Ipamorelin, the first selective growth hormone secretagogue

The development and pharmacology of a new potent growth hormone (GH) secretagogue, ipamorelin, is described. Ipamorelin is a pentapeptide (Aib-His-D-2-Nal-D-Phe-Lys-NH2), which displays high GH releasing potency and efficacy in vitro and in vivo. As an outcome of a major chemistry programme, ipamorelin was identified within a series of compounds lacking the central dipeptide Ala-Trp of growth hormone-releasing peptide (GHRP)-1. In vitro, ipamorelin released GH from primary rat pituitary cells with a potency and efficacy similar to GHRP-6 (ECs) = 1.3+/-0.4nmol/l and Emax = 85+/-5% vs 2.2+/-0.3nmol/l and 100%). A pharmacological profiling using GHRP and growth hormone-releasing hormone (GHRH) antagonists clearly demonstrated that ipamorelin, like GHRP-6, stimulates GH release via a GHRP-like receptor. In pentobarbital anaesthetised rats, ipamorelin released GH with a potency and efficacy comparable to GHRP-6 (ED50 = 80+/-42nmol/kg and Emax = 1545+/-250ng GH/ml vs 115+/-36nmol/kg and 1167+/-120ng GH/ml). In conscious swine, ipamorelin released GH with an ED50 = 2.3+/-0.03 nmol/kg and an Emax = 65+/-0.2 ng GH/ml plasma. Again, this was very similar to GHRP-6 (ED50 = 3.9+/-1.4 nmol/kg and Emax = 74+/-7ng GH/ml plasma). GHRP-2 displayed higher potency but lower efficacy (ED50 = 0.6 nmol/kg and Emax = 56+/-6 ng GH/ml plasma). The specificity for GH release was studied in swine. None of the GH secretagogues tested affected FSH, LH, PRL or TSH plasma levels. Administration of both GHRP-6 and GHRP-2 resulted in increased plasma levels of ACTH and cortisol. Very surprisingly, ipamorelin did not release ACTH or cortisol in levels significantly different from those observed following GHRH stimulation. This lack of effect on ACTH and cortisol plasma levels was evident even at doses more than 200-fold higher than the ED50 for GH release. In conclusion, ipamorelin is the first GHRP-receptor agonist with a selectivity for GH release similar to that displayed by GHRH. The specificity of ipamorelin makes this compound a very interesting candidate for future clinical development.

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Ipamorelin, the first selective growth hormone secretagogue

Kirsten Raun, Birgit Sehested Hansen

, Nils Langeland Johansen

, Henning Thøgersen

, Kjeld Madsen

Michael Ankersen

and Peter Høngaard Andersen

Departments of GH Biology,

Assay and Cell Technology,

Medical Chemistry Research, Health Care Discovery, Novo Nordisk A/S, Novo Nordisk Park,

DK-2760 Ma

̊løv, Denmark

(Correspondence should be addressed to Kirsten Raun, Health Care Discovery, Novo Nordisk A/S, Niels Steensensvej 8, DK-2820 Gentofte, Denmark)

Abstract

The development and pharmacology of a new potent growth hormone (GH) secretagogue, ipamorelin,

is described. Ipamorelin is a pentapeptide (Aib-His-D-2-Nal-D-Phe-Lys-NH

), which displays high GH

releasing potency and efficacy in vitro and in vivo. As an outcome of a major chemistry programme,

ipamorelin was identified within a series of compounds lacking the central dipeptide Ala-Trp of growth

hormone-releasing peptide (GHRP)-1.

In vitro, ipamorelin released GH from primary rat pituitary cells with a potency and efficacy similar

to GHRP-6 (EC

1⁄41.360.4 nmol/l and E

max

1⁄48565% vs 2.260.3 nmol/l and 100%). A

pharmacological profiling using GHRP and growth hormone-releasing hormone (GHRH) antagonists

clearly demonstrated that ipamorelin, like GHRP-6, stimulates GH release via a GHRP-like receptor.

In pentobarbital anaesthetised rats, ipamorelin released GH with a potency and efficacy comparable

to GHRP-6 (ED

1⁄480642 nmol/kg and E

max

1⁄415456250 ng GH/ml vs 115636 nmol/kg and

11676120 ng GH/ml).

In conscious swine, ipamorelin released GH with an ED

1⁄42.360.03 nmol/kg and an E

max

1⁄4

6560.2 ng GH/ml plasma. Again, this was very similar to GHRP-6 (ED

1⁄43.961.4 nmol/kg and

max

1⁄47467 ng GH/ml plasma). GHRP-2 displayed higher potency but lower efficacy (ED

1⁄4

0.6 nmol/kg and E

max

1⁄45666 ng GH/ml plasma).

The specificity for GH release was studied in swine. None of the GH secretagogues tested affected

FSH, LH, PRL or TSH plasma levels. Administration of both GHRP-6 and GHRP-2 resulted in increased

plasma levels of ACTH and cortisol. Very surprisingly, ipamorelin did not release ACTH or cortisol in

levels significantly different from those observed following GHRH stimulation. This lack of effect on

ACTH and cortisol plasma levels was evident even at doses more than 200-fold higher than the ED

for GH release.

In conclusion, ipamorelin is the first GHRP-receptor agonist with a selectivity for GH release similar

to that displayed by GHRH. The specificity of ipamorelin makes this compound a very interesting

candidate for future clinical development.

European Journal of Endocrinology 139 552–561

Introduction

Growth hormone (GH) has been used for treating

various types of growth disorders for a number of years.

Following the availability of recombinant GH in 1985,

the unlimited supply has made it possible to investigate the

more general metabolic effects of GH. Currently, a number

of data suggests benefits of using GH in the frail elderly,

osteoporosis, in catabolic states and wasting conditions,

e.g. following surgery (1, 2). Convincing effects following

GH treatment in obesity have also been reported (3).

Somatotrophs in the anterior pituitary gland release

GH in a pulsatile fashion under the regulation of two

hypothalamic peptides: GH-releasing hormone (GHRH)

and somatostatin. GHRH stimulates GH synthesis and

secretion, while somatostatin inhibits GH release with-

out affecting GH synthesis (4, 5). In addition, a third

system, the GH-releasing peptide (GHRP) system also

seems to be involved in the regulation of GH release (6,

7). Over the past years a growing interest in GHRPs has

evolved. It has been suggested that this novel class of GH

secretagogues, including GHRP-6 (6) and MK-677 (8),

offer an advantage compared with exogenously admin-

istered GH, by increasing plasma GH levels in a more

physiologically relevant, pulsatile fashion (9).

A number of GH secretagogues have proven effica-

cious in elevating plasma GH in animals, e.g. in rats

(10), dogs (11) and swine (12). More recently, GHRP-2

(13), GHRP-6 (14), hexarelin (15), and MK-677 (16)

have also been shown to elevate plasma GH in humans.

All of these GH secretagogues potently release GH and,

in addition, prolactin (PRL) and cortisol, the latter

probably indirectly by stimulating adrenocorticotrophin

(ACTH) (17, 18).

European Journal of Endocrinology (1998) 139 552–561 ISSN 0804-4643

q1998 Society of the European Journal of Endocrinology

In the present study we describe the development of a

novel series of GHRP receptor-active GH secretagogues.

A prototype of this series, ipamorelin, releases GH with

high potency and efficacy without significant effects on

plasma ACTH or cortisol levels, compared with the main

endogenous GH releasing compound, GHRH. Conse-

quently, ipamorelin is the most selective GHRP receptor-

active GH secretagogue described.

Materials and methods

Chemistry

The secretagogues shown in Table 1 were synthesised

stepwise on a solid support using the Boc peptide

synthesis strategy on an Applied Biosystems Model

430A peptide synthesizer or by using the Fmoc strategy

on an Applied Biosystems Model 431A peptide synthe-

sizer. The standard protocols supplied by the manufac-

turer were used. In all cases the crude peptides were

purified using semi-preparative RP-HPLC to a purity of

>95% (based on HPLC with detection at 214 nm). The

compounds were all characterised by analytical HPLC

and by plasma desorption mass spectrometry. The

molecular mass found was in agreement with the

expected value in all cases.

In particular, ipamorelin was prepared from a total of

4.53 g of the peptide resin Boc-Aib-His (Trt)-D-2Nal-D-

Phe-Lys (Boc)-NH-resin according to the Fmoc strategy

on an Applied Biosystems 431A peptide synthesizer in

two identical runs (to obtain sufficient material) in

1 mmol scale using the FastMoc UV protocols supplied

by the manufacturer, that employ HBTU mediated

couplings in NMP and UV monitoring of the deprotec-

tion of the Fmoc protection group.

The starting resin used for the synthesis was

2×1.75 g (4-((20,40-dimethoxyphenyl)-(Fmoc-amino)-

methyl)-phenoxy resin (Rink resin) (Novabiochem,

Bad Soden, Germany, cat. no. 01–64–0013) with a

substitution capacity of 0.55 mmol/g. The protected

amino acid derivatives used were Fmoc-Lys(Boc)-OH,

Fmoc-D-Phe-OH, Boc-D-2Nal-OH, Boc-His(Trt)-OH and

Boc-Aib-OH (Boc-Amino isobutyric acid). The peptide

was cleaved from the 4.53 g peptide resin by treatment

with 54 ml TFA/phenol/ethanedithiol/thioanisole/

water 40:3:1:2:2 for 3 h at room temperature.

Purification was carried out by semi-preparative

RP-HPLC and the final product was characterised by

amino acid analysis, analytical RP-HPLC and by plasma

desorption mass spectroscopy. Amino acid analysis and

mass spectrometry were in agreement with the expected

structure and within the experimental error of the

method (mass spectrometry60.9 amu, amino acid

analysis610%). In addition

H-NMR analysis was

performed and the spectra assigned. The NMR spectra

fully supported the proposed structure.

Ipamorelin is a white amorphous powder isolated as a

trifluoroacetate. Its solubility in distilled water is

>200 mg/ml. The pKas of the compound are 10.3,

7.8, and 6.2.

Rat pituitary cell assay

Sprague-Dawley male albino rats (250625 g) were

purchased from Møllegaard, Lille Skensved, Denmark.

The rats were housed in group cages (four to eight

animals/cage) and placed in rooms with a 12-h light

cycle. The room temperature varied from 19 –24 8C and

the humidity from 30–60%. All media were obtained

from Gibco (Paisley, UK), trypsin from Worthington (NJ,

USA), BSA, DNase, tri-iodothyronine (T

) and dexa-

methasone from Sigma (St Louis, MO, USA).

Cultures of pituitary cells were prepared according to

Heiman et al. (19). Briefly, the rats were decapitated and

the pituitaries dissected. The neurointermediate lobes

were removed and the remaining tissue was immedi-

ately placed in ice-cold isolation buffer (Gey’s medium

(Gibco) supplemented with 0.25% D-glucose, 2% non-

essential amino acids and 1% BSA, pH 7.3). The tissue

was cut into small pieces and transferred to isolation

buffer supplemented with 3.8 mg/ml trypsin and

330 mg/ml DNase. This mixture was incubated at 70

rotations/min for 35 min at 37 8C in a 95%:5% atmo-

sphere of O

:CO

. The tissue was then washed three

EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 Ipamorelin, the first selective GH secretagogue 553

Table 1 Structure–activity relations of some novel pentapeptides on GH release from rat pituitary cells in vitro –

a comparison with GHRP-1, GHRP-2, GHRP-6 and GHRH. The results are shown as means 6S.E.M. (

1⁄43–6

separate experiments).

max

Secretagogue Structure (nmol/l) (%)

GHRP-1 Ala-His-D2-Nal-Ala-Trp-D-Phe-Lys-NH

1.1 60.3 80 65

NNC 26-0039 Ala-His-D2-Nal---------D-Phe-Lys-NH

6.5 61.7 105 613

NNC 26-0133 D-Ala-His-D2-Nal---------D-Phe-Lys-NH

15 686068

Ipamorelin Aib-His-D2-Nal---------D-Phe-Lys-NH

1.3 60.4 85 65

GHRP-2 Ala----- -D-Trp-Ala-Trp-D-Phe-Lys-NH

1.8 60.5 85 68

GHRP-6 His-D-Trp-Ala-Trp-D-Phe-Lys-NH

2.2 60.3 100

GHRH 0.26 60.07 126 67

max

value for GHRP-6 1⁄4231 651ng/ml ð

1⁄46Þ:

times in the above buffer. Using a standard Pasteur

pipette, the tissue was then aspirated into single cells.

After dispersion, cells were filtered through a nylon filter

(160 mm) to remove undigested tissue. The cell suspen-

sion was washed 3 times with isolation buffer supple-

mented with trypsin inhibitor (0.75 mg/ml) and finally

resuspended in culture medium; Dulbecco’s modified

Eagle’s medium supplemented with 25 mmol/l HEPES,

4 mmol/l glutamine, 0.075% sodium bicarbonate, 0.1%

non-essential amino acids, 2.5% fetal calf serum, 3%

horse serum, 10% fresh rat serum, 1 nmol/l T

and

40 mg/l dexa-methasone, pH 7.3, to a density of 2×10

cells/ml. The cells were seeded onto microtitre plates

(Nunc, Roskilde, Denmark), 200ml/well, and cultured

for3daysat378C and 8% CO

Following the culture period the cells were washed

twice with stimulation buffer (HBSS supplemented with

1% BSA, 0.25% D-glucose and 25 mmol/l HEPES, pH

7.3) and preincubated for 1 h at 37 8C and 5% CO

. The

buffer was exchanged with new stimulation buffer

(37 8C). Test compound solution was added and the

plates were incubated for 15 min at 37 8C and 5% CO

The medium was decanted and analysed for released GH.

When testing the effect of antagonists the cells were

incubated with antagonist (final concentration 1 nmol/l

to 100 mmol/l) together with 10 nmol/l ipamorelin,

GHRP-6 or 1 nmol/l GHRH. The medium was collected

following a 15-min incubation period and analysed for

GH content. All assays were performed in triplicate.

Effects in anaesthetised rats

The animals were purchased and housed under the

same conditions as described above. Pentobarbital

dissolved in 0.9% NaCl (final concentration 50 mg/ml)

was administered i.p. (dose volume 1.2 ml/kg) to

Sprague-Dawley male albino rats (250625 g). After

15 min, a blood sample of 0.4 ml was obtained from the

orbital vein (sample 0). Immediately after blood

sampling 2 ml/kg of a test compound solution was

administered i.v. as a bolus injection through the tail

vein. After 10min, a new sample of 0.4 ml was obtained

from the orbital vein (sample 10). The samples were

collected in heparin-coated vials and kept on ice for 15–

20 min prior to centrifugation at 1300 gfor 3 min.

Plasma was frozen and stored at –20 8C until the GH

assay. At least five different doses of each compound

were tested using six rats per dose level.

Effects in conscious swine

Six female, 40 kg slaughter swine of the breed Landrace

Yorkshire cross (Lars Holmenlund, Denmark) were used

for each compound. The swine were housed at the test

facilities at least one week prior to the experiments.

Jugular catheters were inserted and fixed under

general halothane anaesthesia at day zero. Ipamorelin,

GHRP-2 and GHRP-6 were administered as i.v. bolus

injections in increasing doses with 24-h intervals in five

doses per experiment. The dose range was at least a

factor of 100. GHRH was administered at 6 nmol/kg

(30 mg/kg) corresponding to 200–300 times the ED

for GH release. The test compounds were dissolved in

saline containing 0.5% porcine serum albumin and the

dose volume was 0.02ml/kg. Blood samples were

drawn from the jugular catheter at frequent intervals

from 30 min prior to stimulation until 3 h post

stimulation. The blood was centrifuged at 1000 gfor

10 min in heparin-coated tubes and the plasma stored

at –80 8C until GH analysis.

Other hormones besides GH were measured using at

least three different dose levels for the GHRP receptor-

active compounds. The dose of GHRH was 6 nmol/kg.

Blood samples were drawn from the jugular catheter at

0, 10, 30, 45, and 60 min after the bolus injection. The

blood was stabilised with heparin and centrifuged at 1000

gfor 10 min and stored at –80 8 C until analysed for

follicle-stimulating hormone (FSH), luteinizing hormone

(LH), PRL, thyrotrophin (TSH), ACTH and cortisol.

In a control experiment a series of swine was dosed

with isotonic saline. In this experiment no significant

increases in the plasma hormone levels were observed.

Thus, the average zero time plasma levels of ACTH and

cortisol observed in the stimulation experiments were

used as basal levels.

Hormone assays

The rat GH was measured by an in-house-developed

competitive RIA using

125

I-labelled rat GH, rabbit

antibody against rat GH and scintillation proximity

assay particles (SPA-particles, Amersham International,

Amersham, Bucks, UK) coated with antibody against

rabbit antibody. The detection limit was 4 ng/ml plasma

and the intra- and interassay coefficients of variation

were 9.5% and 6.2% respectively.

The porcine (p) GH ELISA was a double monoclonal

sandwich immunoassay using in-house-made antibo-

dies prepared against pGH. The detection limit was 0.3 ng/

ml plasma and the intra- and interassay coefficients of

variation were 4.6% and 2.3% respectively.

ACTH was determined by a two-site immunoradio-

metric assay (IRMA), as described previously (20), using

a commercial test kit (Euro-Diagnostica BV, Apeldoorn,

The Netherlands). The sensitivity of the assay was

0.8 pg/ml plasma. The intra- and interassay coefficients

of variation were 2.6 and 4.7% respectively. FSH and LH

levels were measured by a double-antibody RIA as

described by Van der Meulen (21) using porcine FSH

and LH for standard and for iodination. The sensitivity

of the FSH assay was 1.9 ng/ml plasma at the 90%

B/B

level and that of LH was 0.13 ng/ml plasma. The

intra- and interassay coefficients of variation were 8.2

and 11.0% and 10.6 and 13.8% respectively for the FSH

and LH assays. PRL was analysed by RIA as previously

described (22). The intra- and interassay coefficients of

554 K Raun and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139

variation were 6.9 and 12.3% respectively. TSH was

measured using a double-antibody RIA developed by

Dr F Helmond, The Netherlands (personal communica-

tion) using porcine TSH for standard and for iodination.

The intra-assay coefficient of variation of this assay was

6.0%. Cortisol was measured directly in plasma samples

using a single-antibody RIA technique as described by

Janssens et al. (23). The sensitivity of the cortisol assay

was 0.5 ng/ml plasma at 90% B/B

. The intra- and

interassay coefficients of variation were 4.5 and 11.2%

respectively.

Calculations

The basal GH level for individual swine was calculated

as the average of the three GH values obtained prior to

stimulation. For other hormones the average value

obtained at time zero immediately prior to stimulation

was considered to be the basal value (see ‘Effects in

conscious swine’ for details). Peak hormone levels

adjusted for basal level (C

max

), obtained following

administration of test compounds, the time of C

max

), and the area under the time concentration

curves (AUC) calculated using the trapezoidal rule, were

used to characterize the hormone response of individual

swine. Dose–response curves were constructed using

peak GH levels for rats and C

max

GH plasma concentra-

tions for swine. Fitting to the Hill equation or hyperbolic

Michaelis-Menten equation was performed by nonlinear

regression, from which the efficacy (maximal GH

released, E

max

) was estimated. For the in vitro results

the efficacy was expressed relative to the maximal GH

released by GHRP-6.

Using the E

max

values of the individual compounds,

the potency was calculated as the dose inducing half-

maximal stimulation/inhibition (EC

/ED

/IC

values).

In inhibition studies, the IC

values were converted to

inhibition constant (K

) values using the Cheng-Prusoff

equation (24).

Dose–response curves for all other pituitary hor-

mones and for cortisol were constructed using the same

procedure as for GH in swine.

The results were tested for normal probability

distribution by the Shapiro-Wilk test. Non-parametric

tests were used for data with non-likely normal

probability distribution. All statistical comparisons

were performed using SAS (NC, USA) and Prism

(Graphpad, CA, USA) software.

Results

Rat pituitary cell assay

All secretagogues stimulated GH release from rat

pituitary cells in a dose-dependent manner. GHRP-1,

GHRP-2 and GHRP-6 were approximately equipotent in

this assay. When the central dipeptide Ala-Trp was

deleted (NNC 26–0039) a slight decrease in EC

but no

effect on E

max

as compared with GHRP-1 was observed.

By replacing the N-terminal L-Ala with D-Ala (NNC 26–

0133) a more pronounced decrease in the potency and

a slight decrease in E

max

was observed.

If L-Ala was replaced by Aib (ipamorelin) both the

and E

max

values were restored to the level of GHRP-1

(EC

1⁄41.360.4 nmol/l vs 1.160.3 nmol/l and E

max

1⁄4

8565% vs 8065%). Details of these results can be seen in

Table 1.

To evaluate the pharmacological specificity of ipa-

morelin we investigated whether the GHRP antagonists

((D-Lys

)-GHRP-6, L-692,400 and (D-Arg

,D-Phe

,D-

Trp

7,9

, Leu

)-Substance P) or the GHRH antagonist

(N-Acetyl-Tyr

,D-Arg

)-hGHRH(1–29)NH

) affected the

stimulation. As evident from Table 2, the GHRP

antagonists had no effect on GHRH-induced GH release

whereas the GH releasing effects of both GHRP-6 and

ipamorelin were inhibited with similar potencies. In

contrast, the GHRH antagonist (N-Acetyl-Tyr

,D-Arg

hGHRH(1–29)NH

potently inhibited GHRH-induced

GH release, but had no effect on GH release induced by

either GHRP-6 or ipamorelin. This clearly suggests a

GHRP receptor agonist profile of ipamorelin.

Effects in anaesthetised rats

We evaluated the effect of some of these GH secretago-

gues in anaesthetised rats. The results, listed in Table 3,

EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 Ipamorelin, the first selective GH secretagogue 555

Table 2 Pharmacological profile of ipamorelin-, GHRP-6- and

GHRH-induced GH release in rat pituitary cells in vitro in the

presence of GHRP and GHRH antagonists. The results are shown

as means 6S.E.M. (

1⁄43–6 separate experiments).

(nmol/l)

Secretagogue D-Lys

-GHRP-6 L-692,400 SPA GHRH ant.

Ipamorelin 565 6234 2346 6481 20 64 >17000

GHRP-6 1167 6167 2500 6674 15 67 >17000

GHRH >17000 >10000 >17 000 111 618

SPA,(D-Arg

,D-Phe

,D-Trp

7,9

,Leu

)- Substance P; GHRH ant., (N-

Acetyl-Tyr

,D-Arg

)-hGHRH(1-29)NH

Table 3 The effect of some novel pentapeptides on GH release in

vivo in rat and swine – a comparison with GHRP-2 and GHRP-6.

The results are shown as means 6S.E.M. (from C

max

GH values),

(

1⁄46–18 separate experiments).

Rat Swine

max

Secretagogue (nmol/kg) (ng/ml) (nmol/kg) (ng/ml)

Ipamorelin 80 642 1545 6250 2.3 60.03† 65 60.2

NNC 26-0039 >4300* 0 — —

NNC 26-0133 186 6126 587 693* — —

GHRP-2 30 64 1153 631 0.6 60.2 56 66

GHRP-6 115 636 1167 6120 3.9 61.4† 74 67

<0:05 vs all the secretagogues; †

<0:05 vs GHRP-2

(unpaired

-test).

demonstrated a clear discrepancy between in vitro and

in vivo. Whereas only minor differences were observed in

vitro, the in vivo results demonstrate that GHRP-2 was

more than 100-fold more potent than NNC 26–0039

and four times more potent than GHRP-6. It was very

surprising that NNC 26–0039, in spite of having high

in vitro potency was inactive in vivo. NNC 26–0133, in

which the L-Ala in NNC 26–0039 was substituted with

D-Ala, regained most of its activity as compared with

GHRP-6 (ED

1⁄41866126 nmol/kg vs 115636 nmol/

kg), but with a significantly lower E

max

1⁄4

587693 ng GH/ml plasma vs 11676120 ng GH/ml

plasma). Ipamorelin, where the L-Ala in NNC 26–0133

was replaced by Aib, regained both potency and efficacy

compared with GHRP-6 (ED

1⁄480 nmol/kg642 vs

115636 nmol/kg and E

max

1⁄415456250 ng GH/ml

plasma vs 11676120 ng GH/ml plasma).

Effects in conscious swine

Ipamorelin, GHRP-2 and GHRP-6 all released GH in a

dose-dependent manner. The peak GH response was

obtained within 15 min after stimulation and the GH

plasma level returned to the basal level within 120 min.

The plasma levels of GH versus time following various

doses of ipamorelin are shown in Fig. 1. The ED

and

max

values of all compounds are listed in Table 3. For

the most efficacious dose of each compound and for

GHRH at 6 nmol/kg, mean plasma GH versus time is

shown in Fig. 2 and C

max

, AUC and T

max

values are

listed in Table 4.

GHRP-2 (ED

1⁄40.660.2 nmol/kg) was the most

potent compound tested – more potent than both

ipamorelin (ED

50 1⁄4

2.360.03 nmol/kg) and GHRP-6

(ED

50 1⁄4

3.961.4 nmol/kg). Stimulation with GHRP-6

caused the largest GH release in this study, with an E

max

of 7467 ng GH/ml plasma. This was 12% higher than

for ipamorelin and 25% higher than for GHRP-2. The

AUC values for the three secretagogues demonstrated a

similar picture as the C

max

values.

Stimulation with GHRH (6 nmol/kg) induced GH

release with a C

max

of 2264 ng GH/ml plasma,

significantly lower than found for GHRP-6, ipamorelin

and GHRP-2. The GHRH response reached a peak

approximately 40min after the GHRH injection. A

second, presumably endogenous peak was observed

after 150 min. Even though GHRH had a C

max

less than

half that of GHRP-2, similar AUC values were found for

these two compounds.

To study the specificity of the GH secretagogues, we

evaluated the plasma levels of a number of other pituitary

hormones (ACTH, FSH, LH, PRL, TSH) and cortisol.

None of the GH secretagogues tested affected plasma

levels of FSH, LH, PRL or TSH in any of the administered

doses. Mean basal levels were not significantly different

from the C

max

values obtained after stimulation

(P>0.05) (Table 5).

556 K Raun and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139

Figure 1 Plasma GH levels versus time in swine following i.v.

administration of different doses of ipamorelin: (B) 0.4 nmol/kg; (O)

1.4 nmol/kg; (P) 4.2nmol/kg; (l) 14 nmol/kg; (X) 42nmol/kg. Data,

given as means6S.E.M., are from an experiment using six pigs at

each dose and the basal level was subtracted from the stimulated

values. See Materials and methods for experimental details. ↑

indicates the time of dosing.

Figure 2 Plasma GH levels versus time in swine following i.v.

administration of ipamorelin (X), GHRP-2 (A), GHRP-6 (x) and

GHRH (K) using the most efficacious dose (see Table 4). Data,

given as means6S.E.M., are from an experiment using six pigs at

each dose and the basal level was subtracted from the stimulated

values.For significance see Table 4. See Materialsandmethods for

experimental details. ↑indicates the time of dosing.

Table 4 The effect of different GH secretagogues in swine on

plasma C

max

, AUC and T

max

. The results are shown as means 6

S.E.M. (

1⁄46–18).

max

AUC T

max

Secretagogue (ng/ml) (ng ×min/ml) (min)

Ipamorelin (40nmol/kg) 63 68* 2354 6300 12 61

GHRP-2 (40nmol/kg) 56 66* 1660 6286* 9 64

GHRP-6 (70nmol/kg) 84 63 2687 6244 13 61

GHRH (6nmol/kg) 22 64** 1676 6286* 42 612*

<0:05 vs GHRP-6; **

<0:001 vs ipamorelin, GHRP-2 and

GHRP-6.

As ACTH and cortisol are associated with stress,

plasma samples of these hormones often have a high

degree of variability. To minimize the variation, all

experiments were performed at the same time of the day,

starting at 0900 h. The mean basal plasma level of

ACTH was 15.661.1 pg/ml (mean6S.E.M., n=58, range

7.8–49.7 pg/ml) and of cortisol was 11.961.1 ng/ml

(mean6S.E.M., n=56, range 1.0– 37.0 ng/ml).

In Fig. 3, data are shown of the time dependency from

a single experiment evaluating ACTH and cortisol

plasma levels before and after i.v. injection of saline,

ipamorelin, GHRP-2, GHRP-6 and GHRH. The saline

challenge caused no significant increase in ACTH and

cortisol plasma levels. GHRH showed a slight increase in

both ACTH and cortisol plasma levels, at a dose 200- to

300-fold higher than the ED

for inducing GH release

(6 nmol/kg). On the other hand, both GHRP-6 and to an

even greater extent GHRP-2 induced large and significant

increases in plasma ACTH and cortisol levels. The effect of

GHRP-6 and GHRP-2 was maximal at doses 85- and 45-

fold higher than the ED

for releasing GH.

Very surprisingly, ipamorelin, even at doses more

than 200-fold higher than the ED

value for GH

release, showed very limited effects on ACTH or cortisol

EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 Ipamorelin, the first selective GH secretagogue 557

Figure 3 Plasma ACTH (B) and cortisol (P) levels versus time in swine following i.v. administration of saline, ipamorelin (210 ×GH ED

dose), GHRP-2 (45 ×GH ED

dose), GHRP-6 (85 ×GH ED

dose) and GHRH (200–300 ×GH ED

dose). Data, given as means6S.E.M.,

are from a single experiment carried out in six pigs and the individual basal hormone level is subtracted from the stimulated values. See

Materials and methods for experimental details. C

max

values of ACTH and cortisol for GHRP-2 and GHRP-6 are significantly higher than

those for ipamorelin and GHRH (

P<0.05, unpaired t

-test).

Table 5 The effect of ipamorelin (420nmol/kg), GHRP-2 (270 nmol/kg), and GHRP-6 (340nmol/kg) in swine on plasma levels of FSH, LH,

PRL and TSH. The results are shown as means 6S.E.M. (

1⁄46).

FSH (ng/ml) LH (ng/ml) PRL (ng/ml) TSH (ng/ml)

Secretagogue Basal C

max

Basal C

max

Basal C

max

Basal C

max

Ipamorelin 26.4 66.5 35.9 66.2 5.0 60.6 4.6 60.4 5.1 60.1 5.7 60.2 7.1 61.4 7.3 61.7

GHRP-2 29.3 63.9 33.2 65.1 3.6 60.3 3.7 60.3 5.2 60.3 5.7 60.2 8.4 60.5 8.9 60.5

GHRP-6 26.6 64.6 27.4 64.0 3.7 60.5 3.9 60.4 5.1 60.5 6.2 60.6 8.5 60.7 8.7 61.0

No significant differences between basal and C

max

were obtained.

plasma levels. The effect of ipamorelin was at no point

significantly different from the effect of GHRH on these

hormones.

Average data of the dose–response relationships for

ipamorelin, GHRP-2 and GHRP-6 on ACTH and cortisol

plasma levels are shown in Fig. 4. Evidently, a clear

dose–response relationship exists for GHRP-2 and

GHRP-6, but not for ipamorelin.

No signs of adverse effects were observed with any of

the secretagogues at the doses tested.

Discussion

In this paper we present ipamorelin, the first GHRP

receptor-active compound with selectivity for GH

release.

A number of GHRP receptor-active compounds have

previously been described as releasing GH in several

species, including rats (10), dogs (11), swine (12) and

man (13–16). In our work on identifying novel

small molecules with GH releasing properties, we

hypothesised based on previous work (25) and a super-

position of GHRP-1 and L-692,429, that the central

dipeptide Ala-Trp of GHRP-1 may be redundant (see Fig. 5).

When the central Ala-Trp sequence was deleted from

GHRP-1 (NNC 26–0039), we found the in vitro potency

to be similar to that of GHRP-1. The relatively high

potency found in vitro led us to investigate NNC

26–0039 in male rats to elucidate its in vivo effect on

GH release. Surprisingly however, even at doses of

4300 nmol NNC 26–0039/kg no GH release was

observed. By inverting the stereochemistry at the NNC

26–0039 N-terminal from L-Ala to D-Ala (NNC

26–0133) the in vivo activity in rats was regained.

This observation led us to study further the apparently

crucial role of the N-terminal, and we combined the two

previous N-terminals (L- and D-Ala) to Aib (ipamorelin),

and observed not only an increase in the in vitro activity

but also in the in vivo activity.

To demonstrate that ipamorelin was acting on a

GHRP-like receptor the pharmacological mechanism of

action was investigated in vitro, using previously

described GH secretagogue antagonists. GHRP-6- and

ipamorelin-induced GH release was inhibited with

similar potencies by the GHRP receptor antagonists

(D-Lys

)-GHRP-6 (26), L-692,400 (27) and

(D-Arg

,D-Phe

,D-Trp

7,9

,Leu

)-Substance P (27). On

the other hand, the GHRH antagonist (N-Acetyl-

Tyr

,D-Arg

)-hGHRH(1–29) (28) had no effect on the

GHRP-6- and ipamorelin-induced GH release. As

expected, GHRH-induced GH release demonstrated the

opposite pharmacological profile. These results strongly

suggest that ipamorelin, like GHRP-6, mediates its effect

through a GHRP-like receptor – a receptor distinct from

the GHRH receptor.

These initial promising results with ipamorelin

encouraged us to investigate further this compound as

a novel GH secretagogue in swine. The swine seems to

be a suitable animal model because of its close similarity

to humans in several physiological aspects (29). Swine

have an endogenous GH profile comparable to humans

with respect to basal GH levels, little sex dimorphism, GH

pulse frequency and amplitudes (30). The only major

difference between swine and humans in the GH secretion

pattern is that swine lack a major nocturnal GH pulse –

probably due to the difference in sleeping patterns.

In the present studies, the acute effects of the GH

secretagogues ipamorelin, GHRP-2 and GHRP-6 were

evaluated in conscious swine. To study the specificity,

the possible release of other anterior pituitary hormones

(ACTH, FSH, LH, PRL, and TSH) and cortisol was also

examined. The effects of the GH secretagogues were

compared with that of GHRH, as this was considered to

be the optimal physiological reference GH secretagogue.

In this porcine model, test compounds were given as a

bolus injection and the plasma GH levels were

monitored for three hours. The study was designed as

an open non-balanced dose-escalation study. The

intervals between doses, however, were chosen to be

558 K Raun and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139

Figure 4 Plasma ACTH and cortisol following i.v administration of

increasing doses of ipamorelin (X), GHRP-2 (A) and GHRP-6 (x).

As a reference the corresponding value obtained with GHRH

(6nmol/kg, (K)) is noted in the Figure. Data are given as

means6S.E.M. and are average values from 3–4 separate experi-

ments using six pigs in each experiment. The average basal

hormone level (ACTH, 15.8pg/ml; cortisol, 11.9 ng/ml) has been

subtracted from the stimulated values. See Materials and methods

for further experimental details. Both GHRP-2 and GHRP-6 display

a significant dose–response relationship with ACTH (

<0:004and

<0:007, unpaired

-test) and cortisol (

<0:03 and

<0:08

respectively, unpaired

-test) plasma levels.

24 h, much longer than the plasma half-life of any of the

three secretagogues tested (unpublished observations).

In a pilot study, using this time interval between doses,

we found no variation in C

max

or AUC when GHRP-6

(20 nmol/kg) was administered repeatedly six times in

six swine (31). Therefore, the risk of introducing carry-

over effects in the study was considered negligible.

All three compounds gave rise to an immediate GH

release in a dose-dependent manner, GHRP-2 being the

most potent with an ED

of 0.660.2 nmol/kg. This is

in agreement with the in vivo rat results and also as

described for humans (14). GHRP-6 had the highest

efficacy with an E

max

of 7467 ng GH/ml plasma. The

novel secretagogue ipamorelin had an E

max

of 90% of

the level of GHRP-6, with a peak response eliciting a 20-

to 30-fold increase in basal GH levels. Ipamorelin was

slightly more potent than GHRP-6, but less potent than

GHRP-2. The time for peak response (T

max

) was around

10 min, in agreement with other GH secretagogues in

different species, including man (12, 32–34).

The GH release in swine after maximal GHRH

stimulation (6 nmol/kg) results in a C

max

of 2264ng

GH/ml plasma. This is 3- to 4-fold lower than following

stimulation with the most efficacious doses of the GHRP

receptor-active GH secretagogues. This is in agreement

with previous observations in swine (12) and humans

(34). GHRH gave rise to GH release with a delayed T

max

(Table 4) compared with that of GHRPs, also in

agreement with previously published results for swine

and humans (12, 34). Although GHRH stimulation

exhibits a lower C

max

– less than half that of GHRP-2 –

the protracted profile and additional endogenous GH

pulse cause a relatively greater AUC, resulting in nearly

identical AUCs for GHRH and GHRP-2.

All secretagogues tested were able to release GH

without affecting FSH, LH, PRL and TSH. This is in

agreement with results described in swine with respect

to LH (32) and those described in beagle dogs for PRL

(33), but is in contrast to that found in humans after

hexarelin stimulation with respect to PRL (35). This

might be explained by a different level of mammoso-

matotrophs in various species. Humans have a high

proportion of these cells, which are an intermediate

form between mammotrophs secreting PRL and soma-

totrophs secreting GH (36). Furthermore, it has been

found in rats that the numbers of mammosomatotrophs

increase markedly with age (37). Using young swine in

this study might be part of the reason for not observing

any PRL release.

In vivo experiments describing ACTH and cortisol

plasma levels are often impaired by a large variation,

presumably due to association of these two hormones to

stress. In this study design, a saline challenge had no

significant effect on the basal plasma levels of these two

hormones. When testing GHRH, we observed a slight

increase in both ACTH and cortisol. The finding that

GHRH is not totally specific with respect to GH release is

in agreement with previously described studies in both

swine and humans (12, 38). This slight effect on the

corticotroph system might be an indirect effect of GHRH

on other neurons involved in the hypothalamic–

pituitary–adrenal axis. GHRP-6, and to an even greater

extent GHRP-2, dose-dependently increased both ACTH

and cortisol in a somewhat parallel fashion. The

EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 Ipamorelin, the first selective GH secretagogue 559

Figure 5 Superposition of GHRP-1, L-692,429 and ipamorelin.The primaryamino group in the N-terminal of GHRP-1, L-692,429 and

ipamorelin, the aromatic moiety of D-Nal of GHRP-1 and ipamorelin and the benzolactam moiety of L-692,429, and the aromatic moiety of

D-Phe of GHRP-1 and ipamorelin and the biarylic moiety of L-692,429 have been overlapped.

increase in plasma levels of cortisol stimulated by

GHRP-2 in this study corresponds to the levels achieved

when stimulating swine with 150 mg corticotrophin

releasing hormone (CRH) (12). Also in humans it has

been shown that GHRP-2 increases the plasma level of

cortisol similar to that shown with CRH (17). Admini-

stration of ipamorelin, like GHRH, caused an increase in

the plasma levels of both ACTH and cortisol. However,

to our surprise ipamorelin – even in extreme doses –

was unable to increase ACTH or cortisol plasma concen-

trations to a level significantly different from those

induced by GHRH. Also, ipamorelin showed no dose-

dependent effects on either ACTH or cortisol plasma levels,

contrary to the situation with GHRP-2 and GHRP-6.

These findings are very interesting. First, they show

that GHRP-2 and GHRP-6 dose-dependently cause an

increase in both ACTH and cortisol plasma levels.

Noticeably, GHRP-2 and GHRP-6 produced an ACTH

release of 350% (P<0.05) and 300% (P=0.06) respec-

tively, of GHRH, although they were administered in

doses relatively closer to the respective ED

values

for GH release than were GHRH and ipamorelin.

Consequently, both GHRP-2 and GHRP-6 cannot be

considered as selective GH secretagogues. Secondly,

ipamorelin acts, like GHRH, on the plasma levels of

either hormone. Consequently, ipamorelin is the first

GHRP receptor-active GH secretagogue, with selection

for GH release which does not differ from that of GHRH.

Thirdly, the finding that increase in plasma ACTH was

always followed by a correlated increase in plasma

cortisol suggests that the effect of the GHRPs on cortisol

is mediated via ACTH release. This mechanism of action

is in agreement with previous findings in swine where

the effect on cortisol is abolished after disconnection of

the hypothalamus stalk (12).

Introduction of GHRP receptor-active GH secretago-

gues with selectivity for GH release will be advanta-

geous to the patient. Although this study did not include

human data, the results found in swine indicate that

ipamorelin and other selective GH secretagogues might

be expected to be superior to other GH secretagogues

currently used in clinical trials.

In summary, we have characterised the novel GH

secretagogue ipamorelin in vitro and in vivo.In vitro,

ipamorelin displays a typical GHRP-6-like GHRP recep-

tor agonist profile. However, in vivo ipamorelin had a

very unique profile being the first GHRP receptor-active

GH secretagogue with specificity for GH release.

Consequently, ipamorelin and related secretagogues

hold great promise for the future.

Acknowledgements

We wish to acknowledge the following people for

excellent technical support: Henriette Christensen,

Hasse Fick, Lotte Gotlieb, Anette Heerwagen, Anne-

Grethe Juul Christiansen, Arne Lausen, Jette Møller,

Bolette Nielsen, Vibeke Rode and Pia von Voss.

Part of this work has been published at the Endocrine

Society 79th Annual Meeting in Minneapolis 1997, and

at the IVth European Congress of Endocrinology in

Sevilla 1998.

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Received 16 July 1998

Accepted 19 August 1998

EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 Ipamorelin, the first selective GH secretagogue 561

... For example, incretinomimetic peptides such as GLP-1 agonists and Gastric inhibitory polypeptide (GIP) bind to G protein-coupled receptors (GPCRs) in the pancreas, increase cAMP levels, and promote glucose-dependent insulin release [12]. GH axis peptides (GHRPs) activate the ghrelin receptor (GHS-R1a), elevating intracellular calcium and stimulating GH secretion by somatotrophic cells [15]. Peptides such as Insulin-like Growth Factor 1 (IGF-1) or derivatives activate tyrosine kinase receptors (IGF-1R) and trigger anabolic pathways (PI3K-Akt-mTOR) [5]. ...

... Certain peptides have been formulated for intranasal administration, leveraging the rich vascularization and direct contact with the olfactory cerebrospinal fluid to reach the central nervous system (e.g., Semax and Selank in nasal drops) [17][18][19][20][21]. Peptides are available in topical/transdermal formulations for local action (e.g., GHK-Cu in dermatologic creams). Recently, advances have enabled oral administration of some peptides, including MK-677 (an oral GH secretagogue) and oral semaglutide (a gastric-absorbent formulation) [5,15]. In general, however, the oral bioavailability of peptides is low, and strategies such as encapsulation, lipid conjugation, or intestinal permeation technologies are necessary to make it feasible [22,23] ...

... Final excretion usually occurs by the kidneys (free amino acids or small fragments are filtered out). Important: due to protein degradation, peptides accumulate only minimally in the body; on the other hand, they require frequent or continuous administration for sustained effects, unless their structure is optimized for prolonged duration [15,27]. ...

Guilherme Renke Lucas Chinellato

Therapeutic peptides are short chains of amino acids used to treat metabolic and endocrine conditions such as obesity and type 2 diabetes. While several peptide drugs have undergone rigorous approval processes that evaluate both safety and efficacy, novel, unapproved compounds have emerged and are rapidly expanding into preventive medicine and performance enhancement. Our objective is to present the effects, clinical applications, safety profiles, and regulatory status of prominent peptides used to treat several conditions. We reviewed 106 articles, prioritizing systematic reviews, meta-analyses, and randomized controlled trials in the PubMed, ScienceDirect, and SciELO databases. Our results suggest that therapeutic peptides are a promising tool for treating type 2 diabetes and obesity, for skin rejuvenation, and as hormone analogs for specific diseases and conditions. Although these are strategic and innovative options that can improve health, performance, and longevity, further studies are needed before most new peptides can be used safely in humans.

... Ipamorelin complements CJC-1295 by stimulating GH release through ghrelin receptor activation rather than GHRH pathways (Raun et al., 1998). Its selectivity is remarkable, potent GH release without cortisol or prolactin elevation, even at doses 200-fold above the effective dose, avoiding the adverse endocrine effects seen with earlier GH secretagogues (Raun et al., 1998). ...

Volodymyr Mavrych

Inna Shypilova

Olena Bolgova

Volodymyr Mavrych

Inna Shypilova

Olena Bolgova

Background Peptide therapeutics represent an emerging frontier in gerontological medicine, targeting fundamental hallmarks of aging including metabolic dysfunction, telomere attrition, tissue repair impairment, and hormonal decline. Objective To comprehensively review the mechanisms, clinical applications, evidence base, and safety profiles of therapeutic peptides with demonstrated or potential applications in healthy aging and age-related conditions. Methods A comprehensive narrative review was conducted through systematic searches of PubMed, Scopus, and regulatory databases (FDA, WADA) from inception through January 2026. Search terms included “peptide therapeutics,” “aging,” “gerontology,” “healthspan,” combined with specific peptide names (tirzepatide, epitalon, GHK-Cu, BPC-157, TB-500, Semax, CJC-1295, ipamorelin, bremelanotide). Peer-reviewed articles, clinical trials, regulatory documents, and preclinical studies were evaluated. A total of 20 primary sources were selected based on relevance, methodological quality, and contribution to understanding peptide mechanisms and clinical outcomes in aging populations. Results Nine peptides were identified spanning diverse aging interventions: metabolic restoration (tirzepatide), telomere biology (epitalon), dermal regeneration (GHK-Cu), tissue repair (BPC-157, TB-500), neuroprotection (Semax), growth hormone modulation (CJC-1295, ipamorelin), and sexual function (bremelanotide). FDA-approved agents demonstrated robust safety profiles from large-scale trials. Non-approved peptides showed promising preclinical and limited clinical evidence but lack long-term safety data and systematic validation. Significant knowledge gaps include optimal dosing regimens, combination therapy effects, and biomarkers for monitoring efficacy. Conclusion Therapeutic peptides offer mechanistically diverse approaches to multiple aging hallmarks. While FDA-approved agents demonstrate clinical potential, investigational peptides require rigorous validation through well-designed clinical trials to establish safety and efficacy for healthspan extension.

... Ipamorelin, a selective ghrelin receptor agonist, promotes GH release by inhibiting somatostatin and is noted for its minimal offtarget endocrine effects, particularly on cortisol and prolactin. 25,26 GH secretagogues are generally administered subcutaneously. CJC-1295, a long-acting analog of growth hormone-releasing hormone (GHRH), can be administered with or without a drug affinity compound (DAC) to modulate the timing and release of GH. 27 In combination, ipamorelin and CJC-1295 without DAC (also known as Modified GRF) exhibit a synergistic effect, elevating both GH and IGF-1 levels in a pulsatile, physiologic manner. ...

Omar F. Rahman Steven J. Lee William A. Seeds

Therapeutic peptides are emerging as promising adjuncts in the management of orthopaedic injuries, grounded in their ability to modulate molecular signaling networks central to cellular medicine. By acting on key pathways such as PI3K/Akt, mTOR, MAPK, TGF-β, and AMPK, peptides exert influence over tissue regeneration, inflammation resolution, and neuromuscular recovery. Wound-healing peptides such as BPC-157, TB-500, and GHK-Cu promote angiogenesis, integrin-mediated extracellular matrix remodeling, and fibroblast activation, whereas growth hormone secretagogues like ipamorelin, CJC-1295, tesamorelin, sermorelin, and AOD-9604 activate IGF-1 signaling and satellite cell repair. Recovery-enhancing agents such as epithalon, delta sleep-inducing peptide, and pinealon target circadian and mitochondrial regulators, and neuroactive peptides like selank, semax, and dihexa enhance brain-derived neurotrophic factor and HGF/c-Met pathways critical to neuroplasticity. Although preclinical studies are promising, there is a current lack of clinical trials. This review integrates current mechanistic insights with orthopaedic relevance, emphasizing safety, efficacy, and future directions for responsible integration into musculoskeletal care.

Aleksander Dominikowski

Zofia Rękoś

Michał Olejarz Marek Ruchała

Performance-enhancing drugs (PEDs) marketed as “research compounds” include unregulated peptides intended to modulate the growth hormone–insulin-like growth factor-1 (GH–IGF-1) axis. The agents most commonly encountered in clinical practice and online self-administration protocols include growth hormone-releasing hormone (GHRH) analogues (e.g., sermorelin, tesamorelin, CJC-1295 with Drug Affinity Complex [DAC], CJC-1295 without DAC), growth hormone secretagogues (GHS; e.g., growth hormone-releasing peptide-2 (GHRP-2), growth hormone-releasing peptide-6 (GHRP-6), hexarelin, ipamorelin), the growth hormone (GH) fragment - AOD9604 (hGH 176–191), and insulin-like growth factor-1 (IGF-1) analogues (e.g., pegylated mechano growth factor (PEG-MGF), IGF-1 Long R3 (IGF-1 LR3)). Reported adverse effects span endocrine and metabolic disturbances (including prolactin and cortisol elevations, appetite changes, and dysglycaemia), fluid retention syndromes, musculoskeletal symptoms (myalgia/arthralgia), and injection-site reactions. Given the absence of regulatory approval for physique- or performance-related indications and the uncertainty surrounding product composition, dose, and stacking practices in unregulated supply chains, clinicians increasingly require a pragmatic framework to interpret symptoms and laboratory abnormalities in patients using these compounds. This narrative review contrasts peer-reviewed pharmacokinetic/pharmacodynamic and clinical evidence with commonly encountered online self-administration protocols, stratifying peptides into evidence tiers from regulatory-grade randomized trial data to a complete absence of human studies, and highlights the resulting uncertainty around putative performance and recomposition benefits. We summarise structural characteristics, pharmacologic effects, and commonly reported dosing patterns, and we synthesise clinically relevant adverse effects with particular attention to hormonal imbalance, endocrine–metabolic risk, and biologically plausible but unproven mitogenic concerns. Finally, we propose a clinically oriented assessment algorithm to support exposure history taking, triage of symptom domains, and risk communication without legitimising off-label peptide regimens.

Christopher L Mendias Tariq M. Awan

Peptides are short chains of amino acids with a unique pharmacological niche between small-molecule drugs and large proteins. Their use in sports medicine is rapidly expanding, driven by patient demand for accelerated injury recovery and performance enhancement. While numerous peptide drugs have undergone a rigorous approval process that evaluates both safety and efficacy, a parallel “gray market” of unapproved compounds has emerged, operating largely outside of regulatory oversight. Our objective is to present the pharmacological mechanisms, safety profiles, and regulatory status of prominent approved and unapproved peptides marketed direct to patients, including AOD-9604 (anti-obesity drug 9604), BPC-157 (body protection compound 157), CJC-1295, FS-344 (follistatin-344), GHK-Cu (glycyl-l-histidyl-l-lysine copper), ipamorelin, MOTS-C (mitochondrial ORF of the 12S rRNA type-c), sermorelin, SS-31 (elamipretide), tesamorelin (Egrifta), Tβ4 (thymosin beta-4), and TB-500 (thymosin beta-4 fragment). Many unapproved peptides demonstrate favorable tissue repair and metabolic outcomes in animal models, but rigorous human safety data are scarce, and there is potential for serious harm to patients. This narrative review focuses on the utilization of peptides in sports medicine, and alternative treatments that may be considered. We provide a framework to navigate patient discussions about peptides to better facilitate evidence-based practices for musculoskeletal healing and athletic performance. We also discuss the placebo effect as a mediator of peptide efficacy, and how social media amplifies this effect.

Luis Felipe Duarte Coutinho Lucas Franco de Oliveira Neves Rafael Prates Camilo

The pursuit of pharmacological enhancement in sport has evolved from the widespread use of anabolic-androgenic steroids (AAS) to novel agents such as peptides and peptide analogues. Marketed as more selective and ostensibly safer alternatives, peptides-including growth hormone secretagogues (e.g., Ipamorelin), growth hormone-releasing hormone analogues (e.g., CJC-1295, Sermorelin), and synthetic fragments (e.g., Frag 176-191, KPV)-are promoted for muscle growth, fat metabolism, recovery, and anti-inflammatory effects. Their pharmacological profiles, including enhanced stability and receptor selectivity, have made them attractive in both medical research and bodybuilding communities. Despite their growing popularity, the clinical evidence supporting peptide use in sport is limited. Most published studies examine therapeutic applications under controlled dosing regimens, not the supraphysiological or combined protocols common in bodybuilding. Emerging data highlight potential risks: cardiovascular strain, insulin resistance, dyslipidemia, and psychiatric instability. The largely unregulated supply chain exacerbates these dangers, as products are often mislabeled or contaminated. Regulatory bodies such as the World Anti-Doping Agency (WADA) have responded by expanding detection technologies, yet analytical challenges remain due to peptides’ structural similarity to endogenous hormones and short half-lives. Beyond elite sport, the extent of peptide use in the general population is unknown. Anecdotal reports and widespread promotion on social media suggest growing uptake among recreational gym-goers, including younger individuals, but prevalence studies are lacking. This represents a critical gap in current knowledge. In conclusion, peptides represent a new phase in performance enhancement but remain experimental substances with poorly defined long-term risks. Until longitudinal data clarify their safety and prevalence, peptide use in both competitive and recreational settings should be considered high-risk and ethically problematic.

Cory Mayfield

Ioanna K Bolia

Cailan Feingold

Alexander Weber

Background Therapeutic peptides are short-chain amino acids that regulate cellular functions and facilitate biochemical processes. In recent years, there has been significant growth in the global market for therapeutic peptides and thus its popularity among patients. Given the increase in the development of peptides and increased marketing to patients for orthopaedic injuries, it is critical for orthopaedic surgeons to understand the current evidence behind these therapeutic peptides. Purpose To evaluate the current evidence and applications of injectable peptide therapy, focusing on its potential in regenerative medicine and sports performance, to help orthopaedic providers better understand the current state of different therapeutic peptide approaches. Study Design Narrative review. Methods A comprehensive literature search was conducted using PubMed to identify biochemical and clinical studies on the most popular types of injectable peptide therapy. Key peptides evaluated included BPC-157, TB-4, TB-500, CJC-1295 + ipamorelin, tesamorelin, and GHK-Cu. Results BPC-157 demonstrated potential benefits in tendon and muscle repair, but these findings are largely unvalidated in human trials. A single human case series reported improvements in pain after intra-articular knee injections of BPC-157, although significant methodological flaws and a lack of controls limit its applicability and reliability. TB-4 and its derivative TB-500 promoted angiogenesis and tissue repair in preclinical models, but human orthopaedic data are lacking, and both remain banned substances in sports. CJC-1295 combined with ipamorelin showed significantly improved maximum tetanic tension in murine models with glucocorticoid-induced muscle loss, but these findings are limited to animal studies. Tesamorelin, approved for treating HIV-associated lipodystrophy, has no supporting orthopaedic evidence. GHK-Cu showed promise in wound healing and anti-inflammatory effects, but no clinical data support its use for musculoskeletal conditions. Conclusion While peptide therapy may possess significant therapeutic and regenerative potential, it is critical that orthopaedic and sports medicine providers understand the current lack of evidence to support the clinical use of these peptides. Importantly, information regarding the indications, dosing, frequency, and duration of treatment remains unknown. Despite the popularity of these peptides in mainstream media and among patients, significant research regarding the safety and efficacy of these therapeutic methods is required before definitive recommendations can be made to patients.

Meulen

Frans A Helmond C.P.J. Oudenaarden

Geertruy te Kronnie

In the 1st of 2 experiments, 5 gilts were injected with saline at the onset of standing oestrus (control oestrus), and at the next cycle the same gilts were injected with 100 mu g GnRH analogue. The LH and FSH peaks in the GnRH-treated oestrus were not significantly different from those in the control oestrus, but both peaks were sharper in the GnRH-treated oestrus. In the 2nd experiment, 49 gilts were injected with 100 mu g GnRH at the onset of standing oestrus or were not treated (controls), and slaughtered on day 4, 11, 13 or 35 of pregnancy. For animals slaughtered on day 4, 11 or 13, compared with controls, treated animals had significantly more corpora lutea (16.3 or-2.3 vs. 14.8 or-1.5) and embryos (15.0 or-3.0 vs. 12.1 or-3.4). Embryonic mortality on day 35 was 24.7% in the treated group vs. 11.3% in controls. The av. number of corpora lutea for gilts slaughtered on all 4 days was greater in treated gilts than in controls (16.4 or-2.3 vs. 14.3 or-3.3), but the difference was not significant. A similar trend was observed for number of embryos (15.4 or-2.0 vs. 13.0 or-3.3).

Gerry Hickey Thomas M. Jacks F Judith

Roy G Smith

L-692, 429 is a substituted benzolactam that has recently been shown to stimulate GH secretion from rat pituitary cells in vitro with an ED50 of 60 nM. In the current studies, we evaluated the efficacy and specificity of L-692, 429 as a GH secretagogue in beagles. L-692, 429 at 0.1, 0.25, or 1.0 mg/kg or saline vehicle was administered iv to four male and four female beagles in a balanced cross-over design. Blood samples were collected up to 75 min posttreatment, and serum was assayed for GH, cortisol, PRL, and LH. Mean peak serum GH levels were significantly increased (P < 0.05) by L-692, 429 to 13 +/-2 (mean +/-SEM) ng/ml (0.1 mg/kg), 39 +/-6 ng/ml (0.25 mg/kg), or 71 +/-11 ng/ml (1.0 mg/kg) over the saline control value of 3.6 +/-0.6 ng/ml. Mean peak GH levels occurred at 15 min and had returned to near-baseline levels by 75 min. There was no difference in response between sexes. Mean peak cortisol levels were significantly increased (P < 0.05) by 2.2-, 2.7-, and 3.1-fold above control levels (3.0 +/-0.2 micrograms/dl) at 25-35 min and returned to near-baseline levels by 75 min. PRL was slightly decreased after L-692, 429 treatment, whereas LH was not affected. In a second study, three groups of three male beagles each were administered 5.0 mg/kg L-692, 429, iv; iv saline, or 2.2 U/kg ACTH, im. Blood was collected for 8 h posttreatment and assayed for GH, cortisol, ACTH, aldosterone, PRL, insulin, T3, and T4. L-692, 429 administration significantly increased (P < 0.05) GH over the control level (6.0 +/-3.6 ng/ml) to 133 +/-14 ng/ml by 15 min, with a return to pretreatment levels by 120 min. Cortisol levels were significantly increased (P < 0.05) by 2.0-fold (L-692, 429) or 2.9-fold (ACTH) over the saline control peak concentration of 5.6 +/-1.6 micrograms/dl and were associated with concurrent increases in ACTH levels of 1.2-fold (L-692, 429) or 2.1-fold (ACTH) over the saline control peak concentration of 67 +/-20 pmol/L. Aldosterone, PRL, T3, and T4 were not significantly affected after L-692, 429 administration; however, ACTH treatment significantly increased aldosterone (P < 0.05). These data demonstrate that L-692, 429 is a novel nonpeptidyl secretagogue that stimulates a marked, but transient, increase in serum GH levels in the dog.

J.A. Chowven J. Argente

It is clear that the main hypothalamic regulators of pituitary growth hormone secretion are GHRH neurons of the arcuate nucleus and SS neurons in the periventricular nucleus. Although numerous other neuropeptides, neurotransmitters and locally produced factors may modulate GH secretion, much of this effect is mediated through either GHRH and/or SS neurons. Furthermore, the circulating factors and hormones that modulate GH synthesis and secretion can do so either by a direct effect at the somatotroph, often modulating the somatotroph's response to SS or GHRH, or at the level of the hypothalamus. The physiological role of ghrelin in the regulation of GH secretion, especially at the level of the hypothalamus, remains to be totally clarified. This review gives a bit of historical perspective in the discovery of how the hypothalamus was found to be important in the control of growth and GH secretion, as well as the isolation of the factors involved in this process. In addition, a brief description of the main modulators, both central and peripheral, and their possible mechanisms of action is given.

Michael Ankersen Sehested Hansen

A series of thioureas containing two aromatic groups and one amino group was synthesized as potential growth hormone secretagogues. The growth hormone (GH) releasing properties of these compounds (10–24) were evaluated using primary rat pituitary cells. The most potent compound 20 showed potency with an EC50 of 1.8 μTwo constrained analogs 23 and 24 showed similar activity.

R Claus A. Bingel S Hofäcker

Ulrike Weiler

Diurnal patterns of growth hormone (GH) in peripheral plasma were compared between mature German Landrace (GL) boars (n = 6), GL sows (n = 4) and European wild boars (n = 5). Blood samples were drawn at 20-min intervals through indwelling jugular vein catheters for 24 h. Some of the animals were sampled repeatedly, so twelve 24-h profiles were available for GL boars, six for GL sows and seven for wild boars. The GH concentrations were determined by a specific radioimmunoassay. GL boars revealed significantly higher (P ≤ 0.001) GH concentrations than GL sows (boars: 5.06 ± 2.34 ng ml-1; sows: 3.70 ± 1.01 ng ml-1). In GL boars a clear diurnal pattern with distinct GH pulses during daytime and a less pulsatile GH release during the night was found, whereas in females no diurnal pattern was obvious. Mean GH levels in wild boars (1.97 ± 1.03 ng ml-1) were significantly lower (P < 0.001) than in both groups of GL pigs. The diurnal pattern of GH pulses in wild boars, however, coincided with the pattern of GH release in GL boars (r = 0.26; n = 73; P ≤ 0.05).

Cheng Yung-Chi William H. Prusoff

A theoretical analysis has been made of the relationship between the inhibition constant (KI) of a substance and the (I50) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between KI and I50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I50 values in agreement with the admonitions previously described in the literature. The analysis described shows KI does not equal I50 when competitive inhibition kinetics apply; however, KI is equal to I50 under conditions of either noncompetitive or uncompetitive kinetics.

A A van Landehem D F van de Wiel

A sensitive heterologous radioimmunoassay for porcine prolactin (pPRL) has been developed. Anti-ovine prolactin antibody was raised in rabbits which allowed a final dilution of 1:500 000. The separation of free and antibody bound [125I]pPRL is based on the double antibody solid phase system. The assay is specific for pPRL. There is no cross-reaction with pLH, pFSH and pTSH; little cross-reaction (1.35%) was found with pGH. The smallest detectable amount was 0.08 ng per tube. During lactation high plasma levels are found with great fluctuations. After weaning the plasma PRL levels fall to basal within a few hours. After thyrotrophin-releasing hormone (TRH) administration plasma concentrations increase within a few minutes.

K Cheng Wanda W.-S. Chan Albert Barreto

Roy G Smith

His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) stimulated GH release from rat primary pituitary cells in a time- and dose-dependent manner. Stimulation was observed after a 15-min, but not a 4-h, incubation. The concentrations of GHRP-6 required for half-maximal and maximal stimulation were 7 x 10(-9) and 10(-7) M, respectively. GH release induced by GHRP-6 was not affected by the addition of either naloxone or the GRF antagonist [N-Ac-Tyr1,D-Arg2]GRF-(1-29)-NH2. The latter inhibited GRF-stimulated GH release by shifting the dose-response curve to the right. His-D-Trp-D-Lys-Trp-D-Phe-Lys-NH2, an analog of GHRP-6, inhibited GH release stimulated by GHRP-6 without affecting that induced by GRF. When present together at maximal concentrations, GHRP-6 and GRF produced a synergistic effect on GH release. GHRP-6 had no effect on intracellular cAMP levels, whereas GRF increased intracellular cAMP concentrations by 3-fold. Combined treatment of pituitary cells with GRF and GHRP-6 resulted in a potentiation of the GRF-induced increase in cAMP levels. Basal GH release was reduced by 30% after pretreatment with GHRP-6 (10(-7) M) for 1 h. Pretreatment with GHRP-6 also decreased the subsequent response to GHRP-6, but not GRF. In contrast, pretreatment with GRF for 1 h had no effect on the subsequent action of GHRP-6 or GRF on GH release. The desensitization induced by GHRP-6 was completely reversed within 1 h after removal of the peptide. Results from this study indicate that GHRP-6 and GRF stimulated GH release from somatotrophs via different receptors and through discrete mechanisms.

Mark Heiman Mary V. Nekola William A. Murphy David H. Coy

An improved rat anterior pituitary primary cell culture technique for studying GH-releasing activity of human pancreatic GH-releasing factor (hpGRF) and its analogs is described. Male pituitaries, dispersed by a combination of trypsin digestion and mechanical agitation, were plated at a density of 200,000 cells per well and cultured for 4 days. The attached cells were then stimulated with synthetic hpGRF which was comprised of the first 29 residues of the larger, originally isolated forms and which was amidated at the C-terminal (hpGRF-29). Analogs of hpGRF-29 which were modified in positions 1, 2, 3, or 7, and other secretagogues were similarly tested. Medium was collected after 3 h, and secreted hormone was measured by RIA. The cells were extremely sensitive to hpGRF-29 stimulation, and this effect was specific. The minimal effective dose of hpGRF-29 was an unprecedented 0.4 X 10(-15)M. No stimulation of LH, FSH, or PRL by hpGRF-29 was observed. Bombesin and vasoactive intestinal peptide were ineffective in stimulating GH release. [D-Trp6]LHRH (a potent LHRH agonist), also did not release GH but did stimulate secretion of LH and FSH at doses ranging from 0.4 X 10(-10)M to 1.0 X 10(-9)M. Responses of the cells to hpGRF-29 analogs were characterized by distinct heterologous dose-response curves. [D-Ala2]hpGRF-29 was 50 times more active than its parent 29-amino-acid peptide. [D-Thr7]hpGRF-29, another analog that differed from hpGRF-29 by the insertion of a D-isomer for the naturally occurring L-residue, was about 10,000 times less effective in stimulating GH secretion than was hpGRF-29 itself. Potencies of these and other analogs with respect to GH release in vitro were similar to those estimated in vivo. Thus, this primary cell culture provides an extremely sensitive, selective, and reproducible system for studying hpGRF structure-activity relationships. Further, such tremendous sensitivity to hpGRF can provide a system to study changes in pituitary sensitivity to hpGRF during different physiological states.

Junichi Fukata

Don J Diamond Joseph B. Martin

The effects of rat hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) on the release and biosynthesis of rat GH were studied by RIA and quantitative immunoprecipitation using monolayer cultures of rat anterior pituitary cells. In kinetic studies, GRF stimulation of GH release appeared at the first sampling time (20-min incubation) and the effect began to diminish after 2-h incubation with GRF. On the other hand, total (cell plus medium) content of GH significantly increased only after 24-h incubation. To examine the GH-synthesizing effect of GRF more directly, newly synthesized GH labeled by [35S]methionine during incubation with GRF was quantified by immunoprecipitation. The amount of immunoprecipitable GH increased significantly and specifically (compared with the total amount of labeled proteins) also only after 24-h incubation. When GH pools were labeled with [35S]methionine under different schedules, the basal release of newly synthesized GH, which was labeled for 1 h immediately before chase incubation was lower during the first 15 min than stored GH which had been labeled earlier. Basal newly synthesized GH secretion exceeded stored GH secretion after 30 min. GRF stimulated the release of GH from both pools but the stimulation of stored GH was greater. In this system, SRIF suppressed both the basal and stimulated release of GH but did not modify GH biosynthesis under either condition. Newly synthesized GH showed significant degradation during 24-h incubation; neither GRF nor SRIF affected the rate of GH degradation during the same incubation period. These results indicate that 1) GRF stimulates both release and synthesis of GH; 2) these two effects have different kinetics and different sensitivities to SRIF; and 3) GRF stimulates the release of GH from heterogeneous pools disproportionally.

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