Anti-obesity peptide; The mechanism of action of AOD9604 is unique, as its primary target is not the classic growth hormone receptor. Studies indicate that AOD9604 does not compete for the growth hormone receptor nor induce cell proliferation through it. Its lipolytic action is thought to involve the β3-adrenergic receptor pathway: chronic administration upregulates β3-AR RNA expression in adipocytes, and the weight-loss effect is absent in β3-AR knockout mice, although acute administration still increases energy expenditure, suggesting additional pathways may be involved.

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].

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In vitro studies indicate that AOD9604 lacks the classical growth hormone's proliferative activity. In experiments using BaF-BO3 cells transfected with the human growth hormone receptor, AOD9604 failed to competitively inhibit ¹²⁵I-labeled hGH binding to the receptor or stimulate cell proliferation, in stark contrast to intact hGH. Additionally, in vitro metabolism studies in serum and urine identified six potential metabolites of AOD9604, among which a metabolite consisting of the amino acid sequence CRSVEGSCG was significantly more stable than the parent compound.

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].

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Animal studies demonstrate various in vivo activities of AOD9604. In obese mouse models, administration via mini-osmotic pumps for 14 days significantly reduced body weight gain, increased in vivo fat oxidation, and elevated plasma glycerol levels (an index of lipolysis). Unlike intact hGH, AOD9604 did not induce hyperglycemia or reduce insulin secretion. In a rabbit osteoarthritis model, intra-articular AOD9604 injections enhanced cartilage regeneration and reduced lameness. In a rat osteoporosis model, oral AOD9604 (0.25 mg/kg/day) prevented ovariectomy-induced bone loss and fragility.

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.

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To investigate the binding capacity of AOD9604 to the growth hormone receptor, a radioligand competition binding assay can be performed. Membrane fragments expressing the human growth hormone receptor are incubated with a fixed concentration of ¹²⁵I-labeled growth hormone (as tracer) and increasing concentrations of unlabeled AOD9604 or growth hormone (as positive control) in an appropriate buffer. After incubation to reach equilibrium, bound and free radioligands are separated by rapid vacuum filtration or centrifugation. The radioactivity on the filter or in the pellet is measured using a gamma counter, and the concentration of AOD9604 required to inhibit 50% of tracer binding (IC₅₀) or the inhibition constant (Kᵢ) is calculated.

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.

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To assess whether AOD9604 induces cell proliferation through the growth hormone receptor, cell lines dependent on growth hormone (such as BaF-BO3 cells transfected with the human growth hormone receptor) can be used. Cells are seeded in 96-well plates and starved in growth hormone-free medium for several hours to synchronize the cells. Subsequently, increasing concentrations of AOD9604 or growth hormone (positive control) are added and incubated for 48-72 hours. Cell viability/proliferation is measured using the MTT assay or CellTiter-Glo luminescent assay. Significant cell proliferation is typically observed in the growth hormone-treated group, while no significant proliferative effect is observed in the AOD9604-treated group.

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 using blank urine samples from 8 subjects. No peaks of the folded or reduced form of AOD9604, the internal standard, or the shortest metabolite described below were detected at the same retention time. Matrix interference was determined by adding AOD9604 and the internal standard to extracted urine or Tris buffer from 8 subjects, with 50 µg/mL bovine insulin as the carrier protein. The mean matrix inhibition rate of AOD9604 and the internal standard was -59% when comparing peak areas. However, the matrix inhibition rate was not the same for different samples due to the different retention times. Therefore, the peak area ratios were different. The degree of matrix inhibition was acceptable because this method is designed for the qualitative identification of synthetic peptides and has a good detection limit. [3]

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Post-extraction stability[3]
Post-extraction stability of the reduced peptide was determined after incubation at 10 °C for 0 h and 24 h in an autosampler due to the reversibility of the peptide disulfide bond. The peak area comparison of AOD9604 and the internal standard showed that the samples retained 101% and 89% of the signal, respectively, after 24 hours (Table 2). Although it is recommended to analyze the sample immediately after reduction, detection or reanalysis within 24 hours will not result in significant loss of the reduced peptide.

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In vitro metabolism[3]
Although the oral form of AOD9604 has been granted GRAS (Generally Recognized As Safe) status by the U.S. Food and Drug Administration (FDA), the peptide has not been approved for human use. 8 Since the oral formulation of the peptide has not been marketed and the injectable formulation has not been approved by the FDA, it is not possible to conduct metabolic experiments in humans. Therefore, we used in vitro metabolism experiments, incubating the peptide with serum and urine, respectively. This method has been successfully used to identify potential metabolites of GHRP peptide and long-chain R3-IGF-1. 16, 23 To identify potential metabolites, we incubated the peptide in serum with and without AOD9604 for 0, 10, 20 and 60 minutes, respectively. After the metabolic reaction was terminated, we added acetonitrile to remove high molecular weight proteins. Metabolites were identified by comparing high-resolution full scan and product ion scan data of serum with and without peptides. As shown in Table 3, several metabolites were identified after incubation in serum. We observed proteolytic degradation at the N-terminus of the peptide, but the degradation did not exceed the disulfide-linked cysteine residues. No metabolites of C-terminal degradation of the peptide were identified. Although N-terminal and C-terminal specific exopeptidases are present in serum, the hairpin structure of the C-terminus may prevent proteolytic cleavage. A. Thomas et al. showed that the GHRP metabolites identified after in vitro incubation in human serum were highly consistent with the metabolites (in vivo) injected into mice and detected in urine. [3]

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Considering the 13% measurement uncertainty of this detection method, the proportion of SerumMix samples containing recombinant hGH did not change after the addition of peptide AOD-9604, and the concentrations measured by rec- and pit- detection methods of kit 1 and kit 2 did not increase or decrease. The addition of AOD-9604 to serum samples containing recombinant growth hormone (GH) did not change the concentration of GH (Kit 1: 5.45 ng/ml before addition, average 5.35 ng/ml after addition; Kit 2: 5.12 ng/ml before addition, average 5.55 ng/ml after addition). Gonadotropin-releasing hormone (Pit) levels also remained unchanged (Kit 1: 1.66 ng/ml before addition, average 1.74 ng/ml after addition; Kit 2: 1.67 ng/ml before addition, average 1.75 ng/ml after addition). All serum samples containing GH showed significantly positive results before and after the addition of AOD-9604 (Kit 1 and Kit 2), with no false negatives. Preliminary results indicate that although only a small number of samples were tested, AOD-9604 itself did not affect the differential immunoassay for WADA hGH isotypes. Because AOD-9604 was recently classified as an S0 substance, it is banned in sports and will become a target for routine doping tests. This may require further investigation into its pharmacokinetics in humans, as well as the detection of the intact drug or its metabolites in urine or plasma. If the candidate drug is excreted in urine, it could be considered for inclusion in existing peptide drug screening programs.

The toxicological profile of AOD9604 is primarily characterized by its advantages over intact growth hormone. Animal studies demonstrate that AOD9604 does not cause the typical growth hormone-related adverse effects such as hyperglycemia and inhibition of insulin secretion. Regarding liver safety, one mouse study showed that 8 weeks of AOD9604 combined with exercise significantly reduced alanine aminotransferase levels, whereas growth hormone injection led to elevations in certain liver damage markers (e.g., cytokeratin 18). Currently, publicly available systematic evaluation data on the long-term toxicity profile of AOD9604, as well as its potential reproductive toxicity, carcinogenicity, and other toxicological endpoints, are limited.

[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 increasing prevalence of mature systems and strategies in the field of biotechnology, and the deepening understanding of cellular processes and related biomolecules, the detection of sports drugs has become a highly complex topic in analytical chemistry. To combat cheating among athletes, it is necessary to demonstrate the source of peptide drugs and their analogues at extremely low concentration levels in biological samples with limited sample sizes (typically blood, serum, and urine). Therefore, chromatography-mass spectrometry, electrophoresis, immunology, and combined detection methods have emerged and developed accordingly. These methods can detect the misuse of low molecular weight (e.g., growth hormone-releasing peptide, ARA-290, TB-500, AOD-9604, CJC-1295, desmopressin, luteinizing hormone-releasing hormone, corticotropin, etc.), medium molecular weight (e.g., insulin, IGF-1 and its analogues, "full-length" mechanotrophic factor, growth hormone, human chorionic gonadotropin, erythropoietin, etc.), and high molecular weight (e.g., stalmumab) peptide compounds with the required specificity and sensitivity. However, a gap remains between technically feasible detection methods and routine analytical practices, which needs to be further bridged. 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.)