Immunoaffinity purification of peptide hormones prior to liquid chromatography-mass spectrometry in doping controls.

For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (∼25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled.

Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly.

A 44 amino acid peptide with growth hormone-releasing activity has been isolated from a human tumor of the pancreas that had caused acromegaly. The primary structure of the tumor-derived peptide is H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala- Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly -Ala-Arg-Ala-Arg-Leu-NH2. The synthetic replicate has full biological activity in vitro and in vivo specifically to stimulate the secretion of immunoreactive growth hormone. The tumor-derived peptide is identical in biological activity and similar in physiochemical properties to the still uncharacterized growth hormone-releasing factor present in extracts of hypothalamic tissues.

Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(3-44)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).

Partial purification and characterization of a peptide with growth hormone-releasing activity from extrapituitary tumors in patients with acromegaly.

Growth hormone (GH)-releasing activity has been detected in extracts of carcinoid and pancreatic islet tumors from three patients with GH-secreting pituitary tumors and acromegaly. Bioactivity was demonstrated in 2 N acetic acid extracts of the tumors using dispersed rat adenohypophyseal cells in primary monolayer culture and a rat anterior pituitary perifusion system. The GH-releasing effect was dose responsive and the greatest activity was present in the pancreatic islet tumor. Small amounts of activity were also found in two other tumors (carcinoid and small cell carcinoma of lung) unassociated with GH hypersecretion. Each of the tumors contained somatostatin-like immunoreactivity but the levels did not correlate with the net biologic expression of the tumor. Sephadex G-75 gel filtration indicated the GH-releasing activity to have an apparent molecular size of slightly greater than 6,000 daltons. The GH-releasing activity was adsorbed onto DEAE-cellulose at neutral pH and low ionic strength, from which it could be eluted by increasing ionic strength. The GH-releasing activity was further purified by high pressure liquid chromatography using an acetonitrile gradient on a cyanopropyl column to yield a preparation that was active at 40 ng protein/ml. Partially purified GH-releasing activity, from which most of the bioactive somatostatin had been removed, increased GH release by pituitary monolayer cultures to five times base line. Enzymatic hydrolysis studies revealed that the GH-releasing activity was resistant to carboxypeptidase, leucine-aminopeptidase, and pyroglutamate-amino-peptidase but was destroyed by trypsin and chymotrypsin, indicating that internal lysine and/or arginine and aromatic amino acid residues are required for biologic activity and that the NH2-terminus and CO9H-terminus are either blocked or not essential. The results provide an explanation for the presence of GH-secreting tumors in some patients with the multiple endocrine neoplasia syndrome, type I, and warrant the addition of GH-releasing activity to the growing list of hormones secreted by tumors of amine precursor uptake and decarboxylation cell types.

Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS.

A complete analytical protocol for the determination of 25 doping-related peptidic drugs and 3 metabolites in urine was developed by means of accurate-mass quadrupole time-of-flight (Q-TOF) LC-MS analysis following solid-phase extraction (SPE) on microplates and conventional SPE pre-treatment for initial testing and confirmation, respectively. These substances included growth hormone releasing factors, gonadotropin releasing factors and anti-diuretic hormones, with molecular weights ranging from 540 to 1320Da. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. Weak cation exchange SPE followed by C18 HPLC chromatography and accurate mass detection provided the required sensitivity and selectivity for all the target peptides under study. 2mg SPE on 96-well microplates can be used in combination with full scan MS detection for the initial testing, thus providing a fast, cost-effective and high-throughput protocol for the processing of a large batch of samples simultaneously. On the other hand, extraction on 30mg SPE cartridges and subsequent target MS/MS determination was the protocol of choice for confirmatory purposes. The methodology was validated in terms of selectivity, recovery, matrix effect, precision, sensitivity (limit of detection, LOD), cross contamination, carryover, robustness and stability. Recoveries ranged from 6 to 70% (microplates) and 17-95% (cartridges), with LODs from 0.1 to 1ng/mL. The suitability of the method was assessed by analyzing different spiked or excreted urines containing some of the target substances.

Partial isolation and characterization of testicular GnRH-like factors.

We report here partial isolation and characterization of at least two GnRH-receptor binding factors from the ethanol: chloroform: acetic acid (ECA) extracts of rat testis. The displacement curve of defatted, steroid-free and desalted ECA extract was parallel to that of D-(leu)6-des (Gly)10-GnRH-EA in a GnRH-radioreceptor assay. Immunoaffinity chromatography on cyanogen bromide-activated Sepharose 4B beads covalently bound to an antibody raised against d-(lys)6-GnRH resulted in more than a hundredfold increase in receptor binding specific activity. Equivalent amounts of kidney extract after affinity chromatography showed no significant activity. Coincubation of the material purified by affinity chromatography with the labeled ligand did not result in significant peptidase degradation of the label, indicating that apparent displacement of the label in the receptor assay was not the result of cleavage of the ligand. HPLC of the material partially purified by affinity chromatography on a reverse phase 5 micron ODS column revealed two peaks of receptor binding activity. Preliminary estimates of molecular weights of these factors based on SDS-PAGE and gel filtration are 68,000 and 6,000 respectively. We conclude that there are at least two factors in rat testis with GnRH-receptor-binding properties that are chemically distinct from the native decapeptide.

Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H](2+)), and their sequences, SIPNLPQRF-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH(2) (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10(-7) M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Presence of growth hormone-releasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties.

The distribution and physical and biological properties of GH-releasing hormone-like immunoreactivity (GHRH-IR) in human tissues and tumors was investigated using a specific GHRH RIA, gel chromatography, immunoaffinity chromatography, and bioassay with cultured rat anterior pituitary cells. Variable concentrations of GHRH-IR, ranging from 1.4-39.0 ng/g wet wt, were found in normal liver, lung, placenta, and pancreas. In the latter tissue, however, a different chromatographic profile and a marked decrease in GHRH-IR after immunoaffinity occurred, suggesting that GHRH-IR in pancreatic extracts is not native GHRH. In all tumors examined (n = 35) GHRH-IR could be detected, and four tumors (three carcinoids and one jejunal carcinoma) contained a very high amount of GHRH-IR (greater than 1000 ng/g wet wt). Affinity chromatography of tumor extracts led to a significant loss (greater than 50%) of GHRH-IR in nine tumors. The four tumors containing large amounts of GHRH-IR were obtained from two patients with active acromegaly and two patients who had no clinical evidence of acromegaly. Using antibodies with different specificities for GHRH-(1-44) and GHRH shortened at the C-terminus, varying concentrations of GHRH-(1-44) in these tumors were found, ranging from 10-87% of the total GHRH-IR. The biological activity of GHRH in the four tumor extracts was similar to that of synthetic GHRH alone or GHRH added to control tissue subjected to extraction. These results demonstrate the presence of GHRH-IR in the majority of normal tissues and tumors, which, though they may produce large amounts of biologically active GHRH, do not always lead to acromegaly.

Developmental changes in the expression of growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide in zebrafish.

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) are structurally and functionally related members of the glucagon superfamily, a group of hormones important in development, growth, and metabolism. Our objectives were to determine the developmental expression pattern of the ghrh-pacap1 gene using the zebrafish model. The temporal and spatial expression pattern of the ghrh-pacap1 gene was examined by RT-PCR and in situ hybridization. In zebrafish, the ghrh-pacap1 mRNA transcript was expressed throughout development beginning at the transition between the blastula and gastrula periods. During midgastrulation, alternative splicing resulted in the generation of a novel transcript lacking the cryptic peptide. During the segmentation period, expression was localized to the neural tube, developing eye, and neural crest; strong expression was found in the developing cerebellum. Later in development, expression was localized in the hatching gland and developing pharyngeal arches. The temporal and spatial expression pattern of the ghrh-pacap1 transcript suggests that these hormones may modulate patterning during development.

Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.

Partial purification and characterization of a novel growth hormone-releasing factor (GRF) from teleost brain related to the rat hypothalamic peptide.

A growth hormone-releasing factor (GRF)-like molecule has been partially purified and characterized from acid extracts of codfish (Gadhus morhua) brain using immunoaffinity and gel chromatography, followed by HPLC. This material has a mol.wt. which is similar to known mammalian forms of GRF but is immunologically and/or chromatographically distinct from previously described GRF peptides. However, it is related to rat(r) GRF(1-43) since it causes marked displacement in the rGRF RIA. Codfish GRF is a highly specific and potent hypophysiotropic factor as shown by its ability to stimulate the release of GH, but no other hormone, from rat anterior pituitary cells in vitro. These findings suggest that, phylogenetically, GRF is an ancient molecule with its biologic activity and certain immunoreactive domain(s) conserved, at least, from teleost to mammal.

Primary structure of neuropeptide Y from brains of the American alligator (Alligator mississippiensis).

The purification of NPY from brains of the American alligator (Alligator mississippiensis) was achieved using reverse-phase high performance liquid chromatography (HPLC). The amino acid sequence was determined using automated Edman degradation as Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu- Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile- Asn-Leu - Ile-Thr-Arg-Gln-Arg-Tyr. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The conservation of alligator NPY suggests that serine in position 7 of chicken NPY evolved after the birds and reptiles diverged from a common Archosaurian ancestor. Furthermore, the sequence identity between alligator and human NPY suggests this sequence is the same as the ancestral amniote NPY.

Human growth hormone releasing factor and somatostatin from two pancreatic tumors: isolation and characterization.

Peptides with high intrinsic activity to release growth hormone from pituitary cells in tissue cultures were isolated from two different human pancreatic tumors that had caused acromegaly. Homogeneous peptides were obtained after gel filtration and two steps of reverse-phase high-performance liquid chromatography. From one tumor a 44-residue peptide (human pancreas growth hormone releasing factor, hpGRF-44) was isolated, together with two shorter fragments of reduced bioactivity having 40 and 37 amino acid residues (hpGRF-40, hpGRF-37). In contrast, the other tumor contained only one form of GRF which proved to be identical to hpGRF-40. These hpGRFs are indistinguishable from partially purified preparations of hypothalamic growth hormone releasing factor of human, porcine and murine origins with respect to biological activity and are very similar in their physicochemical properties (molecular weight, retention behavior on reverse-phase HPLC, absence of sulfhydryl groups). One of the pancreatic tumors also contained two forms of immunoreactive somatostatin. One form, after isolation and partial microsequencing, was identified as somatostatin-14 with a structure identical to that of the peptide found in other species. The second form has tentatively been identified as somatostatin-28 on the basis of chromatographic behavior.

Radioimmunoassay of growth hormone-releasing hormone (GHRH) with a polyclonal antibody against synthetic GHRH(1-29)-Gly4-Cys-NH2: method and clinical studies.

A radioimmunoassay (RIA) for growth hormone-releasing hormone (GHRH) using a polyclonal antibody against synthetic GHRH(1-29)-Gly4-Cys-NH2 has been developed. The antiserum (RBM105) showed full cross-reactivity with GHRH-(1-44)NH2, GHRH-(1-40)OH, GHRH-(1-37)OH and GHRH-(3-44)NH2, and probably recognized the region of Ala4 to Lys12 of GHRH. Since the sensitivity of the GHRH RIA was 1.5 pg/tube, the lowest detectable plasma level was 5 ng/l when an extract of 0.3 ml of plasma per tube was used. On gelfiltration chromatography, the GHRH immunoreactivity of normal plasma was eluted in the same position as synthetic GHRH. The plasma GHRH concentration in healthy subjects was 20.5 +/- 6.5 ng/l (mean +/- SD), and in patients with hypothalamic disorders was 17.4 +/- 2.0 ng/l. In contrast, the plasma GHRH level in hemodialysis-dependent, chronic renal failure (CRF-HD) patients (38.7 +/- 13.1 ng/l) was significantly higher than normal. The acromegalic patients were 24.3 +/- 11.9 ng/l, except for one patient with ectopic GHRH syndrome (990 ng/l): his plasma GHRH level reached 7,100 ng/l during operation, and then decreased logarithmically to 70 ng/l after 6 h. Somatostatin at concentrations of 10 and 1,000 nmol/l significantly suppressed (GHRH release) from primary culture cells of the GHRH-producing tumor from 17.3 +/- 0.92 ng/2 x 10(5) cells to 9.98 +/- 3.61 and 4.32 +/- 1.01 ng/2 x 10(5) cells, respectively after 48 h. These data indicate that this GHRH RIA is useful for determining the plasma GHRH concentration in normal and diseased states and also for in vitro studies of GHRH release.

Set-up of large laboratory-scale chromatographic separations of poly(ethylene glycol) derivatives of the growth hormone-releasing factor 1-29 analogue.

In this paper we report the scale-up of the purification of poly(ethylene glycol) (PEG) derivatives of the growth hormone-releasing factor 1-29, from laboratory scale (100 mg of bulk starting material) to larger scale (3 g of bulk), through the use of a cation-exchange TSK-SP-5PW chromatographic column. A one-step purification process capable of purifying large amounts of mono-PEGylated GRF species from the crude reaction mixture was developed. A simple, straightforward stepwise gradient elution separation was developed at laboratory scale and then scaled up with a larger column packed with a chromatographic resin with the same chemistry which maintained the laboratory-scale separation profile. Active material recovery and material purity remained constant through the scale-up from the 13-microm stationary phase to the 25-microm larger column. Overall, the gram GRF equivalent/batch process scale showed to be quite reproducible, and could be considered as a good platform for scale up to production scale.

Comparison of separation and detection techniques for human growth hormone releasing factor (hGRF) and the products derived from deamidation.

Separation of the deamidation products, Asp8 Leu27 hGRF(1-32)NH2 (MH+ = 3654) and isoAsp8 Leu27 hGRF(1-32)NH2 (MH+ = 3654), from the parent analogue Leu27 hGRF(1-32)NH2 (MH+ = 3653) was achieved by reversed-phase LC and CE, where the retention order was seen to change from tr isoAsp8 hGRF < tr Asn8 hGRF < tr Asp8 hGRF to tr Asn8 hGRF < tr Asp8 hGRF < tr isoAsp8 hGRF, respectively. Both reversed-phase LC and CE gave adequate separations, limits of detection and standard curves. However, CE was preferred due to shorter analysis time, better separation and a smaller demand for material. Packed capillary LC with ESI-MS was then compared with UV detection. On-line LC-MS was found to offer the most efficient approach to detection and identification of hGRF analogues within a single methodology. Identification of Asn8 hGRF from the isobaric deamidation products was achieved from analysis of the triply charged states, where the species were separated by 0.5 amu. LC-MS separation and identification of degradation products offers a viable alternative to fraction collection and subsequent sequencing or enzymatic identification methods. The method becomes increasingly useful for such cases as trace degradation product identification, minimal sample availability or instability of resulting degradation products.

[Purification and activity assay of recombinant growth hormone-releasing hormone].

OBJECTIVE: To express recombinant growth hormone-releasing hormone (GHRH) peptide in E.coli by genetic engineering and examine its biological activity. METHOD: GHRH peptide was purified to homogeneity by means of cell lysis, washing, ethanol precipitation, acid hydrolysis, SP-Sephadex C25 and Sephadex G-25 column chromatographies. RESULT: SDS-PAGE showed that the recombinant plasmid pET-28a /L-ansB-GHRH in E.coli BL21(DE3) expressed the fusion protein under the induction with IPTG. The fusion protein was expressed in the form of inclusion body, accounting for 30% of the total bacterial protein. After the purification procedures, the peptide was purified about 147-fold with a peptide yield of 0.68%. The molecular mass of the peptide was 5 235 Da as determined by electrospray ionization (ESI) mass spectrum, in agreement with the predicted value, and SDS-PAGE presented a single peak in assessment of the purity. Experiment showed that there were significant differences in the growth hormone released by the peptide between the dose groups and the blank control group, and the differences tended to be more obvious with the increase of the doses. CONCLUSION: The recombinant GHRH peptide possesses good biological activities.

Acromegaly induced by ectopic secretion of GHRH: a review 30 years after GHRH discovery.

Ectopic acromegaly is very rare and since the discovery of growth hormone-releasing hormone (GHRH), 30 years ago, only 74 cases have been reported in the literature. Except for a recent French series of 21 cases, most of them were case reports. The present review summarizes the current knowledge on clinical presentation, diagnosis and prognosis. Tumors secreting GHRH are neuroendocrine tumors, usually well differentiated and mainly from pancreatic or bronchial origin. They are usually large and easy to localize using TDM and somatostatin receptor scintigraphy. Clinical presentation is an acromegaly of variable intensity, whose features are similar to that of a somatotropic adenoma. Pituitary may be normal or enlarged at MRI which may be difficult to interpret especially in MEN1 patients where the association of a microprolactinoma to a pancreatic tumor secreting GHRH may be misleading. GHRH plasmatic measurement has an excellent specificity for the diagnosis, using a threshold of 250 to 300ng/L and is a good tool for follow-up of patients after treatment. These tumors have a good overall prognosis, even in metastatic forms which represent 50% of cases. Surgical approach is recommended and, when a complete tumoral resection is feasible, results, in most patients, in long-lasting remission. In such cases, GHRH concentration is normalized and its increase is an accurate indicator of recurrence. In uncured patients, somatostatin analogs control GH secretion but inhibit, only partially, GHRH secretion. MEN1 mutation should be systematically investigated in patients with a pancreatic tumor.

Identification of CJC-1295, a growth-hormone-releasing peptide, in an unknown pharmaceutical preparation.

Several peptide drugs are being manufactured illicitly, and in some cases they are being made available to the public before entering or completing clinical trials. At the request of Norwegian police and customs authorities, unknown pharmaceutical preparations suspected to contain peptide drugs are regularly subjected to analysis. In 2009, an unknown pharmaceutical preparation was submitted for analysis by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). The preparation was found to contain a 29 amino acid peptide with a C-terminal amide function. Based on the interpretation of mass spectrometric data, an amino acid sequence was proposed. The sequence is consistent with a peptide currently marketed under the name CJC-1295. CJC-1295 is a releasing factor for growth hormone and is therefore considered a Prohibited Substance under Section S2 of the WADA Prohibited List. This substance has potential performance-enhancing effects, it is readily available, and there is reason to believe that it is being used within the bodybuilding community.