AOD9604 acetate

Anti-obesity peptide

AOD9604是一种肽，由来自氨基酸177-191的人类生长激素的C末端片段组成，在肽的N末端有一个额外的酪氨酸残基。据报道，它模拟了生长激素的脂解特性，没有致糖尿病的副作用。因此，AOD9604可能被用作提高成绩的药物，并被世界反兴奋剂机构（WADA）禁止使用。该肽可在几个互联网网站上找到，最近在美国没收的小瓶中被发现。为了检测运动员对该肽的滥用，在尿液中验证了一种固相萃取方法，检测限为50 pg/mL。该方法具有良好的线性、精密度（<20%）、特异性和回收率（62%）。AOD9604在血清和尿液中孵育后，鉴定出该肽的六种潜在代谢产物。血清中代谢物的定量鉴定出一种由氨基酸CRSVEGSCG组成的单一代谢物，其稳定性明显高于其他代谢物或母体化合物。AOD9604和稳定代谢物的筛选可能会增加检测窗口[2]。

在这项研究中，AOD9604的剂量为0.25mg，与之前关于GH在促进骨关节炎兔胶原酶模型中恢复正常行走和关节修复方面的剂量相当。AOD9604是GH的一个片段；因此，所使用的AOD9604剂量是先前研究使用的活性GH剂量的摩尔当量。以0.6ml关节内注射量给予3mg人生长激素。以摩尔计，3mg GH相当于0.25mg AOD9604。此外，已发表的数据表明，关节炎兔的滑液体积约为0.7 ml。结合注射体积，总体积为1.3 ml，因此AOD9604的初始浓度为0.19 mg/ml。在之前对比格犬关节内注射GH后的研究中，研究人员注射了1.5 mg 0.15 ml体积的GH水溶液。水性制剂在滑液中的初始浓度约为200-300 ug/mL。按摩尔当量计算，这相当于0.11mg/mL的AOD9604，接近本研究中使用的值。 结论：超声引导下关节内注射AOD9604可促进软骨再生，在胶原酶诱导的膝OA兔模型中，联合注射AOD9600和HA比单独注射HA或AOD9604更有效。[1]

AOD9604在血浆和尿液中的代谢[1]
将100μL的人血清以2µg/mL的浓度添加和不添加AOD9604，并在37°C下孵育0，10, 20, 60分钟或0，1.2.6.24小时（n=4）。在每个时间点，加入200μL 1%乙酸和400μL乙腈，摇动溶液10分钟。通过在20 000 x g下离心5分钟去除沉淀物，并在50°C的空气流下将上清液蒸发至干。将样品重新悬浮在100μL 0.1%甲酸中。使用安捷伦6550 QTOF的高分辨率全扫描分析，比较有和没有肽的血清，鉴定代谢物。然后收集每种代谢物的高分辨率产物离子扫描数据。如表4所示，选择了三种产物离子用于MRM转换。血清样本用0.1%甲酸稀释1:10，在Waters Xevo TQ-S上进行代谢物定量。最大百分比定义为每个时间点的平均峰面积除以每种代谢物的最高平均峰面积x 100%。 将1000μL尿液以2µg/mL的浓度添加和不添加AOD9604进行强化，并在室温下孵育24小时。按照所述提取和减少尿液。通过安捷伦6550 QTOF的高分辨率全扫描分析，比较有和没有肽的尿液样本，鉴定了代谢物。收集AOD9604代谢物的产物离子扫描数据。

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特异性和基质抑制[1]
从八名个体中收集空白尿液样本，如上所述提取，并对AOD9604的折叠和线性形式、内标和代谢物（CRSVEGSCG）进行MRM定量。在用DTT还原之前，用2.5 ng/mL AOD9604和内标对来自同一8名个体的第二组提取尿液进行强化。无基质对照含有2.5 ng/mL AOD9604或牛胰岛素内标（50µg/mL），50 mM Tris，pH 8.0。用DTT减少强化尿液和基质对照并测量。基质干扰的百分比计算为：（尿液峰面积-缓冲液峰面积）/缓冲液峰区域x 100%。

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稳定性[1]
用0.3ng/mL和3.0ng/mL的AOD9604强化尿液（n=5），并在室温下孵育，4°C和-20°C，持续0、1、3和8天。按照所述提取尿液，并测量AOD9604和代谢物（CRSVEGSCG）。如果超过50%的样本的信噪比为3或更高，则AOD9604和代谢物被描述为“检测到”，否则被描述为未检测到。

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提取后稳定性[1]
如所述，提取用AOD9604以3.0 ng/mL强化的尿液，并用二硫苏糖醇还原。将样品置于10°C的自动取样器中，并在还原后0和24小时进行测量。 血清样本分析[3]
为了评估hGH片段AOD-9604对常规使用的hGH兴奋剂检测的影响，用Genotropin®（重组hGH；5 ng/ml水溶液）和0、250、500或1000 ng/ml的AOD-9602强化了五个血清样本（SerumMix；来自男性运动员）的混合物。用500 ng/ml的AOD-9604水溶液（100µg/ml）强化人零血清（由试剂盒制造商提供，通常用于试剂盒对照品的重建）和一份阴性兴奋剂常规血清样本（来自一名女运动员）。按照制造商的说明，用试剂盒1和试剂盒2对样品进行分析：将血清样品等分到分析管中，然后加入缓冲液并在室温下孵育2小时。洗涤步骤后，加入检测抗体并在室温下孵育2 h。在另一个洗涤步骤后，在配备LBIS软件版本3.3的Luminometer AutoLumat plus 953上进行了三次分析。试剂盒1和试剂盒2的定量限（LOQ）分别为0.05 ng/ml。

Thirty-two rabbits were divided into 4 equal groups. Four different solutions, including saline, HA, AOD9604, and AOD9604 with HA, were injected in each group on a weekly basis for 4–7 weeks after the first collagenase injection. Group 1 received intra-articular saline injection (0.6 mL). Group 2 received intra-articular HA, (Hyruan-plus®; LG Life Science, Daejeon, Korea) injection (6 mg). The molecular weight of HA was measured at 3.0×106 Da, and it was prepared to a 10 mg/mL concentration. Group 3 received intra-articular AOD9604 (Metabolic pharmaceuticals, Melbourne, Australia) injection (0.25 mg per 0.6 mL). Group 4 received combined intra-articular AOD9604 (0.25 mg) and HA (6 mg) injections. All injections were administered by a physiatrist, using a commercially available ultrasound system with 3–12 MHz multi-frequency linear transducer (E-CUBE 15®; Alpionion Medical Systems, Seoul, Korea) under general anesthesia and under sterile conditions (Figure 1). No medication was administered after the injection. The rabbits were euthanized by CO inhalation 9 weeks after the first collagenase injection. [1]

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Mature New Zealand white rabbits (n=32) were randomly administered 2 mg collagenase type II twice in each knee joint. Weekly injections of 0.6 mL saline (Group 1), 6 mg HA (Group 2), 0.25 mg AOD9604 (Group 3), and 0.25 mg AOD9604 with 6 mg HA (Group 4) were administered for 4-7 weeks after the first intra-articular collagenase injection. The degree of cartilage degeneration was assessed using morphological and histopathological findings, and the degree of lameness was observed at 8 weeks after the first collagenase injection. [1]

Specificity was determined in blank urine from eight individuals. No peaks were detected at the same retention time for the folded or reduced form of AOD9604, the internal standard, or the shortest metabolite, described below. Matrix interference was measured after addition of AOD9604 and the internal standard to extracted urine from eight individuals or Tris buffer using bovine insulin, 50 µg/mL, as a carrier protein. When comparing peak area, the average matrix suppression was −59% for AOD9604 and −59% for the internal standard. However, due to the difference in retention time between the two, the matrix suppression of individual samples was not the same. Therefore, differences between the peak area ratios were present. Since this method is intended to provide a qualitative identification of a synthetic peptide and has a good limit of detection, the level of matrix suppression is acceptable.[3]

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Post-extraction stability[3]
Due to the reversible nature of peptide disulfide bonds, the post-extraction stability of the reduced peptide was determined after incubation in an autosampler at 10 °C for 0 and 24 h. Comparison of the peak area for AOD9604 and the internal standard show that after 24 h, the samples retain 101% and 89% signal, respectively (Table 2). While it is recommended that samples are analyzed immediately after reduction, detection or re-analysis is possible for up to 24 h without significant loss of the reduced peptide.

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In vitro metabolism[3]
AOD9604 is not approved for use in humans although the oral form of the peptide has been granted GRAS status or ‘generally recognized as safe’ by the US Food and Drug Administration (FDA). 8 Since the oral form of the peptide is not available commercially and injection of the peptide is not approved by the FDA, no metabolism experiments could be performed in humans. Therefore, in vitro metabolism experiments were performed by incubation of the peptide in serum and urine. This method has been successfully used to identify potential metabolites for GHRP peptides and Long R3-IGF-1.16, 23 To identify potential metabolites, serum was fortified with and without AOD9604 and incubated at 0, 10, 20, and 60 min. The metabolism was stopped and high molecular weight proteins were removed by addition of acetonitrile. Metabolites were identified by comparing high resolution full scan and product ion scan data from serum with and without the peptide. Several metabolites were identified after incubation in serum as listed in Table 3. Proteolytic degradation was observed at the N-terminal of the peptide which did not proceed beyond the disulfide bonded cysteine residue. Metabolites with degradation at the C-terminus of the peptide were not identified. While both N-terminal and C-terminal specific exopeptidases are present in serum, the folded, hairpin structure of the C-terminal may prevent proteolytic cleavage. Studies performed by A. Thomas et al. demonstrated that the GHRP metabolites identified after in vitro incubation in human serum agreed well with those identified after injection in mice and detected in urine (in vivo). [3]

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Under consideration of the assay's measurement uncertainty of 13%, the ratios of SerumMix samples containing recombinant hGH did not change after addition of the peptide AOD-9604 either and the concentrations determined using the rec- and the pit-assay of Kit 1 and Kit 2 did not increase or decrease. After addition of AOD-9604 to the serum samples fortified with recombinant GH, the concentration of rec-assay did not change (Kit 1: 5.45 ng/ml before and averagely 5.35 ng/ml after addition; Kit 2: 5.12 ng/ml before and averagely 5.55 ng/ml after addition). Also the pit-assay results did not change (Kit 1: 1.66 ng/ml before and averagely 1.74 ng/ml after addition; Kit 2: 1.67 ng/ml before and averagely 1.75 ng/ml after addition). All of the ratios of the SerumMix samples containing recombinant GH showed a clearly ′positive′ result (Kit 1 and Kit 2) before and after addition of AOD-9604 and no ′false negative′ GH finding was generated by the addition of the substance. These first results demonstrate that AOD-9604 itself has no influence on the WADA hGH isoform differential immunoassay, although only a small number of samples were investigated. Since AOD-9604 has recently been categorized as an S0 substance, its use is prohibited in sports and the substance will be the subject of analyses in routine doping controls, which might necessitate further investigations concerning pharmacokinetics in humans and the detection of the intact drug or its metabolites from urine or plasma. If the drug candidate is excreted into urine, implementation into existing peptide screening procedures is conceivable.

[1]. Effect of Intra-articular Injection of AOD9604 with or without Hyaluronic Acid in Rabbit Osteoarthritis Model. Ann Clin Lab Sci. 2015 Summer;45(4):426-32.
[2]. Detection and in vitro metabolism of AOD9604. Drug Test Anal. 2015 Jan;7(1):31-8.
[3]. AOD-9604 does not influence the WADA hGH isoform immunoassay. Drug Test Anal. 2013 Nov-Dec;5(11-12):850-2.

With the growing availability of mature systems and strategies in biotechnology and the continuously expanding knowledge of cellular processes and involved biomolecules, human sports drug testing has become a considerably complex field in the arena of analytical chemistry. Proving the exogenous origin of peptidic drugs and respective analogs at lowest concentration levels in biological specimens (commonly blood, serum and urine) of rather limited volume is required to pursue an action against cheating athletes. Therefore, approaches employing chromatographic-mass spectrometric, electrophoretic, immunological and combined test methods have been required and developed. These allow detecting the misuse of peptidic compounds of lower (such as growth hormone-releasing peptides, ARA-290, TB-500, AOD-9604, CJC-1295, desmopressin, luteinizing hormone-releasing hormones, synacthen, etc.), intermediate (e.g., insulins, IGF-1 and analogs, 'full-length' mechano growth factor, growth hormone, chorionic gonadotropin, erythropoietin, etc.) and higher (e.g., stamulumab) molecular mass with desired specificity and sensitivity. A gap between the technically possible detection and the day-to-day analytical practice, however, still needs to be closed. https://pubmed.ncbi.nlm.nih.gov/25382550/

C78H123N23O23S2.CH3COOH

1875.13 (acetate); 1815.10 (free base)

1813.8603

C, 51.61; H, 6.83; N, 17.75; O, 20.27; S, 3.53

221231-10-3

71300630

H-Tyr-Leu-Arg-1le-Val-GIn-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-OH (Disulfide bond Cys&Cys)

YLRIVQCRSVEGSCGF

White to off white powder or loose lump

1.5±0.1 g/cm3

1.667

-3.46

815.3

28

28

45

126

3710

15

S1C[C@@H](C(N[C@H](C(N[C@@H](CO)C(N[C@H](C(N[C@H](C(NCC(N[C@@H](CO)C(N[C@H](C(NCC(N[C@H](C(=O)O)CC2C=CC=CC=2)=O)=O)CS1)=O)=O)=O)CCC(=O)O)=O)C(C)C)=O)=O)CCCNC(=N)N)=O)NC([C@H](CCC(N)=O)NC([C@H](C(C)C)NC([C@H]([C@@H](C)CC)NC([C@H](CCCNC(=N)N)NC([C@H](CC(C)C)NC([C@H](CC1C=CC(=CC=1)O)N)=O)=O)=O)=O)=O)=O

GVIYUKXRXPXMQM-BPXGDYAESA-N

InChI=1S/C78H123N23O23S2/c1-9-41(8)62(101-68(115)47(18-14-28-86-78(83)84)91-69(116)50(29-38(2)3)95-63(110)45(79)30-43-19-21-44(104)22-20-43)75(122)100-61(40(6)7)74(121)94-49(23-25-56(80)105)67(114)98-55-37-126-125-36-54(65(112)88-32-57(106)89-51(76(123)124)31-42-15-11-10-12-16-42)97-70(117)52(34-102)90-58(107)33-87-64(111)48(24-26-59(108)109)93-73(120)60(39(4)5)99-71(118)53(35-103)96-66(113)46(92-72(55)119)17-13-27-85-77(81)82/h10-12,15-16,19-22,38-41,45-55,60-62,102-104H,9,13-14,17-18,23-37,79H2,1-8H3,(H2,80,105)(H,87,111)(H,88,112)(H,89,106)(H,90,107)(H,91,116)(H,92,119)(H,93,120)(H,94,121)(H,95,110)(H,96,113)(H,97,117)(H,98,114)(H,99,118)(H,100,122)(H,101,115)(H,108,109)(H,123,124)(H4,81,82,85)(H4,83,84,86)/t41-,45-,46-,47-,48-,49-,50-,51-,52-,53-,54-,55-,60-,61-,62-/m0/s1

(2S)-2-[[2-[[(4R,7S,13S,16S,19S,22S,25R)-25-[[(2S)-5-amino-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylpentanoyl]amino]-3-methylbutanoyl]amino]-5-oxopentanoyl]amino]-22-(3-carbamimidamidopropyl)-13-(2-carboxyethyl)-7,19-bis(hydroxymethyl)-6,9,12,15,18,21,24-heptaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23-heptazacyclohexacosane-4-carbonyl]amino]acetyl]amino]-3-phenylpropanoic acid

AOD 9604; 221231-10-3; UNII-7UP768IP4M; AOD-9604; 7UP768IP4M; AOD9604 acetate; Tyr-somatostatin (177-191);

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)

H2O：＞1mg/ml.

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。