CJC-1295 With and Without DAC: Chemistry and the Research Literature

Research-use-only context. This article summarizes published third-party scientific literature and laboratory practice. It is not medical advice, not a therapeutic or performance claim, and not a usage guide. US Prime Peptides products are supplied strictly for in vitro laboratory research and are not for human or veterinary use.

This article explains the two molecules sold under the name CJC-1295, what each of the four amino acid substitutions in the GHRH(1-29) frame does, what the Drug Affinity Complex adds and how it binds albumin, what the rat and human-volunteer literature reports about growth hormone secretion, why ConjuChem's development program ended, and how a certificate of analysis tells the two forms apart.

One name, two molecules

"CJC-1295" refers to two related peptides, and a research buyer needs to know which one is in the vial. The base molecule is a 29-residue analog of growth hormone-releasing hormone, GHRH(1-29)-NH2, carrying four substitutions: D-alanine at position 2, glutamine at position 8, alanine at position 15 and leucine at position 27. Vendors call this "modified GRF(1-29)," "Mod GRF 1-29" or "CJC-1295 without DAC." Its formula is C152H252N44O42 and its molecular weight is about 3,367.9 Da. The second molecule adds a thirtieth residue, a lysine whose side-chain amine carries a 3-maleimidopropionyl group, and is the compound ConjuChem actually named CJC-1295; it is sold as "CJC-1295 with DAC" and has a molecular weight of about 3,647 Da. DAC stands for Drug Affinity Complex, ConjuChem's term for the maleimide linker.

The unmodified 29-residue parent is sermorelin, covered in the sermorelin overview, and TH9507 takes the alternative route of capping the N-terminus rather than substituting residues.

What the four substitutions do, with the papers behind each

Each substitution answers a specific degradation route documented in the GHRH analog literature of the 1980s and 1990s. The D-alanine at position 2 addresses dipeptidyl peptidase IV. Frohman, Downs, Heimer and Felix showed in a 1989 Journal of Clinical Investigation paper that plasma DPP-IV cleaves native GHRH and its 1-29 fragment at the Ala2-Asp3 bond, and that a D-amino acid at position 1 or 2 prevents the cleavage. Heiman and colleagues had already reported in a 1985 Endocrinology paper that [D-Ala2]hpGRF-29 was about fifty times more active than the parent fragment in dispersed rat pituitary cells, so the substitution improves potency as well as stability.

The glutamine at position 8 and the leucine at position 27 address chemical rather than enzymatic decay. Campbell and colleagues, in a 1994 Peptides paper, documented that asparagine 8 in the human sequence rearranges to aspartate and isoaspartate through an aminosuccinimide intermediate in aqueous solution, and that methionine 27 oxidizes to the sulfoxide; both changes reduce activity. Their analogs used Gln8 and Leu27, and gained stability in vitro without losing activity in rat pituitary cell culture. The alanine at position 15 comes from the same lineage: Kubiak and colleagues, in a 1993 Journal of Medicinal Chemistry paper on bovine GRF analogs, used Ala15 in place of Gly15 to improve bioactivity, and Campbell's paper describes the Ala15 substitution as previously shown to enhance receptor binding affinity.

Taken together, these four changes produce a 29-residue peptide that survives plasma DPP-IV and resists the two main solution-phase chemical reactions of the native sequence. That is the whole of what "modified GRF(1-29)" is; nothing in the base molecule extends its circulation beyond what a stabilized 3.4-kDa peptide would normally have.

What the Drug Affinity Complex adds

The DAC is a maleimide that forms a covalent bond with the free thiol of cysteine 34 on serum albumin, so that the peptide circulates attached to a 66-kDa carrier protein. Jette and colleagues at ConjuChem laid this out in a 2005 Endocrinology paper. They synthesized three maleimido derivatives of hGRF(1-29), conjugated each to human serum albumin ex vivo, and found that all three conjugates resisted DPP-IV in vitro and released growth hormone from cultured rat anterior pituitary cells. In male Sprague-Dawley rats, the derivative they named CJC-1295 produced a four-fold larger growth hormone area under the curve over two hours than unmodified hGRF(1-29), was still measurable in plasma beyond 72 hours, and appeared on Western blots as an immunoreactive species at the albumin band from 15 minutes to beyond 24 hours after exposure.

The chemistry has two consequences for a laboratory. First, the maleimide is a reactive Michael acceptor: in any solution that contains free thiols, whether albumin in serum-containing culture medium or reducing agents such as dithiothreitol or 2-mercaptoethanol, the peptide will attach to them, which is the designed behavior and must be accounted for in experimental design. Second, maleimides slowly hydrolyze in aqueous buffer to a ring-opened maleamic acid that no longer reacts with thiols, so the reactive form is only guaranteed in freshly dissolved material. Timms, Ganio and colleagues describe the analytical side of the same chemistry in two 2019 Drug Testing and Analysis papers on equine plasma: once conjugated, the peptide becomes a macromolecule of undefined molecular weight that evades standard peptide mass-spectrometry screens, which is why they developed an immuno-PCR screen sensitive to 0.8 pg/mL and a confirmation method using immunoaffinity capture, tryptic digestion and LC-MS/MS down to 180 pg/mL.

What the human volunteer studies measured

The DAC form was studied in healthy adults in two ConjuChem-sponsored trials. Teichman and colleagues reported both in a 2006 Journal of Clinical Endocrinology and Metabolism paper: randomized, placebo-controlled, double-blind, ascending-amount designs lasting 28 and 49 days. After a single exposure, mean plasma growth hormone rose two- to ten-fold for six days or more and mean IGF-I rose 1.5- to three-fold for nine to eleven days; the estimated half-life of the conjugated peptide was 5.8 to 8.1 days, and after repeated exposures IGF-I remained above baseline for up to 28 days.

Ionescu and Frohman asked a more specific physiological question in a second 2006 Journal of Clinical Endocrinology and Metabolism paper: what happens to the normal pulsatile pattern of growth hormone secretion when the pituitary receives continuous GHRH-receptor stimulation? They sampled healthy men every 20 minutes overnight before and one week after a single exposure. Pulse frequency and pulse magnitude were unchanged; what rose was the trough level between pulses, by about 7.5-fold, which lifted mean overnight growth hormone by about 46 percent and IGF-I by about 45 percent. The paper's conclusion is that pulsatility persists during continuous stimulation, with the gain coming from the baseline rather than the peaks.

Sackmann-Sala, Ding, Frohman and Kopchick used sera from eleven of those volunteers in a 2009 Growth Hormone and IGF Research paper to look for proteomic markers of the raised growth hormone and IGF-I; two-dimensional gel electrophoresis followed by mass spectrometry identified decreased apolipoprotein A1 and transthyretin isoforms and increased beta-hemoglobin and albumin fragments. These three papers are the entire peer-reviewed human literature on the compound.

Development history and regulatory status

CJC-1295 is not an approved drug in any jurisdiction, and its development program ended in 2006. ConjuChem, a Montreal company, had advanced the DAC form into phase 2 for HIV-associated lipodystrophy. In 2006 the company disclosed that a participant in one of its studies had died; the company stated that the event had not been established as related to the compound, and development of CJC-1295 was discontinued afterward as the company redirected its resources. That history exists in company disclosures and secondary accounts rather than in a peer-reviewed report, and should be cited as such. Teichman's 2006 paper, written before the program closed, reported no serious adverse reactions in its own two trials.

Since then the compound's literature has been almost entirely about detection. Henninge and colleagues, in a 2010 Drug Testing and Analysis paper, analyzed an unknown preparation seized by Norwegian police and customs by high-resolution LC-MS/MS and found a 29-residue amidated peptide whose proposed sequence matched the product being marketed as CJC-1295, a reminder that what is sold under the name is often the DAC-free base molecule. Both forms are listed under section S2 of the World Anti-Doping Agency Prohibited List, and Memdouh and colleagues' 2021 Drug Testing and Analysis method covers sermorelin, TH9507, CJC-1295 and CJC-1295 with DAC together, with nineteen synthesized in vitro metabolites as reference standards. A research vial is not a drug product of any kind and carries none of the clinical history above.

Telling the two forms apart on a certificate

The mass spectrum is the line that settles which CJC-1295 is in the vial, because the two forms differ by 279 Da. The DAC-free base should deconvolute to about 3,368 Da, typically observed as 3+ and 4+ ions near m/z 1,123 and 843; the DAC form should deconvolute to about 3,647 Da. A certificate that reports a mass near 3,368 for a product labeled "with DAC" is describing a different molecule from the one on the label. A third mass is worth knowing: the maleimide ring opens on hydrolysis with a gain of 18 Da, so a DAC-form lot showing a significant peak near 3,665 Da contains material that has lost its thiol reactivity. The interpretation of charge states and deconvolution is covered in LC-MS identity confirmation.

HPLC purity should be reported as area percent with the chromatogram attached; the Leu27 and Gln8 substitutions remove the two degradation peaks that complicate sermorelin chromatograms, so a clean main peak is a fair expectation for the base form. Net peptide content, endotoxin by LAL, sterility and ICP-MS metals complete the panel. CJC-1295 is supplied by US Prime Peptides only in two-component vials, CJC-1295 / Ipamorelin 5 mg / 5 mg and CJC-1295 / GHRP-6 5 mg / 5 mg, and the lot-matched certificate for each must identify and quantify both components separately, which is straightforward here because the GHRH analog and the secretagogue differ in mass by more than 2,000 Da and elute far apart. How to read such a document is explained in reading a blend certificate, and the pharmacological reason the pairing is studied, two different receptors on the same cell, is in CJC-1295 vs ipamorelin.

Frequently Asked Questions

What is the difference between CJC-1295 with DAC and without DAC?

Both share a 29-residue GHRH(1-29) frame with D-Ala2, Gln8, Ala15 and Leu27 substitutions. The DAC form adds a thirtieth residue, a lysine carrying a maleimidopropionyl group, that bonds covalently to cysteine 34 of albumin in plasma. The DAC-free form is about 3,368 Da; the DAC form is about 3,647 Da.

Is CJC-1295 approved by the FDA?

No. CJC-1295 has never been approved by the FDA or any other regulator. ConjuChem discontinued development in 2006 after reporting a death in its phase 2 program, an event the company stated was not established as related to the compound, and it is listed on the WADA Prohibited List.

What did the CJC-1295 human studies find?

Teichman and colleagues (2006) reported growth hormone elevations lasting six days or more and IGF-I elevations lasting nine to eleven days after a single exposure in healthy adults, with a half-life of 5.8 to 8.1 days. Ionescu and Frohman (2006) found that growth hormone pulse frequency and size were unchanged while trough levels rose about 7.5-fold.

Why does CJC-1295 bind albumin?

The maleimide group on the DAC form is a Michael acceptor that reacts with the single free thiol on albumin, Cys34, forming a stable covalent bond. Jette and colleagues showed in 2005 that the conjugated peptide appeared at the albumin band on Western blots of rat plasma within 15 minutes and persisted beyond 24 hours.

Citations

1. Heiman ML, Nekola MV, Murphy WA, Lance VA, Coy DH. An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormone-releasing factor analogs. Endocrinology. 1985;116:410-415. PMID 2856874
2. Frohman LA, Downs TR, Heimer EP, Felix AM. Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma. J Clin Invest. 1989;83:1533-1540. PMID 2565342
3. Kubiak TM, Friedman AR, Martin RA, et al. Position 2 and position 2/Ala15-substituted analogs of bovine growth hormone-releasing factor (bGRF) with enhanced metabolic stability and improved in vivo bioactivity. J Med Chem. 1993;36:888-897. PMID 8464043
4. Campbell RM, Stricker P, Miller R, et al. Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences. Peptides. 1994;15:489-495. PMID 7937325
5. Jette L, Leger R, Thibaudeau K, et al. Human growth hormone-releasing factor (hGRF)1-29-albumin bioconjugates activate the GRF receptor on the anterior pituitary in rats: identification of CJC-1295 as a long-lasting GRF analog. Endocrinology. 2005;146:3052-3058. PMID 15817669
6. Teichman SL, Neale A, Lawrence B, et al. Prolonged stimulation of growth hormone (GH) and insulin-like growth factor I secretion by CJC-1295, a long-acting analog of GH-releasing hormone, in healthy adults. J Clin Endocrinol Metab. 2006;91:799-805. PMID 16352683
7. Ionescu M, Frohman LA. Pulsatile secretion of growth hormone (GH) persists during continuous stimulation by CJC-1295, a long-acting GH-releasing hormone analog. J Clin Endocrinol Metab. 2006;91:4792-4797. PMID 17018654
8. Sackmann-Sala L, Ding J, Frohman LA, Kopchick JJ. Activation of the GH/IGF-1 axis by CJC-1295, a long-acting GHRH analog, results in serum protein profile changes in normal adult subjects. Growth Horm IGF Res. 2009;19:471-477. PMID 19386527
9. Henninge J, Pepaj M, Hullstein I, Hemmersbach P. Identification of CJC-1295, a growth-hormone-releasing peptide, in an unknown pharmaceutical preparation. Drug Test Anal. 2010;2:647-650. PMID 21204297
10. Timms M, Ganio K, Forbes G, Bailey S, Steel R. An immuno polymerase chain reaction screen for the detection of CJC-1295 and other growth-hormone-releasing hormone analogs in equine plasma. Drug Test Anal. 2019;11:804-812. PMID 30489688
11. Timms M, Ganio K, Steel R. A method for confirming CJC-1295 abuse in equine plasma samples by LC-MS/MS. Drug Test Anal. 2019;11:1248-1257. PMID 30938069
12. Memdouh S, Gavrilovic I, Ng K, et al. Advances in the detection of growth hormone releasing hormone synthetic analogs. Drug Test Anal. 2021;13:1871-1887. PMID 34665524

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