-amino-2-methylpropanamide;(2R,3R)-2,3-dihydroxybutanedioic acid

Capromorelin; 193273-66-4; Capimorelin; Capromorelin [INN]; CP-424391; CP-424,391; UNII-0MQ44VUN84; 0MQ44VUN84;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。