Anti-obesity peptide

AOD9604 is a peptide consisting of the C-terminal fragment of human growth hormone from amino acids 177-191 with an additional tyrosine residue at the N-terminus of the peptide. It is reported to mimic the lipolytic properties of growth hormone without the diabetogenic side effects. Therefore, AOD9604 may be used as a performance enhancing drug and is banned by the World Anti-doping Agency (WADA). The peptide is available on several Internet websites and was recently identified in confiscated vials in the USA. To detect abuse of the peptide in athletes, a solid-phase extraction method was validated in urine with a limit of detection of 50 pg/mL. The method has good linearity, precision (<20%), specificity and recovery (62%). Six potential metabolites of the peptide were identified after incubation of AOD9604 in serum and urine. Quantification of the metabolites in serum identified a single metabolite, consisting of amino acids CRSVEGSCG, which is significantly more stable than the other metabolites or the parent compound. Screening for AOD9604 and the stable metabolite may potentially allow an increased window of detection [2].

In this study, AOD9604 was given in a dose of 0.25 mg that is comparable to the dose used in a previous report on GH in promoting recovery to normal walking and in joint repair in the rabbit collagenase model of osteoarthritis. AOD9604 is a fragment of GH; therefore the dose of AOD9604 used was the molar equivalent of the active GH dose that the previous study used. Human GH was given as 3 mg in 0.6 ml intra-articular injection volume. On a molar basis, 3 mg of GH equates to 0.25 mg of AOD9604. In addition, published data suggest that the volume of synovial fluid in an arthritic rabbit is approximately 0.7 ml. Combined with the injection volume, this gives a total volume of 1.3 ml and therefore an initial concentration of AOD9604 of 0.19 mg/ml. In a previous study of GH in the beagle after intra-articular injection, researchers injected 1.5 mg of GH in aqueous solution in 0.15 ml volume. The aqueous formulation gave an initial concentration of approximately 200–300 ug/mL in the synovial fluid. On a molar equivalent basis this equates to 0.11 mg/mL of AOD9604, which is close to the value used in this study. Conclusion: Intra-articular AOD9604 injections using ultrasound guidance enhanced cartilage regeneration, and combined AOD9604 and HA injections were more effective than HA or AOD9604 injections alone in the collagenase-induced knee OA rabbit model [1].

AOD9604 metabolism in plasma and urine [1]
One hundred μL of human serum was fortified with and without AOD9604 at 2 µg/mL and incubated at 37 °C for 0, 10, 20, 60 min, or 0, 1, 2, 6, 24 h (n = 4). At each time point, 200 μL of 1% acetic acid and 400 μL of acetonitrile were added and solution was incubated with shaking for 10 min. The precipitate was removed by centrifugation at 20 000 x g for 5 min and the supernatant was evaporated to dryness under a stream of air at 50 °C. The samples were resuspended in 100 μL of 0.1% formic acid. Metabolites were identified using high resolution full scan analysis on the Agilent 6550 QTOF comparing serum with and without the peptide. High resolution product ion scan data was then collected for each metabolite. Three product ions were selected for MRM transitions as listed in Table 4. Serum samples were diluted 1:10 with 0.1% formic acid and metabolite quantification was performed on the Waters Xevo TQ-S. The percent of maximum is defined as the average peak area at each time point divided by the highest average peak area for each metabolite x 100%. One thousand μL of urine was fortified with and without AOD9604 at 2 µg/mL and incubated at room temperature for 24 h. Urine was extracted and reduced as described. Metabolites were identified by high resolution full scan analysis on the Agilent 6550 QTOF comparing urine samples with and without the peptide. Product ion scan data was collected for AOD9604 metabolites.

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Specificity and matrix suppression [1]
Blank urine samples were collected from eight individuals, extracted as described above, and MRM quantification was performed for the folded and linear form of AOD9604, the internal standard and the metabolite (CRSVEGSCG). A second set of extracted urine from the same eight individuals was fortified with 2.5 ng/mL AOD9604 and internal standard prior to reduction with DTT. The matrix-free control contained 2.5 ng/mL AOD9604 or internal standard in bovine insulin (50 µg/mL), 50 mM Tris, pH 8.0. Fortified urine and matrix controls were reduced with DTT and measured. The % of matrix interference was calculated as: (peak area in urine – peak area in buffer)/peak area in buffer x 100%.

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Stability [1]
Urine was fortified with AOD9604 at 0.3 ng/mL and 3.0 ng/mL (n = 5) and incubated at room temperature, 4 °C, and −20 °C for 0, 1, 3, and 8 days. The urine was extracted as described and AOD9604 and the metabolite (CRSVEGSCG) were measured. AOD9604 and the metabolite were described as ‘detected’ if greater than 50% of the samples were detected with a signal-to-noise of 3 or greater, otherwise they were described as not detected.

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Post-extraction stability [1]
Urine fortified with AOD9604 at 3.0 ng/mL was extracted and reduced with dithiothreitol as described. The samples were placed in an autosampler at 10 °C and measured at 0 and 24 h post-reduction.

Analysis of serum samples [3]
In order to estimate the effect of the hGH-fragment AOD-9604 on the routinely used doping control assay for hGH , a mixture of five serum samples (SerumMix; from male athletes) was fortified with Genotropin® (recombinant hGH; 5 ng/ml aqueous solution) and 0, 250, 500, or 1000 ng/ml of AOD-9604. Human zero serum (supplied by the kit manufacturer, usually used for reconstitution of the controls of the kits) and one negative doping routine serum sample (from a female athlete) was fortified with 500 ng/ml of an aqueous solution of AOD-9604 (100 µg/ml). Samples were assayed with Kit 1 and Kit 2 as given in the manufacturer's instructions: Aliquotation of the serum samples to the assay tubes is followed by addition of buffer and incubation at room temperature for 2 h. After a washing step the detection antibody is added and incubation was preformed for 2 h at room temperature. After another washing step analysis was performed in triplicates on a Luminometer AutoLumat plus 953 equipped with LBIS software version 3.3 . Assay limit of quantification (LOQ) for Kit 1 and Kit 2 is 0.05 ng/ml for rec- and pit-assay, respectively.

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

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.

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)