1. The pharmacokinetics of three new peptidyl growth hormone secretagogues, ipamorelin (NNC 26-0161), NNC 26-0194 and NNC 26-0235, were compared with two well-known hexapeptides, GHRP-2 and GHRP-6, in the male rat following different routes of administration. 2. Following i.v. bolus injection, plasma concentrations of the peptides declined biexponentially. Ipamorelin differed markedly from the other peptides investigated, demonstrating a systemic plasma clearance 5-fold lower than that of GHRP-6. Ipamorelin was mainly excreted in the urine, whereas GHRP-6 was predominantly excreted in the bile. NNC 26-0194 and NNC 26-0235 also showed high biliary excretions. Ipamorelin and the two NNC peptides were moderately resistant towards metabolism as 60-80% of the administered dose could be recovered from bile and urine as intact peptide. 3. After intranasal application, the bioavailability of ipamorelin was estimated at approximately 20%. Higher bioavailabilities of approximately 50% were determined for NNC 26-0235, NNC 26-0194 and GHRP-2, whereas the nasal absorption of GHRP-6 was somewhat lower. Thus, the peptides could be easily transported across the nasal epithelium suggesting that the nasal route seems promising for systemic delivery of this family of peptidyl growth hormone secretagogues.
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x e n o b i o t i c a , 199 8, vol . 28, n o . 11, 10 83± 1092
P ha rm aco ki ne tic ev a lua ti on o f ip am o rel in a nd ot he r
p ep tid yl g ro wt h hor m on e se cr et ag og ue s w ith
e m ph a si s on n a sa l ab so r pti on
P. B. JOHA NS EN *, K . T. HANS EN , J. V. ANDER SE N
and N. L. J OH ANS EN
Dep artme nts of Growth Hormone Pha rmac ology, Drug M etabolism and Med Che m
Res earch IV , Nov o Nordisk A}S, Novo Alle!, D-2860 Ba gsvaerd, Denmark
Rece ived 1 6 May 1998
1. T he pha rmac okinetics o f three new peptidyl grow th hor mone sec retag ogues,
ipamorelin (NNC 26-0161), NNC 26-0194 and NNC 26-0235, were compared with two
well-known hexapeptides, GHRP-2 and GH RP-6, in the male rat following diå erent
routes of administration.
2. Following i.v. bolus injection, plasma concentrations of the peptides declined
biexponentially. Ipamorelin diå ered markedly from the other peptides investigated,
dem onstra ting a systemic pla sma clearance 5-fo ld lower tha n that of GH RP-6 . Ipa more lin
was mainly excreted in the urine, whereas GH RP-6 was predomina ntly excreted in the bile.
NN C 26-0194 and N NC 2 6-02 35 als o showed high bi liary excretions. Ip amo relin a nd the
two NNC peptides w ere moderately resistant tow ards metabolism a s 60± 80 % of the
administered dose could be recovered from bile and urine as intact peptide.
3. A fter intranasal application, the bioavailability of ipamorelin w as estimated at C
20%. Higher bioavailabilities of C50 % we re d eterm ine d fo r N NC 26 -023 5, N NC 26 -
0194 and GHRP-2, whereas the nasal absorption of GHRP-6 was somewhat lower. T hus,
the peptides could be easily transported across the nasal epithelium suggesting that the
nasa l route see ms promising for systemic delivery o f this fa mily of pe ptidy l growth
horm one sec retag ogues.
In t ro d u ct io n
The growth horm one (G H) secretagogue era started in the m id- 197 0s when
Bo we rs et al. (1977) discovered the GH-r eleasing property of m et-enkephalin.
Introduction of d-for ms o f am ino acids into the met-enkeph alin template led to the
discovery of GHRP-6 (H-His-D-Trp-Ala-Trp-D-Phe-Lys-NH#) (Bowers et al.
1984, Momany et al. 1984). Subsequent studies in the rat demonstrated that the GH
release obtained by GH secretagogues was accomplished through diå erent mech-
anisms than that of growth hormone-releasing hormone (GHRH) (McCormick et
al. 1985 , Sartor et a l. 1985). Recently, the mechanism o f ac tion has been determined
for th e GH se cretagogues with the identi® cation of a new G -protein-coupled
recep tor isolated from rat and porcine pituitaries (Pong et al. 1996). The GH
secretagogues are believed to mimic a so far unidenti® ed natural hormone.
The issue of the pharmacokinetics of peptidyl GH secretagogues, however, is
only sparsely reported in the literature. Until now, only one comprehensive animal
study appears to have investigated plasma concentrations and routes of excretion of
GHRP-6 in dog and rat follo wing i.v. a nd s. c. in jec tions (Davis et al. 1994). It w as
demonstrated that GHR P-6 is predo minan tly e liminated u nchan ged in the bile,
* Author for correspondence.
0049± 8254}98 $ 12.00 ’199 8 Taylor & Fran cis Ltd
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1084 P.B.Joh ansen et al.
indicating highly improved resistance towards enzymatic degradation as compared
with met-enkephalin, which is readily degraded by peptidases when introduced into
the blood stream (Dupont et al. 1977). Furthermore, a clinical phase I study showed
tha t even thoug h similar GH re lea se could be obtained a fter i.v. and oral
admin ist ration of GH RP-6, the oral administration required ext rem ely high dos es,
suggesting a systemic availability of C0 .3 % by the o ral ro ute (H ar tman et al. 1992).
Therefore, o the r routes of administrat ion have been att empted. Of th ese, th e nasa l
route seems promising. In the dog, the nasal bioavailability of GHRP-6 has been
estimated ind ire ctly fr om the G H release to be 34± 56 % of the administered dose
(N els on et a l. 1991). I n addition, long-term in tranasal adm inis tration o f the G HRP-
6 analogue h exa relin three times daily ha s shown an a ccelerate d growth velocity in
childr en w ith familial short stature (L aron e t al. 1995).
During the past 5 years intensive research programmes initiated by several
pharmaceutical companies have led to discovery of many new exciting GH
secre tago gues (S mith et al 1997, Camanni et al. 1998). We have synthesized
hundreds of small molecules and screened them for GH-releasing activity in vitro
and in vivo. R ecently, ipamore lin w as reporte d to be a potent, se lec tive a nd
eæ cacious GH secretagogue in several animal species (Raun et al. 199 8). In parallel
with the screening programme, the pharmacokinetic behaviour of many of these
peptid es was inves tigated in the rat based on their diver sity in mol ecular structure.
Here we present pharmacokinetic data on ipamorelin and two other compounds,
demonstrating th at changes primarily in the N -terminal lead to m arked changes in
the pharmacokinetic pro® le. Because this category of peptides was expected to show
a low oral bioavaila bility, we were interested in evaluating the bioavailab ility after
nasal application. In the present study, we used a reversed-phase HPLC method for
determination of th e GH se cretag ogu es in biological ̄ uid s.
M a ter ia ls a nd m e th od s
The s tructu res of the GH se creta gogues inv estiga ted are shown in table 1. The G H secretagogue s
wer e synth esize d at N ovo No rdisk A}S and w ere used as their acetate salts. For i.v. injections, intranasal
app licati ons a nd oral gavage, test so lutio ns of 1, 10 and 40 m g}ml calculated as free base were prepared
by d issolv ing the peptides in sal ine.
Animals
Male Sprague-Da wley rats weighing C250 g were obtained from Mù llegaard’ s Breeding & Research
Cen tre (Denma rk). T hey w ere acclimatized f or at least 7 day s and w ere fe d conv entio nal ra t chow and
drinking water ad libitum. T he animals were housed under controlled conditions. Feed was withdrawn at
07 : 00 h o n the day of the exp erime nt.
Plasma concentrations
During halothane anaesthesia, a catheter was inserted into the carotid artery, after wh ich the rat was
placed in a restrainer where it regained consciousness and remained for the entire study. The test
solutions were a dministered intravenously, intranasally or by oral gavage; the dose volum es being 1, 0.1
and 1 0 ml}kg , respectively. Intravenous injection was p erformed in a tail vein. Intranasal application wa s
performed by removing the rat from the restrainer and by applying the test solution with a pipette during
ultrashort halothan anaesthesia with the rat in the dorsal supine position. The rat w as returned to the
restra iner w here it reg aine d con sciousne ss within a few minutes. Ora l gavage w as performed with a blu nt
syringe. Blood samples of C500 ll were drawn into hepa rin stabilized test tubes placed on ice before
injection and at diå erent times following injection. Plasma were obtained after centrifugation and stored
at
–
18 Ê
C until analysis.
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Evaluation of peptidyl growth hormone secretagogues 1085
Table 1. Chemical structures of the peptidyl GH secretagogues investigated.
Pep tide Stru cture M .W.
GH RP -6 H-H is-D -Trp - Ala -Trp -D -Phe -L ys-N H #872.6
GH RP-2 H-D -Ala -D -2Na l-Al a-Tr p-D- Phe-L y s-NH #817.2
Ipa mor elin H-A ib-H is-D -2 Nal -D-P he- Lys- NH #(NN C26-0161) 711.2
NNC 26-0194 3-(4-imidazolyl)propionyl-D-Phe-A la-Trp- D-Phew(C H#NH )-Lys-ol 7 92.0
NN C 26-02 35 3-am inom ethyl-benzoy l-D-2 Nal-N -M e-D- Phe-L ys-NH#636.4
Ex cre tion in bile an d urine
The rats were anaesthetized with pentobarbital i.p. (60 mg}kg). Brie ̄ y, polyethylene tubes were
plac ed in th e ju gula r v ein a nd com mon bile duc t (p olysta n ; i .d. 0 .28 mm , o .d. 0.61 m m). The form er was
® lled with heparin solution (50 IE}ml). I n additio n, a polyethy lene tube wa s placed in th e urin e bladder
(polystan ; i.d. 0.76 mm, o.d. 1.22 mm) to allow collection of urine samples. Bo dy tempe rature was kept
constant a t about 37 Ê
C using a rectal thermometer and a heating pad. During the experiment a co-
solution of pentobarbital (1.2 mg }ml) and mannitol (150 mg}ml) w as infu sed to m aintai n anae sthesia and
urine ̄ ow. Following a 15-min recovery, the test compo und was a dministered as a bolus i.v. injection
(1 mg }kg) and bile and urine samples we re collected every 30 min for 4 h.
Ph arm acokinetic analyses
A two-compartment open mo del was ® tted to the observed plasma conc entrations follow ing i.v.
injection:
whe re k" $ a nd k$ " a re the transfer rate constants and kethe elimination rate constant. T he pharmacokinetic
analyses were performed in TOPFIT (Heinzel et al. 1993), using a relative weighting function of 1}C#
for all concentrations within a data set, and a lag time of zero. Thus, the individual plasma concentrations
could be described by the following biexponential equation:
Cpl
=
A[eÕat1B[eÕb[t,
whe re Cpl is the p lasma c oncen tratio n, Aand Bthe intercepts on the ordinate, a nd aand bthe distribution
and e limi natio n coe æ c ients.
The bioavailability following intranasal and oral application w as estimated b y use of a deconvolution
analysis program (Gillespie 1991). The means of the estimated pharmacokinetic parameters (A,B,a,b)
obtained after i.v. injection were used as the impulse function and the spline-® tted curves following
intranasal and oral adm inistration as the input function. The cumulative input, Wjin, was c alcu lated until
the last sampling point using the analytical deconvolution algorithms previously described (Gillespie and
Ven g-Pedersen 1985), a fter w hich the absorbed frac tion was cal culated by div iding Wji n with th e nasal ly
or or ally administere d dose .
HPLC analysis
The plasma concentrations of the peptides were determined by a gradient, reversed-phase HPLC
method using Waters LC mod ule 1 and 470 ̄ uorescence detector. The data acquisition, processing and
result calculations were handled by use of M illennium 2010 Chromatography Manager. In general the
exc itatio n and emission wave leng ths we re set at 280 and 3 50 nm, re spectively . The pe ptide N NC 26 -
023 5, how ever, was analysed by UV at a wave le ngth of 224 nm . The co lumn system c onsisted of a g uard
and an analytical column (C" ) , 3.93150 mm, 60 A
c, 4 lm , WAT 037520). The mobile phases consisted of
(A) ac eton itrile : amm oniu m sulp hate 0 .1 m20 : 8 0 v }v% , p H 2.5 , an d (B) ac eton itrile : a mm oniu m
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1086 P.B.Joh ansen et al.
Figure 1. Plasma concentrations of diå erent peptidyl GH secretagog ues following single i.v. injections
of 1 mg}kg to the male rat. Da ta are the mea ns‰SE of four to six anim als. Ipamorelin (y),
GH RP-2 (_), NNC 26-0235 ( U), GH RP-6 (+), N NC 26-0 194 (E).
sulphate 0.1 m40 : 60 v }v%, pH 2.5. U sing the two mobile phases and using gradient pro® le 3, a curve-
linear gradient was run from 20 to 30 % acetonitrile over 10 min. Thereafter, the column m aterial was
̄ us hed w ith mo bile phase B for 5 min and was equilibrated with mobile phase A for 10 min befo re the
nex t injec tion, re sultin g in a run time of 25 min. The ̄ ow ra te was 1 ml}min . The analysis was ru n at
ambient temperature. T he solid-phase extraction procedure used SPEC‡C" ) -A R mic ro-co lumns
(Kingo Diag nostics, Denmark) and a 16-sam ple Waters Vacuum Manifold. After conditioning the
col umns with 40 0 ll methanol followed by 400 ll pho sphate buå er 0 .1 mpH 7, 200 ll plasma 1100 ll
internal standard 2 lg}m l (G HR P-6 o r GHRP-2) 1200 ll buå er were added and aspirated. After
was hing the columns w ith 1 m l wate r and 1 m l 20 % m ethanol, th e pepti de was eluted twice with 0 .5 ml
TF A 1 % in me thano l. Of w ater, 1 00 ll wa s adde d to the eluent, which subsequentl y was evaporated to
abo ut 10 0 ll in a vacuum centrifug e. A fter additio n of 100 ll m obile phas e A, 5 0 ll of each sample was
injected into the liquid chromatograph. T he concentrations of the individual peptides were qu anti® ed by
use of a calibration line obtained by spiking of plasma with six diå erent concentrations of the respective
peptides, ranging from 50 ng}m l to 20 lg}ml.
The re tention times of the GH secretagogues are given in pare nthes is : Ipam orel in (3 m in), NN C 26 -
019 4 (7), NNC 2 6-02 35 (14), GH RP- 6 (7) and GH RP-2 (1 0). Th us, the pea ks of the anal yte and the
chosen internal standard could be clearly separated in the chrom atogram. The reco very was determined
by sp iking of six plasma samp les with the re spec tive peptides in a co ncen tration of 10 lg}ml. GHR P-6
and G HR P-2 showed a lmost c omplete recovery, whe reas ipamo relin, NNC 2 6-01 94 and N NC 2 6-02 35
show ed mea n rec ove ries of 67, 7 1 an d 78 % , resp ectiv ely . The prec ision wa s dete rmi ned b y spi king of
eight plasma samples w ith the individual peptides in a concentration of 10 lg}ml and either internal
stan dard. The coe æ c ient of varia tion ranged betw een 15 and 20 %. The li mit of quanti® c ation w as
arbi trary set a t 50 ng}ml.
Res ults
The pla sma concentra tions of the peptides after i.v. injections a re shown in ® gur e
1 and the es tim ated pharmacokine tic p arame ter s are shown in table 2. Th e plasma
concentrations of all peptides could be described by use of a biexponentialequation.
The blood samples were only drawn during 40 min after injection, which does not
exclude th at the term inal elimination ha lf-lives could be longer. Co nse quently, the
AUC would become larger and the clearances accordingly lower. As seen, however,
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Evaluation of peptidyl growth hormone secretagogues 1087
Tab le 2. Pharma cok inetic p aram eters in the male rat in jecte d intra veno usly with 1 m g}kg of diå erent
peptidyl GH secretagogues. Parameters were estimated by ® tting a two-compartment open model
to the plasma concentration± time data. Da ta are the means and SE.
Parameters
GH RP-2
(n
=
6)
GHR P-6
(n
=
5)
Ipamorelin
(n
=
6)
NN C 26-0 194
(n
=
4)
NN C 26-02 35
(n
=
4)
CLB(ml}m in}kg) 24 ‰1.3 25 ‰1.9 5.2 ‰0.6 40 ‰6.3 22‰2.3
T"
#b(min ) 9.7 ‰0.7 26 ‰5.3 27 ‰4.3 14 ‰1.0 11‰1.4
Vss (ml}k g) 203 ‰8 415 ‰88 199 ‰21 465‰87 25 4‰43
ke(min Õ") 0.197‰0.0 15 0.130‰0.014 0.0 59‰0.006 0.1 76‰0.012 0.142‰0.015
A(ng}ml) 6765 ‰451 439 2‰500 5583 ‰678 4048‰767 47 28‰918
B(ng}ml) 1664‰198 7 95‰185 4532 ‰671 67 2‰37 21 32‰553
a(min Õ") 0.344‰0.0 45 0.0303 ‰0.047 0 .436 ‰0.089 0.313‰0.046 0.3 09‰0.046
b(min Õ") 0.074‰0.0 05 0.032‰0.007 0.0 29‰0.005 0.0 52‰0.004 0.065‰0.007
Figure 2. Excretion pro® les of diå erent peptidyl G H secretagogues following a single i.v. injections of
1 mg}k g to the male rat. Da ta are the m eans of two to fo ur animals. B ile is closed symb ols, urine
is op en sym bols. Ipa more lin (y), N NC 26-0235 (U), G HRP -6 (+), NNC 2 6-019 4 (E).
ipamorelin showed a signi® cantly lower systemic clearance when compared with the
oth er pept ide s investiga ted. Th is diå erence in sy ste mic clear ance wa s subsequently
demonstrated to be re ̄ ected in the excre tion pro® le .
GHRP-6 was predominantly excreted in the bile as the intact peptide. The
ex cretion w as r apid w ith 47 .3 % ‰1. 5 % o f th e ad min iste red do se re cover ed af ter t he
® r st 3 0-m in sam pl ing. T he c um ulat ive bil iar y e xcr etion a fter 4 h w as 70 .7 % ‰
1.8 %. The e xcret ion in urine, howeve r, was much less, after 4 h accounting for
8.9 % ‰0.5 % o f th e ad min ist ered d ose. T otal ly, t he cu mul ativ e ex cre tion am oun ted
to 7 9.7 % ‰1.2 % . Also, NNC 26-0194 an d NN C 26-0235 were mainly excreted via
the biliary route, whereas for ipamorelin urinary excretion was more important.
Also, like GHR P-6, ipa mo relin an d the two N NC pep tid es were mainly excreted
uncha nged. T he excretion pr o® les are show n in ® gu re 2. The biliary excretion of
GHRP-2 cou ld not be dete rmined due to metabolite interference in the HP LC
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1088 P.B.Joh ansen et al.
Fig ure 3. Plas ma co ncentrations of d iå erent pept idyl G H sec retag ogues follo wing a single intranasal
app licati on of 1 mg}kg to the male rat. Data are the means ‰SE, n
=
4± 8. Ipamorelin (y),
GH RP-2 (_), NNC 26-0235 ( U), GH RP-6 (+), N NC 26-0 194 (E).
Table 3. Mean bioavailability of diå erent peptidyl GH secretago gues follow ing intranasal
app licati on of 1 mg}k g to the m ale rat.
Peptide
Bioavailability
(% ) 95 % C I (% ) n
Ipa more lin 21 14± 27 8
NN C 26-01 94 46 22± 68 6
NN C 26-02 35 54 29± 79 6
GH RP-2 53 38± 67 4
GH RP-6 30 15± 45 5
chromatogram. However, the renal excretion of this peptide (not shown) w as similar
to th at of N NC 26-0194, NNC 26-0235 and GHR P-6, sugge sting that the rem ain ing
part of th e peptid e wa s in fact eliminated via the liver .
The plasma concentrations of the peptides following intranasal administration
are sho wn in ® gure 3. The absorption pro® les of the peptides d iå e red signi® cantly.
The pe ak conc entration occu rre d rapidly following application of GH RP -2 and
NNC 26-0235 ; the p las ma concentration-tim e pr o® le looking a lmost as if the
peptid es had be en injected int raveno usly. T he re was, how ever, a very distinct pea k
follow ing ip am orelin , N NC 26-0194 and G HR P-6 occur ring 15± 30 min after
applic ation. The mean a bsolute bioavailability o f the peptides w as foun d by
deconvolution analysis to be between 20 and 55 % of the administered dose (table 3).
GHRP-6 wa s the only peptide inve stigated following oral administrat ion . The
bio ava ilab ility fol low ing t his r oute of a dmi nis tratio n w as lo w, na me ly 1 %
(0. 8± 1.4 % ).
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Evaluation of peptidyl growth hormone secretagogues 1089
D is cu ss io n
The pharm ac okinetic da ta obta ine d wi th G HRP-6 in the pres ent s tud y ac cor d
with a previous ly published st udy in rat which reported a rapid decline in the
systemic circulat ion w ith a clearance of 26 m l}min}kg , a terminal elimination half-
life of 28 min, and a predominant biliary excretion (Davis et al. 199 4). It was fu rth er
demonstrated that the excretion pro® les of NNC 26-0194 and NNC 26-0235 were
very similar to that of GHRP-6, whereas ipamorelin had a more even excretion
between bile and ur ine . It ha s thus been possible to obtain a p eptide with a
signi® cantly diå erent pharmacokinetic pro® le without loss of GH releasing
potential.
The areas under the plasm a concentr ati on-tim e curves of the G H se cretagogues
investigated in the present study were much larger when compared with other native
peptid yl c ompounds. F or e xam ple, m et-en kaphalin, w hich w as used as a t em pla te
for the synthesis of G HRP-6 an d leu-enkephalin , were r apidly cleared from the
systemic circulation in rats with plasma half-lives of 2± 4 s and no detectable intact
pentapeptide remained 2 min after injection (Dupont et al. 1977). Considerably
increased metabolic resistance could be demonstrated for the GH secretagogues by
the high recovery of intact pep tid e from u rin e and bile. Thus, b y introduction of d-
am ino a cid s into the molecules, rapid metabolic degradation h as b een cir cumvented.
Extensive biliary excretion has been demonstrated for other peptide-mimicking
drugs, suggesting that carrier-mediated mechanisms are involved in their rapid
extraction from portal blood (Ziegler et al. 1996). Re cently, the e nzymatically stable
model opioid peptide [D-pen#,&] Enkephalin w as r eporte d to p ossess a biliary
excretion pro® le very similar to that found for GHRP-6 in the present study (Chen
and Po llack 1997). Also, for this op ioid pe ptide, the majority of th e administered
dose (C80 %) wa s recovered from the b ile a s the intact peptid e. The disposition w as
best de scribed by a tw o-c om partm ent model w ith M ichaelis-Menten elimination
from the central compartment into bile, also suggestive of involvement of an active
hepatic transport system . In contrast, hex arelin, a methylated GHR P-6 analogue,
ha s de mon stra ted a low 20 % he pati c ex tra ction r atio ( ER) in the in situ isolated
perfused r at liver (R oumi e t al. 1997). Since the ER can be expressed as th e ratio
between clearance and hepatic blood ̄ ow, the ER o f GH RP -6 can be estimated from
the p resent study to be in the vicinity of 0.5 , assuming a rat hepatic blo od ̄ ow of
1.2 5 ml}min}g liver (Gores et al. 1986), a hematocrit of 0.4 , a liver weight of 10 g
and taking into account the renal excretion and the total recovery. Diå erences in the
aæ nity from the presumed hepatocellular transport system or in protein binding
could be possible explanations for the apparent diå erence between hexarelin and
GHRP-6, but could of course also be ascribed to the diå erent methodologies used.
The high ER of G HRP-6 co uld be the cause for the low oral bi oav ailability. We
have e vidence, howeve r, th at the low bioava ila bility of not only G HRP-6 but also
the other peptides investigated is caused by a poor transepithelial transport across
the intestinal tract (data not shown). Interestingly, however, it has been possible in
both healthy volunteers and children with short stature to obtain a GH secretion
after oral administration equivalent to that seen after i.v. injection (Hartman et al.
1992, B ellone et al. 1995). The doses used were 300 lg}kg orally and 1 lg}kg
int ravenously, suggesting a systemic a vailability in ma n in the vicinity of 0.3 %. In
the pres ent study, the oral bioavailability of G HRP-6 was C1 % in r at. D ue t o th is,
other routes of delivery have come into focus and among these the nasal route has
already been used clinically. For example, the signi® cant GH response seen after
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1090 P.B.Joh ansen et al.
admin ist ration o f GHRP-2 suggests th at it ma y be used as a diagnostic tool in
asses sing pituitar y GH function (P iho ker e t al. 199 5). Also, GH RP-6 and h exarelin
have been shown to elicit a prom pt GH response after nasal administration (Hayashi
et al. 1991, G higo e t al. 1994, Laron et al. 1994). In a clinical p hase II study, La ron
et al . (1995) reported that nasally administered hexarelin in a dose of 60 lg}kg three
tim es d aily for up to 8 m on ths ind uced a signi® c ant increase of linear gr owth
velocity.
The nasal bioava ilability of GHR P-6 has ind ire ctly been e sti mated in th e
conscious dog to be 34± 45 % by using the i.v. and intranasal GH response data
(N els on et a l. 1991). This is in close agreement with th e bioavailability e sti mated in
the present study by direct determination of the plasma concentrations of GHRP-
6 follow ing the two ro ute s of admi nis tration . In m an, the bi oav ailability of hex arelin
fol low ing in tra nasa l ap plic atio n h as be en fou nd to b e 4± 6 % as e stim ate d fr om th e
GH response (Ghigo et al. 1 994 ). Also, in an animal study, the in ̄ ue nce of
for mu lat ion v ariables on pl asma G H l eve ls w as investigated follo wing intranasal
admin ist ration of GHRP-6 in th e anaesthetized rat. I n this latter study , the
bio availa bility w as also e valuated indirectly by meas urement of the GH release
(Vora et al. 1993). A hypotonic peptide formulation achieved the highest bio-
availa bility of 6 %, which is much low er th an the bioavailability found in t he dog
stu dy and in the prese nt study. The authors discuss, how ever, the pr oblem w ith
using the GH release for estimation of bioavailability, and conclude that direct
measurement of plasma G HRP-6 levels is a mo re relevant indicator of peptide
absorption. In the present study, which used direct measurements, the mean
bioavailabilities of the selected peptides for nasal evaluation were between 21 and
54 % .
It should also b e noticed that the pe ptides inv estigated were not form ulated
pharm aceutically, but were me rely dissolved in saline and a pplied into the nostril by
use of a pipette during ultrashort halothane anaesthesia. In this experimental
system, some of the test solution m ay have be en q uic kly removed by mucociliary
cle ara nce me chanisms . It could be a rgued that the m odel is closer to the clinical
sit uation as com pared with the experime nta l system, where the passage of the
nasopalatine tract is sealed to prevent drainage of drug solution from the nasal cavity
int o the mouth (H ussain et al. 1980, S u et a l. 1984). It is noteworthy, however, that
the relative ly high n asal b ioav ailabilities w ere obta ined witho ut use of an y abs orption
enhancers which usually are considered toxic and may in ̄ ict severe damage to the
nasal epithelium (Agerholm et al. 1994).
The ® nding that the peptides can be adm inistered intranasa lly w ith r ela tive ly
high absorption fractions was not unexpected as the molecular weights of the
peptid es investigated were !1000 Daltons, the limit where nasal permeability has
been shown to decrease rapidly for hydrophilic molecules (McMartin et al. 1987).
The peptides, however, had very diverse molecular structures and physicochemical
properties which made any correlation between the observed nasal absorption and
the m olecu lar w eight impossible. Even th ough th e molecular weight of ipam orelin
is m uc h lower th an th at of GH RP-6, the na sal bioava ilability w as not increased.
This is believed to be caused by the extr emely hydrophilic nature of ipamorelin. It
is also not known how the NNC peptides are transported across the nasal epithelium,
but the transport of GHRP-6 across ovine nasal epithelium has been shown to occur
by a pa ssive diå us ive p rocess. Evidence for th is is base d on the fa ct that both the
mucosal-serosal and the serosal-mucosal ̄ uxes were equal (Readon et al. 1993).
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Evaluation of peptidyl growth hormone secretagogues 1091
In conclusion, this study has demonstrated distinct diå erences in pharma-
cokinetic pro® les and has further substantiated the nasal delivery as an attractive
route o f admin ist ration for this family of p eptidy l GH se cretagogues.
A ck no w le dg em e n ts
The skilful techn ical assistance of E lse Bentsen, H anne Jepsen, Helle F. P etersen
and Anne Trolle-Christensen is gratefully acknowledged. The study was conducted
under the Aarhus University-Novo Nordisk Center for Research in Growth and
Regeneration.
R ef er e n ce s
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... Ipamorelin affects ACTH/cortisol levels less than GHRP-2 or GHRP-6 [84]. It has a slower plasma clearance than GHRP-6 and is moderately resistant towards metabolism because 60-80 % of the peptide is excreted unchanged in the urine [95]. Its intranasal bioavailability is approximately 20 %, which is less than GHRP-2 and more than GHRP-6 [95]. ...
... It has a slower plasma clearance than GHRP-6 and is moderately resistant towards metabolism because 60-80 % of the peptide is excreted unchanged in the urine [95]. Its intranasal bioavailability is approximately 20 %, which is less than GHRP-2 and more than GHRP-6 [95]. Ipamorelin has completed 2 phase II trials for recovery from surgeries of the gastrointestinal tract (clinicaltrials.gov; ...
Miha Vodnik Borut Strukelj
Mojca Lunder
In the recent decades, great progress has been made in the development of ghrelin receptor ligands. The discovery of the first in vitro only active peptide growth hormone secretagogue derived from Met-enkephalin was the foundation for later discoveries of the receptor and the endogenous ligand ghrelin. Since then, the scope of peptides, peptidomimetics, and small-molecules targeting the ghrelin receptor, GHS-R1a, has expanded dramatically. Numerous agonists have been tested in animals and several in humans, and a handful have progressed to clinical trials for indications such as growth hormone release, gastric emptying, and cachexia. However, with the exception of the approval of GHRP-2 for diagnostic purposes in Japan, none of the candidates have been successfully introduced into the market. More recently, the attention of researchers has been concentrated on developing antagonists and inverse agonists for pharmacological treatment of the ever-expanding obese and overweight population. In this review, we describe the development of GHS-R1a targeting agonists, antagonists, and inverse agonists. We focus on current and completed clinical trials and the therapeutic potential of currently available ligands. © Georg Thieme Verlag KG Stuttgart · New York.
... GHRPs stimulate GH release and have been regarded as an alternative to support diagnostics and treat diseases related to GH deficiency [3,4]. Their effects have been described in vivo and in vitro, in both animals and humans, using various doses and administration routes [5][6][7][8][9][10]. However, only the peptide GHRP-2 (also known as pralmorelin) has been clinically tested and approved in Japan for diagnostic purposes [11], while other GHRPs have not passed clinical trials or are still in different stages of development. ...
Pedro Ferro Grigory Krotov Irina Zvereva
Jordi Segura
The use of small peptides for medical purposes has gained great importance in the last decade. Among these molecules are the growth hormone releasing peptides (GHRPs). These substances are highly sought by cheating athletes because of their ergogenic effects. Thus, GHRPs are listed in the World Antidoping Agency Prohibited List and several methods have been developed for their detection in urine samples. Serum is another useful matrix frequently used for anti-doping control. An experimental adaptation of a competitive methodology for GHRPs wide-group detection, previously developed for urine, is reported here using a serum matrix instead. The modifications have been mainly addressed the need to remove endogenous ghrelin (always present in serum) to avoid interference in the competitive binding assay. The modified test was first evaluated using serum spiked with different pure GHRPs (i.e., GHRP-2, GHRP-6 and Hexarelin), then using serum samples from pilot studies in which healthy subjects were treated intravenously with GHRP-2 and intranasally with GHRP-2 and GHRP-6. The results showed the reliability of the modified methodology for the detection of GHRPs in a serum matrix.
... The different effects reported on IGF-I may be caused by differences in pharmacokinetics between the GH secretagogues in question. Thus, the half-life of ipamorelin in rats has been found to be short, about 30-60 min 34 . A longer halflife may affect the endogenous GH profile to a higher extent than a drug with a short half-life. ...
Peter B Johansen Jette Nowak
Christian Skjaerbaek Hans Ørskov
Ipamorelin is a new and potent synthetic pentapeptide which has distinct and specific growth hormone (GH)-releasing properties. With the objective of investigating the effects on longitudinal bone growth rate (LGR), body weight (BW), and GH release, ipamorelin in different doses (0, 18, 90 and 450 microg/day) was injected s.c. three times daily for 15 days to adult female rats. After intravital tetracycline labelling on days 0, 6, and 13, LGR was determined by measuring the distance between the respective fluorescent bands in the proximal tibia metaphysis. Ipamorelin dose-dependently increased LGR from 42 microm/day in the vehicle group to 44, 50, and 52 microm/day in the treatment groups (P<0.0001). There was also a pronounced and dose-dependent effect on BW gain. The treatment did not affect total IGF-I levels, IGFBPs, or serum markers of bone formation and resorption. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the metaphysis of the tibia did not change significantly with treatment. The responsiveness of the pituitary to a provocative i.v. dose of ipamorelin or GHRH showed that the plasma GH response was marginally reduced (P<0.03) after ipamorelin, but unchanged after GHRH. The pituitary GH content was unchanged by ipamorelin treatment. Whether ipamorelin or other GH secretagogues may have a place in the treatment of children with growth retardation requires demonstration in future clinical studies.
... administration. 43 In conclusion, the decrease in bone formation found in GC-injected rats can be counteracted by administration of GHS. ...
N B Andersen
Kjell Malmlöf
Peter B Johansen H Oxlund
The ability of the growth hormone secretagogue (GHS) Ipamorelin to counteract the catabolic effects of glucocorticoid (GC) on skeletal muscles and bone was investigated in vivo in an adult rat model. Groups of 8-month-old female rats were injected subcutaneously for 3 months with GC (methylprednisolone) 9 mg/kg/day or GHS (Ipamorelin) 100 microg/kg three times daily, or both GC and GHS in combination. The maximum tetanic tension of the calf muscles was determined in vivo in a materials testing machine. The maximum tetanic tension was increased significantly, and the periosteal bone formation rate increased four-fold in animals injected with GC and GHS in combination, compared with the group injected with GC alone. In conclusion, the decrease in muscle strength and bone formation found in GC-injected rats was counteracted by simultaneous administration of the growth hormone secretagogue.
Ekaterina Semenistaya Irina Zvereva Andreas Thomas Grigory Rodchenkov
Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
Jogarao Gobburu
Henrik Agersø William J. Jusko
Lars Ynddal
To examine the pharmacokinetics (PK) and pharmacodynamics (PD) of ipamorelin, a growth hormone (GH) releasing peptide, in healthy volunteers. A trial was conducted with a dose escalation design comprising 5 different infusion rates (4.21, 14.02, 42.13, 84.27 and 140.45 nmol/kg over 15 minutes) with eight healthy male subjects at each dose level. Concentrations of ipamorelin and growth hormone were measured. The PK parameters showed dose-proportionality, with a short terminal half-life of 2 hours, a clearance of 0.078 L/h/kg and a volume of distribution at steady-state of 0.22 L/kg. The time course of GH stimulation by ipamorelin showed a single episode of GH release with a peak at 0.67 hours and an exponential decline to negligible GH concentration at all doses. The ipamorelin-GH concentration relationship was characterized using an indirect response model and population fitting. The model employed a zero-order GH release rate over a finite duration of time to describe the episodic release of GH. Ipamorelin induces the release of GH at all dose levels with the concentration (SC50) required for half-maximal GH stimulation of 214 nmol/L and a maximal GH production rate of 694 mIU/L/h. The inter-individual variability of the PD parameters was larger than that of the PK parameters. The proposed PK/PD model provides a useful characterization of ipamorelin disposition and GH responses across a range of doses.
Catherine Pihoker R Middleton
Gemma Reynolds T M Badger
GH secretion is primarily regulated by the hypothalamic-releasing hormones GHRH and somatostatin. Additionally, several neurotransmitters act at the hypothalamus and pituitary to modulate GH release. The agents commonly used in clinical practice to diagnose GH deficiency, such as arginine, insulin and L-dopa, act through the neural GH network. Many children with a poor GH response to conventional agents have a significant serum GH response to iv GHRH. GH-releasing peptides (GHRPs) are synthetic peptides that like GHRH act directly on pituitary somatotrophs to stimulate GH release. GHRP-2, an investigational drug, is one of the most potent members of the GHRP family. It has been shown to be effective in adults via the oral and intranasal as well as the iv route of administration. In this study, GH responses to GHRP-2 were compared with GH responses to other provocative agents in children of short stature. GHRP-2 was administered iv or intranasally to children with short stature. In the same subjects, GHRP-...
A Dupont
Leonello Cusan M Garon
Fernand Labrie
Thirty sec after the intrajugular injection of [3H] methionine-enkephalin (met-enkephalin) in the rat, the radioactivity was already distributed in an apparent volume of 53 ml and the metabolic clearance rate calculated from the characteristics of the plasma disappearance curve was 10 ml/min. As shown by partition chromatography plasma extracts obtained 15 sec after injection of [3H] met-enkephalin, only 5% of the total radioactivity migrated as the intact pentapeptide, while no detectable intact pentapeptide remained 2 min after injection, thus indicating a half-life of [3H] met-enkephalin of the order of 2 to 4 sec. Incubation of rat cerebral tissue with [3H] met-enkephalin indicates that the first step in the breakdown of met-enkephalin in both plasma and brain tissue is cleavage of the Tyr-Gly amide bond. These data offer an explanation for the low activity of met-enkephalin after intraventricular or intravenous administration.
Kenneth S. E. Su Kristina M. Campanale Christian L. Gries
The blood levels of the [14C]clofilium ion in rats after various routes of administration of clofilium tosylate were compared. The results indicate that the blood levels after nasal administration were not statistically different from levels after intravenous administration (p > 0.05). Administration by the oral route resulted in considerably lower blood levels. Nasal administration of clofilium tosylate appeared to be superior to oral administration. Histological examinations of nasal mucosa were conducted. At the lower concentration, mild necrosis was observed, and large areas of mucosa were unaffected. However, necrosis of large areas of mucosa occurred after exposure to the higher concentration. Levels of radioactivity in heart, liver, lung, and kidney tissue, as a function of time, were also studied. Unlike the blood levels after nasal administration, the levels of radioactivity were persistent in heart tissue. The data suggest that the [14C]clofilium ion and/or metabolite concentrate in the heart and that blood levels of radioactivity may not be an accurate index of cardiac levels or biological response.
Zvi Laron Jenny Frenkel R. Deghenghl A. Siibergeld
OBJECTIVE Hexarelin is a recently synthesized small growth hormone releasing peptide (GHRP) (His-D-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2). It Is actlve by intra-venous, oral and intranasal administration In animals and man. The aim of this study was to find out whether longterm administration of this peptlde would promote growth In short chlldren. DESIGN AND PATIENTS intranasal hexareiin was administered in a dose of 60 pglkg thrice dally to 8 prepubertal short children aged 4-11-6 years for perlods of up to months. RESULTS Hexarelln treatment stimulated Insulin-like growth factor-i (IGF-I) secretion ralsing the level from 10.4 ± 3.9 (SD) to 141 f 46 nmoill (P < 0.004). The rise IGF-l led to a significant Increase In the mean (±SD) linear growth velocity from 53 ± 08 to 8 3 ± 1.7 cm/year (P < 0.0001). There was also a slgniflcant decrease skinfold thickness despite Increase in body weight and an Increase in head circumference. Additional flndlngs were a rlse in serum phosphate from 1–5 ± 01 to 1–8 ± 01 mmoi/l (P < 0004) and of aikallne phosphatase from 219 ± 74 to 261 ± 75 U/l (P < 005). CONCLUSIONS The long-term GHIIGF-I stimulating, anabolic and growth promoting effects achieved by intranasai administration of this hexapeptide, seemingly without undeslrable slde-effects, suggests clinical potential for this new class of drugs.
William R Gillespie
Peter Veng-Pedersen
A new deconvolution algorithm (DCON) suitable for pharmacokinetic applications is presented. It requires that both the impulse and input responses, typically systemic drug levels, be well described by polyexponential equations. DCON has a wider range of applications than an earlier method (DECONV) from which it is derived. A FORTRAN program is provided, making implementation of the technique a simple matter. DCON is demonstrated to evaluate the GI bioavailability, defined as the rate and the extent of gastrointestinal drug release, of various ibuprofen dosage forms. The GI drug release kinetics exemplifies a pharmacokinetic system which cannot be evaluated using the previous deconvolution algorithm (DECONV) because of an initial zero drug level response. This limitation is not found in DCON. It is also demonstrated how the mean in vivo dissolution time MDT can be evaluated by deconvolution.
Marie Roumi
Sylvie Marleau M F Boghen
Huy Ong
Purpose. To assess the hepatic extraction of hexarelin (HEX), a novel peptidyl GH secretagogue, in the isolated perfused rat model and document the in vitro binding of HEX to plasma proteins using plasma from rats, dogs, pigs, and humans. Methods. Rat liver was perfused in situ using a recirculating system. The recirculating perfusate consisted of a Krebs Henseleit buffer containing 20% (v/v) prewashed bovine red blood cells, 1 % albumin, and 1g/L dextrose. Three HEX concentrations of 5, 50, and 500 ng/ml were examined. In vitro plasma binding was determined by the ultrafiltration method. Results. The disappearance rate constant (K), half-life (t1/2), clearance (Cl), and hepatic extraction ratio (E) were: K = 0.013–0.014 min–1, t1/2 = 45–55 min, Cl = 0.345–0.401 ml/min/g liver, and E = 19-21% for the different concentrations of HEX. A linear increase in AUC (270-24334 min pmol/ml) was observed with increasing concentrations. Binding of HEX to plasma proteins of rats, dogs, pigs, and humans was 68.7 0.8%, 78.7 0.6%, 67.3 0.7%, and 65.2 0.6% respectively. Plasma binding was concentration-independent in the range between 0.003–3 M for the four species examined. Conclusions. These results show that 1) the hepatic extraction of HEX is low, 2) the hepatic clearance is concentration independent up to 500 ng HEX/ml of perfusate, and 3) the plasma protein binding of HEX is significant over the dose range studied. HEX exhibits a low hepatic extraction ratio, allowing us to predict that its hepatic clearance may be limited upon HEX protein binding.
Jayesh K. Vora Gregory A. Christensen Mauricio J. Reginato Choon K. Oh
Growth hormone releasing peptide (GHRP-6) is a synthetic hexapeptide that stimulates the release of growth hormone in several animal species. The feasibility of developing an efficacious intranasal formulation of GHRP-6 is currently under consideration for clinical and commercial applications. In this study, the influence of several formulation variables on growth hormone (GH) release following intranasal administration of GHRP-6 was examined in anesthetized rats. Pharmacologie availability of the peptide was significantly increased by reducing formulation tonicity (hypotonie solution) and incorporation of the preservative benzalkonium chloride. Alteration of formulation pH from 5 to 7 did not demonstrate significant changes in pharmacologie response. GH release decreased as solution viscosity was increased, suggesting a slower rate of peptide absorption across the nasal epithelium.