sermorelin acetate

STATEMENTS NOT REVIEWED BY FDA Pricing: $650 per month, including all supplies

New Age Medical Clinic, PA. www.GrowthHormoneNJ.com (908) 598-0509

Sermorelin

Growth hormone-releasing hormone (GHRH)

Losing muscle mass and tone? Feeling fatigued?

Gaining weight? Low sex drive?

SERMORELIN IS A GROWTH RELEASING HORMONE THAT CAN HELP YOU LOOK AND FEEL BETTER



Contents:

1. Overview: What is Sermorelin?

a. Benefits

b. Side effects

2. Administration and Dosing

a. Instructions for Administration

3. Studies



What is Sermorelin?

Increase Human Growth Hormone Safely and More Effectively

New Age Medical restores optimal levels of Human Growth Hormone using a more effective approach. We

combine an effective dosage of Sermorelin with bio-identical hormones like testosterone to increase lean

muscle tissue, reduce body fat, and improve overall health and longevity. Our medical team also utilizes

amino acids and peptides to help improve results.

What is Sermorelin Acetate? Sermorelin(GHRH) is a bio-identical hormone that has been genetically engineered to stimulate the

secretion of Growth Hormone Releasing Hormone (GHRH) from the hypothalamus, a gland adjacent to the

pituitary gland. GHRH is a peptide that contains the first 29 amino acids of our own GH. These 29 amino

acids are the active amino acids of GHRH. It is GHRH that stimulates the pituitary glands to release GH.

As we get older, the hormones produced by the anterior pituitary are depleted. It has now been shown that

GHRH can restore the GH-RNA to a youthful level causing elevation of levels of IGF-1.

How do I take sermorelin?

Sermorelin is a self-administered injection taken nightly. Please review the dosing and administration

information in this packet.

Why would I take sermorelin? What are the benefits? Human Growth Hormone (HGH) produced by recombinant gene technology has been used extensively for

anti-aging therapy during the past decade. Some of the symptoms of low HGH levels, include a declining

sex drive, lower energy levels, drowsy, sleepiness and fatigue, an accumulation of body fat and adipose

tissue around the mid section, decreased mental clarity, skin wrinkling on the face, neck and hands, and

declining immune system. HGH (Human Growth Hormone) is mostly secreted at night during REM sleep.

HGH is critical in maintaining the organs and tissues in our body. HGH levels are highest during youth and

puberty, but then decline once our body has reached it mature size, and HGH production summits in our

youth. When we reach 30 to 40 years of age, the decline of HGH is approximately 1 to 2 percent each

calendar year that passes. At the age of 40, our HGH levels are less than half of what they were when we

were in our early 20s.

Many people can benefit from regular sermorelin use. If you have trouble sleeping, difficulty losing weight,

decreased strength and muscle mass, and certain medical problems, sermorelin can help. Studies show that

sermorelin may:



Increase development of leanbody mass

Reduce body fat Increase energy and vitality Increase strength Increase endurance Accelerate healing Strengthens the heart Enhances the immune system Increases IGF-1 production

Improves sleep quality Increase calcium retention Increases protein synthesis and

stimulates the growth of allinternal organs except the brain.

Plays a role in fuel homeostasis. Promote liver glucogenesis. Contributes to the maintenance

and function of pancreatic islets.

The most outward visible benefits of sermorelin treatments include tighter, softer and plumper,

supple skin (your skin will look more like it did when you were younger). With Sermorelin

injections wrinkles decrease, skin sagging is reduced and thin skin firms up and holds more

water, as your skin cells did when you were younger.

People often report a renewed sense of vitality and mental clarity with sermorelin as well as relief

from many of the health problems associated with aging. Weight loss, and muscle development

can be benefits as well as it causes your metabolism to increase thereby allowing you to burn fat

more easily and to develop muscle. That benefit increases even more when exercise is added to

the program.

Are there side effects?

The most common treatment-related adverse event (occurring in about 1 patient in 6) is local

injection reaction characterized by pain, swelling or redness. Of 350 patients exposed to Other

treatment-related adverse events had individual occurrence rates of less than 1% and include:

headache, flushing, dysphagia, dizziness, hyperactivity, somnolence and urticarial.



Administration and Dosing

How does Sermorelin come? Sermorelin is presented in a multi-dosed, injectable vial. Each vial contains a powder disc which contains

15mg (15,000 micrograms) of lyophilized Sermorelin Acetate. The vial is vacuum sealed by the pharmacy

for your protection and for the preservation of the hormone peptide. Each Sermorelin vial also comes with

a bottle of Bacteriostatic Water as a diluent. The Bacteriostatic Water is mixed with the Sermorelin to

provide solution for injection. Administration and storage instructions will be provided with your

prescription.

How do I take Sermorelin? Sermorelin is injected into the body fat, subcutaneously, using a very small needle similar to what a

diabetic uses to inject insulin. Injections are initially prescribed for every day and are decreased in

frequency over time.

When do I take Sermorelin? The best time to take Sermorelin is prior to bedtime. Growth Hormone is primarily released during sleep

and most beneficial to the body’s recovery and repair during this time. Sermorelin has a promoting effect

on sleep and can therefore make you tired if taken during the day.

How do you measure the effectiveness of Sermorelin? Due to the pulsatile nature of both endogenous HGH and IGF-1, a single blood draw is not sufficient for

accurate measurement. Most physicians who prescribe Sermorelin and similar peptides measure

effectiveness in patients through symptomology (the study of your symptoms-see benefits); physical

appearance and measurements; and more frequent blood analysis.

How will I know its working? After 4 years of observing patients taking Sermorelin, I have noticed that patients usually report improved

sleep within the first few weeks of therapy. Of course, this is only noticed in patients who have trouble

sleeping in the first place, however most patients at least notice an increase in sleep quality. This is usually

concurrent with increased energy levels and improved mood. After 3-6 months of therapy patients start

reporting noticeable or significant body changes, such as increase in muscle tone and a leaner physique.

Over time patients will also notice a significant improvement in skin tone and health.

How long does it take to work? Just like most peptide hormones, Sermorelin usually has a “loading” period of 3-6 months before full

effects are noticed. Once injected, both Sermorelin and rHGH are eliminated from then body very quickly

and therefore need to be injected frequently. Its actions are dependent on a chain reaction of biological

processes which result in elevated and sustained HGH and growth factors. It takes some time for levels to

become optimal and initiate the benefits we are seeking to achieve.

Do I need to take Sermorelin forever to keep seeing results? Actually, no. Sermorelin has an ongoing effect in which optimal HGH levels can be sustained long after

the last injection. Just like synthetic HGH, Sermorelin initially must be injected every day. Unlike



synthetic HGH, once optimal levels are sustained with Sermorelin injection frequencies can be decreased or

stopped altogether. Once results are achieved, patients are then switched to a maintenance protocol

eliminating the need for ongoing daily injections and reducing the total cost of therapy.



Instructions for Subcutaneous Administration

Step-By-Step instructions for Reconstitution and Administration of Sermorelin Injection

We prescribe 15mg Sermorelin per month mixed with bacteriostatic water. Be sure to come in to office

one week prior to the end of your supplies running out to be evaluated for continuing the program.

Administer subcutaneous .3 ml with insulin syringe in belly fat before bed daily.

Do not eat 2 hours before or after administration. If redness or nervousness should occur; cut dose in half

for two days or until the symptoms resolve.

Possible sites for subcutaneous injections:

Stomach

Thigh

Buttocks

Step-By-Step Instructions for Administration

1. Wash your hands with an antibacterial soap and use alcohol to clean the area you will be working on.

Have these supplies ready:

§ A vial of sermorelin 15mg and a vial of sterile water for injection

(diluent)

§ A syringe and sterile needle

§ Alcohol pads, rubbing alcohol, and gauze

§ A needle disposal container 2. Preparing your medicine and filling the syringe

Remove the syringe and needle from the wrapper. While holding the protective cap, twist needle

clockwise to make sure needle is secure. Set the syringe aside

Remove the plastic caps from the tops of the vials

Wipe the tops of the vials with alcohol. Don't touch the tops of the vials once you have wiped

them.

Uncap the needle by carefully twisting the needle cap clockwise and pulling the cap upward.

Avoid twisting the needle counterclockwise, as this can cause the needle to separate from the

syringe.

Insert the needle through the rubber stopper of the sterile water vial.

Do not tap the point of the needle against the sides or bottom of the vial because it may dull or

bend the tip.



Tip the sterile diluent vial and, with the needle in the fluid, pull back on the plunger to withdraw

3ml of the sterile water into the syringe.

Withdraw the needle from the sterile water vial. Slowly inject the sterile water into the vial

containing the sermorelin powder, aiming the sterile diluent at the side of the vial to avoid creating

bubbles.

The Sermorelin powder will dissolve quickly. Gently swirl until the powder is completely

dissolved. Do not shake the vial because this will create bubbles.

Wait until all the powder has completely dissolved. Then withdraw the specified amount for

injection by turning the vial upside down and pull back on the plunger to withdraw the solution as

you slowly lower the needle.

Hold syringe straight up. Draw back slightly on plunger and tap syringe so that any air bubbles

rise to top. Slowly press plunger until all air is out of syringe and a small drop of solution forms at

tip of needle.

Tap the syringe to remove the drop of solution at the tip of the needle.

Carefully recap needle to keep it sterile.

The Sermorelin solution is now ready for injection.

3 Preparing the injection site

Select your site and clean it using the alcohol pad.

Take a large pinch of skin to pull the fatty tissue away from the muscle underneath it.

Holding the syringe like a dart, quickly insert the needle at a 90-degree angle to the skin.

Slowly inject the medication.

Release the pinch of skin, and then withdraw the needle.



Studies

Growth hormone (GH)–releasing hormone and GH secretagogues in normal aging:

Fountain of Youth or Pool of Tantalus?

Sermorelin: A better approach to management of adult-onset growth hormone

insufficiency?

Use of growth-hormone-releasing peptide-6 (GHRP-6) for the prevention of multiple

organ failure

© 2008 Dove Medical Press Limited. All rights reservedClinical Interventions in Aging 2008:3(1) 121–129 121

R E V I E W

Growth hormone (GH)–releasing hormoneand GH secretagogues in normal aging: Fountainof Youth or Pool of Tantalus?

Elizabeth C HerschGeorge R Merriam

VA Puget Sound Health Care System and University of Washington School of Medicine, Tacoma and Seattle, Washington USA

Correspondence: George R MerriamResearch and Medicine Services(A–151) VA Puget Sound Health CareSystem, 9600 Veterans Drive SW, Tacoma,WA 98493, USATel +1 253 582 8440 ext 76172Fax +1 253 589 4105Email [email protected]

Abstract: Although growth hormone (GH) is primarily associated with linear growth in

childhood, it continues to have important metabolic functions in adult life. Adult GH defi ciency

(AGHD) is a distinct clinical entity, and GH replacement in AGHD can improve body composi-

tion, strength, aerobic capacity, and mood, and may reduce vascular disease risk. While there

are some hormone-related side effects, the balance of benefi ts and risks is generally favorable,

and several countries have approved GH for clinical use in AGHD. GH secretion declines pro-

gressively and markedly with aging, and many age-related changes resemble those of partial

AGHD. This suggests that replacing GH, or stimulating GH with GH-releasing hormone or a

GH secretagogue could confer benefi ts in normal aging similar to those observed in AGHD

– in particular, could reduce the loss of muscle mass, strength, and exercise capacity leading

to frailty, thereby prolonging the ability to live independently. However, while most GH stud-

ies have shown body composition effects similar to those in AGHD, functional changes have

been much less inconsistent, and older adults are more sensitive to GH side effects. Preliminary

reports of improved cognition are encouraging, but the overall balance of benefi ts and risks of

GH supplementation in normal aging remains uncertain.

Keywords: growth hormone, growth hormone-releasing hormone, growth hormone

secretagogues, aging, sarcopenia, frailty

IntroductionFrailty in the elderly is a syndrome of progressive loss of strength and aerobic capac-

ity that can increase the risk of falls and their complications, and leads in part to this

functional decline. The result is the need for costly home-based or institutional sup-

port in the rapidly growing part of the population older than 80 years (Merriam et al

2002, 2003). Sarcopenia, or loss of muscle mass, leads to this progressive functional

decline. Growth hormone (GH) also declines with age, and the fi ndings in frail elders

are similar in many ways to those signs and symptoms found in younger adults with

GH defi ciency (AGHD). Replacement of GH or stimulation of GH secretion with GH-

releasing hormone (GHRH) or other GH secretagogues (GHS) would thus seem to be an

appealing option to delay the onset of frailty in older adults and to prolong the capacity

for independent living; but the balance of pros and cons is not necessarily the same

as in AGHD. This review describes the components of the GH axis and their actions,

compares and contrasts normal aging with AGHD; and summarizes GH replacement

and the use of GHRH and GHS in these contexts.

Principal components of the growth hormone axisGH is the most abundant pituitary hormone, accounting for 10% of pituitary dry

weight (Merriam et al 2002). It plays an important metabolic role in adult life as

Clinical Interventions in Aging 2008:3(1)122

Hersch and Merriam

a partitioning hormone, regulating body composition and

function (Merriam and Cummings 2003). GH is a 191 amino

acid protein whose secretion depends on stimulation by the

hypothalamus and is regulated by tissue responses (Merriam

et al 2003). There are three hypothalamic factors or peptide

systems that regulate GH synthesis and secretion (Figure 1):

somatostatin (SRIF), GHRH, and ghrelin (Anawalt and

Merriam 2001; Melmed 2006). Somatostatin, a family of

14 and 28 amino acid peptides, is a potent noncompetitive

inhibitor of the release of GH and other hormones. It modu-

lates the pituitary GH response to GHRH. GHRH, a 44 amino

acid peptide, is the principal stimulator of GH synthesis and

secretion. Ghrelin, discovered in 1999 by Kojima and col-

leagues (Merriam and Cummings 2003), is an endogenous

ligand for a previously described GHS receptor. While the

abbreviation GHS technically could be applied to any growth

hormone secretagogue, it is generally used to refer to ghrelin

and its mimetics rather than to GHRH. Ghrelin is secreted in

large quantities by the stomach, and circulates systemically

at levels high enough to stimulate central GHS receptors,

with access facilitated by its unique lipophilic octanoyl side

group, which is also required for binding to the GHS recep-

tor (Merriam 2002). Ghrelin also has appetite-stimulating

activities distinct from its GH-stimulating effects (Anawalt

and Merriam 2001).

All of these peptides respond to a variety of stimuli and

inhibitors, such as sleep, stress, exercise, food intake and

body composition, and interact to generate the physiologi-

cal pattern of pulsatile GH secretion (Anawalt and Merriam

2001). There are approximately 10 pulses of GH secretion

Ghrelin

GHRH SRIF

Ghrelin

HYPOTHALAMUS

PITUITARY

STOMACH

LIVER

GH

IGF-I

?

?

?

??

? Ghrelin

GHRH SRIF

Ghrelin

HYPOTHALAMUS

PITUITARY

STOMACH

LIVER

GH

IGF-I

?

?

?

??

?

Figure 1 Major components of the GH neuroregulatory system. Question marks on the arrows leading from the stomach indicate uncertainty about the physiological role of gastric ghrelin in the regulation of GH; and on arrows from ghrelin in the hypothalamus indicate uncertainty as to whether ghrelin found in the hypothalamus is synthesized in neurons there, or is synthesized elsewhere and acts at hypothalamic or pituitary levels. IGF-1 is synthesized in many GH target tissues, but more than 85% of circulating IGF-1 is liver-derived. From Anawalt and Merriam 2001.

Clinical Interventions in Aging 2008:3(1) 123

GH secretagogues in normal aging

per day, each lasting about 90 minutes and separated by

120 minutes. Peak GH secretory activity occurs within an

hour after the onset of deep sleep (Melmed 2006). With

increasing age, GH pulse amplitude is markedly reduced, and

there is a loss of the nocturnal GH increase, but the number

of GH pulses does not change greatly (Ho et al 1987). This

secretion is modifi ed by age and sex in addition to the stimuli

mentioned above (Molitch et al 2006). GH, in turn, stimulates

the synthesis of insulin-like growth factor-I (IGF-I), which

mediates many of GH’s effects and is a potent inhibitor of

GH secretion (Merriam 2002). GH has some direct effects

as well via GH receptors present on the surface of many

cell types (Cummings and Merriam 2003). Circulating

IGF-I is synthesized mainly in the liver, but IGF-I is also

locally generated in target tissues. The inhibition of IGF-I

production can create a syndrome of relative GH resistance,

causing increased GH secretion with decreased GH effects.

Examples include fasting, malnutrition, and oral estrogen

therapy (Merriam 2002).

GH promotes lipolysis and inhibits lipogenesis, with a

resultant redistribution of fat. It inhibits the conversion of

cortisone to the active glucocorticoid cortisol, accelerates

the conversion of l-thyroxine to the more biologically active

triiodothyronine (Cummings and Merriam 2003), and exerts

antinatriuretic effects by stimulating renal tubular sodium-

potassium pumps and facilitating the renin-angiotensin-

aldosterone system (Merriam and Cummings 2003).

GH infl uences bone physiology after linear bone growth

has ceased, and is anabolic toward bone and muscle. It

contributes to an increase in overall energy expenditure by

stimulating protein synthesis and fat oxidation (Cummings

and Merriam 2003). GH also enhances intestinal absorption

of calcium and phosphate, vitamin D activity, renal tubular

phosphate reabsorption, osteoblast proliferation, and synthe-

sis of DNA and procollagen mRNA in bone (Merriam and

Cummings 2003).

Normal aging vs adult growth hormone defi ciencyGH secretion rates decline exponentially from a peak of

about 150 µg/Kg/day during puberty to about 25 µg/Kg/day

by age 55 (Melmed 2006). In this process there is a reduc-

tion in GH pulse amplitude, but little change in GH pulse

frequency (Merriam et al 2003). There is a particularly

marked decline in sleep-related GH secretion, resulting

in loss of the nocturnal pulsatile GH secretion seen in

younger individuals and lack of a clear night-day GH

rhythm (Figure 2) (Ho et al 1987; Merriam et al 2000).

It seems that the age-related decline in GH is not the

cause of the decline in slow-wave sleep (SWS), however,

since in most studies administering GH or GHRH does

not enhance SWS in seniors (Vitiello et al 2001). The

decline in GH production parallels the age-related decline

in body mass index and is associated with alterations

Figure 2 Patterns of GH secretion in younger and older women and men. There is a marked age-related decline in GH secretion in both sexes and a loss of the nighttime enhancement of GH secretion seen during deep (slow-wave) sleep. This decrease is primarily due to a reduction in GH pulse amplitude, with little change in pulse frequency. L = large GH pulses, S = small GH pulses. From Ho et al 1987.


Hersch and Merriam

in body composition, hormonal status, and functional capacity

that mimic the changes seen in AGHD or partial hypogonad-

ism (Merriam et al 1997). In addition to deteriorating memory

and cognitive function, the changes in body composition that

are most pronounced in normal aging include a reduction in

bone density and in muscle mass and strength, an increase in

body fat, and adverse changes in lipoprotein profi les (Anawalt

and Merriam 2001; Merriam and Cummings 2003). While the

aging pituitary remains responsive to GH, GHRH, and GHS,

it is less responsive to stimuli such as exercise. This decline in

GH production is initially clinically silent, but may contribute

over time to sarcopenia and frailty.

The decline in GH may also play a role in age-associated

changes in cognition. While there are many systems for clas-

sifying different cognitive domains, often they are grouped

as “crystallized” vs “fl uid” intelligence. The former includes

vocabulary and long-term memory; the latter includes short-

term memory and active problem-solving and declines more

markedly with aging. A number of studies have shown that

in older adults there is a signifi cant correlation between per-

formance on tests of fl uid intelligence and circulating levels

of IGF-I (Aleman et al 1999), suggesting that GH may play

a role in maintenance of fl uid intelligence.

Several possible mechanisms for the age-related decline

in GH secretion have been postulated: loss of (or decline

in) pituitary responsiveness to GHS, increased sensitivity

to the negative feedback by IGF-I, decline in hypothalamic

stimulation, and increase in somatostatin inhibition of GH

(Anawalt and Merriam 2001; Merriam and Cummings 2003).

Published studies have pointed against the fi rst two of these

mechanisms as major factors (Pavlov et al 1996) (Figure 3).

The precise mix of the latter two factors, and of any others, is

still not completely understood. Given that the aging pituitary

can still respond to GHS, that there is no change in sensitiv-

ity to IGF-I, and that there may be some relative defi ciency

of GHRH and possibly ghrelin, it seems reasonable to infer

that the cause of the overall decline of GH secretion with age

is multifactorial and arises above the level of the pituitary

(Merriam and Cummings 2003).

Aging is not a disease. Rather, it is a physiological state

of relative GH defi ciency. This is demonstrated by higher GH

secretion and physiological responses seen in older adults

when compared with AGHD patients of similar age (Merriam

et al 2002). It is important to distinguish true AGHD from nor-

mal aging, since the consequences of the two states differ.

Since all biochemical tests for GHD are imperfect,

and their accuracy is strongly affected by the pre-test

probability of the condition, the most important indicator of

the likelihood of GHD is the clinical context (Merriam and

Cummings 2003). Among adults with AGHD, 85% acquire

the defi ciency as an adult, mostly from pituitary tumors

or their treatment with radiation or surgery (Merriam and

Cummings 2003). Impairment of the hypothalamus may be

present due to similar processes; although in the presence

of pituitary damage, which renders them unresponsive to

GHRH or GHS, this is more diffi cult to gauge. Traumatic

brain injury is also becoming more frequently recognized

as a cause of GHD in adults (Merriam and Wyatt 2006;

Molitch et al 2006), and may produce defi ciencies in other

pituitary hormones as well. Studies have shown that adults

with hypopituitarism have increased mortality compared

with nonhypopituitary populations adjusted for age and

sex. The main causes of the excess mortality were cardio-

vascular and cerebrovascular disease (Molitch et al 2006).

Patients who acquire GHD in adult life also have an increase

in cardiovascular and cerebrovascular mortality and have

clinically signifi cant abnormalities in hormone profi les, body

composition, and physical and mental functions (Merriam

and Cummings 2003).

GHD adults are physically and emotionally less healthy

than their age-matched peers (Table 1). Their skin is cool,

dry, and thin. They suffer psychological and social diffi cul-

ties and cognitive impairment. Fat mass is increased by

7%−10%, with much of the excess located in the visceral

compartment of the abdomen. Lean body mass is decreased

by 7%−8% and skeletal muscle volume is diminished by

up to 15% (Cummings and Merriam 2003; Merriam and

Cummings 2003). Cardiac muscle is also lost, with impaired

ventricular function and cardiac capacity as a result. Hyper-

tension is more common, thrombogenic blood components

are increased, and an atherogenic lipid profi le exists. All of

this contributes to the cardiovascular (and cerebrovascular)

disease seen in AGHD (Merriam et al 2000; Cummings and

Merriam 2003; Merriam and Cummings 2003; Merriam and

Wyatt 2006).

Growth hormone replacementand its side effectsWhile a single case study in 1962 described improved vigor,

ambition and well-being in a 35 year old hypopituitary adult

who received GH, large-scale trials of GH replacement in

AGHD could not be conducted with scarce extracted pituitary

GH. With the availability of synthetic GH in unlimited quali-

ties, clinical trials in AGHD were begun soon after recombi-

nant GH was approved for pediatric use in 1985, and results

of these studies began to appear in the late 1980’s. In 1996



the FDA approved the use of GH in GHD adults (Merriam

and Cummings 2003; Molitch et al 2006). GH replacement in

AGHD has been successful in reversing many structural and

functional abnormalities in the condition (Table 2) (Merriam

2002; Molitch et al 2006). The benefi ts and risks of GH

replacement in AGHD have been documented in more than

1000 publications (Cummings and Merriam 2003; Merriam

et al 2003). While dosing was initially derived from pediatric

practice, doses appropriate for growing children produced

severe side effects in adults and were rapidly reduced. Over

time, weight-based dosing as used in pediatrics gave way

to the current adult practice of beginning with a low fi xed

dose unlikely to produce side effects, with subsequent dose

titration until either an age- and gender-appropriate level of

IGF-I or side effects are encountered. This titration must

be conducted particularly carefully in older adults, who are

more susceptible to adverse effects.

Since aging is a milder GH-defi cient state than AGHD,

GH replacement seems a potentially reasonable approach to

prevention or even reversal of the frailty symptoms of aging.

The fi rst studies in non-GHD older adults took place soon

after its effects in AGHD were published. In a widely cited

Figure 3 Effects of a single intravenous bolus of GHRH on GH secretion in healthy subjects of different ages. While the highest responses are seen in young adults, there is no signifi cant decrease with aging, and pituitary GH responses are well preserved even in the oldest subjects. From Pavlov et al 1986.


Hersch and Merriam

study by Rudman et al (1990), healthy men over 60 years old

responded to 6 months’ GH treatment with an 8.8% increase

in lean body mass, a 14.4% decrease in adipose tissue mass,

and a 1.6% increase in vertebral bone mineral density (BMD).

Since most studies of AGHD have required 12–18 months of

treatment to show an improvement in BMD, this improve-

ment was especially remarkable. Although the Rudman study

did not include any functional measures, given these results, it

was postulated that GH treatment might also improve muscle

strength and functional performance. Studies of physical

functional effects, however, have been generally disappoint-

ing and inconsistent. Papadakis et al (1996) tried to determine

whether GH treatment would improve functional ability in

older men. The authors concluded that GH supplementation

improved body composition but not functional status. Since

the subjects were generally very fi t and functional scores

were close to the maximum at the beginning of the trial, it is

not clear whether this was a true negative result or a “ceiling

effect” related to the testing measures used.

Despite this lack of demonstrated functional effi cacy, a

number of clinics began to offer GH treatment to otherwise

healthy older men and women. Faced with this growing

practice and dearth of information, the NIH National Insti-

tute on Aging issued a call for applications in 1991 to study

trophic factors in aging. Several studies of GH, either alone

or in combination with sex steroids, IGF-I, or exercise con-

ditioning, and one study of GHRH were funded and have

now been completed. While a comprehensive review of the

fi ndings of these studies is beyond the scope of this article,

there is a general consensus among these reports that GH

replacement in normal seniors can elevate levels of IGF-I

to the young adult normal range. While attempts to repro-

duce the doses used by Rudman and colleagues encountered

severe side effects, forcing their reduction to 50% or less of

those he used, target IGF-I levels could usually be reached

at lower doses with tolerable short-term side effects. There

is also a general consensus that GH treatment increases

lean body mass and reduces body fat, especially abdominal

visceral fat (Blackman et al 2002). The studies that included

exercise conditioning confi rmed its benefi cial effects, but

GH did not augment exercise effects and there was no clear

improvement in strength or aerobic capacity with GH alone.

Studies published to date also provided no defi nitive proof

that GH treatment could improve sleep or mood impairment

(Merriam 2002).

All of these studies were conducted for 6–12 months at

a single site, and so only short-term intermediate outcomes

and side effects, not long-term risks, could be observed. Their

results provide no guidance on the effects of GH on long-term

clinical outcomes or risks such as falls or fractures, maintenance

of functional status, or effects on cardiovascular morbidity and

mortality – factors that would establish more defi nitively the

rationale for GH treatment in normal aging (Cummings and

Merriam 2003; Merriam and Cummings 2003). And while few

long-term risks have been observed, this refl ects more a lack

of information than a demonstration of safety.

Table 1 Clinical features of the adult GHD syndrome

↑ Fat mass (especially abdominal fat)↓ Lean body mass↓ Muscle strength↓ Cardiac capacity↓ RBC volume↓ Exercise performance↓ Bone mineral densityAtherogenic lipid profi leThin, dry skin; poor venous accessImpaired sweatingPsychosocial problems Low self-esteem Depression Anxiety Fatigue/listlessness Sleep disturbances Emotional lability and impaired self-control Social isolation Poor marital and socioeconomic performance

Note: From Merriam and Cummings 2003.

Table 2 Effects of GH replacement in GHD adults

↓ Fat mass (especially abdominal fat)↑ Lean body mass↑ Total-body water and plasma volume↑ Muscle mass strength↑ Improved cardiac capacity↑ Red blood cell volume↑ Skin thickness↑ Sweating↑ Exercise capacity↑ Resting energy expenditure↑ Bone mineral density (after 1 yr of treatment)Altered lipid profi le Decreased total cholesterol Decreased LDL-C Decreased Apo B Decreased triglycerides (if initially elevated) Increased HDL-C (not seen in all studies) Increased Lp(a)↓↑ Insulin sensitivity (↓ acutely, ↑ after changes in body composition)Common side effects Fluid retention; edema Arthralgias Carpal tunnel syndrome Decreased insulin sensitivity (acutely); hyperglycemia

Note: From Merriam and Cummings 2003.



Since elders are more sensitive to replacement with GH

(and GH resistance may not be uniform in all tissues), they are

also more susceptible to the side effects of therapy. The side

effects are due to the hormonal effects of over-replacement,

so careful dose titration is extremely important. Patients

who are older, heavier, or female are more prone to develop

complications (Molitch et al 2006). Common side effects

of GH replacement include fl uid retention, with peripheral

edema (40% of patients), arthralgias (20% of patients), and

carpal tunnel syndrome (10% of patients) (Anawalt and

Merriam 2001; Cummings and Merriam 2003; Merriam

and Cummings 2003). Studies have also shown increased

fasting glucose levels. Although these levels generally return

toward normal with the improvement in body composition

and reduced insulin resistance, some studies have found a

persistent increase in fasting glucose and insulin with chronic

GH treatment, even after body composition changes have

stabilized. Other less frequently reported side effects include

headache, tinnitus, and benign intracranial hypertension

(Merriam and Cummings 2003; Merriam and Wyatt 2006).

GH can accelerate both the clearance of thyroxine and

promote its conversion to triiodothyronine, and so can have

variable effects in hypothyroid patients on fi xed replacement

doses. Since GH and IGF-I are growth factors, there are

concerns for promotion of cancer cell growth, but studies to

date have not demonstrated this (Merriam 2002).

Besides these increased vulnerabilities in older patients,

which are common to the use of GH both in GHD and in

normal aging, there are concerns specifi c to the use of GH

in non-GHD elders. In treatment of GHD, the target for

dosing is replacement to age-appropriate normal levels. In

anti-aging therapy, age-appropriate normal levels are the

starting point, not the target; rather, the target is the normal

range for young adults, and the balance of benefi cial effects

vs adverse effects and risks may thus be quite different in

these two contexts. The ongoing controversy over the pros

and cons of postmenopausal estrogen therapy, despite a large

literature, should raise cautions that only studies conducted

with the specifi c dosing targets and in the specifi c population

for which the use is being proposed can adequately assess

those benefi ts and risks.

Growth hormone-releasing hormone and growth hormone secretagoguesGrowth hormone secretagogues such as GHRH, ghrelin,

and their mimetics stimulate the secretion of GH, if the

pituitary is intact and responsive. Since most AGHD is

due to hypopituitarism, and these patients – unlike normal

elders – are thus unresponsive to GHRH or GHS, there are

not many studies of GHS replacement effects, and the use of

GHS in normal aging has not been approved by regulatory

authorities in any jurisdiction (Merriam et al 2002, 2003).

In principle, treatment with GHS should offer a more physi-

ologic approach to GH replacement, leading to a pulsatile

rather than prolonged elevation in GH and preserving the

capability for negative feedback inhibition of GH by rising

levels of IGF-I (Merriam et al 2000; Merriam 2002). GHS

effects are infl uenced by the same factors which modulate

endogenous GHRH secretion, such as negative feedback

by somatostatin. This normal negative feedback regulation

offers some buffering against overdose (Merriam et al 2002).

The side effects of GHRH treatment are similar in character

to GH treatment, but are milder and less frequent. And,

since the GHS are smaller molecules than GH, they can be

administered orally, transdermally, or nasally (Merriam et al

2003; Merriam and Cummings 2003).

Once daily GHRH injections can stimulate increases

in GH and IGF-I at least to the lower part of the young

adult normal range (Merriam et al 2000). The University of

Washington study of 6 months treatment with daily bedtime

subcutaneous injections of GHRH(1–29)NH2, alone or in

combination with supervised exercise conditioning, was

begun in response to the NIH initiative (Merriam et al 2002,

2003). IGF-I levels rose approximately 35%. As with GH,

subjects showed an increase in lean body mass and decrease

in body fat (particularly abdominal visceral fat). However,

there was no improvement in strength or aerobic fi tness

associated with GHRH injections. Testing again confi rmed

the benefi ts of exercise but showed no effect upon IGF-I

levels; thus it appears that GH/GHRH and exercise work

through different mechanisms (Vitiello et al 1997). Subjects

receiving GHRH also showed no change in scores on an

integrated physical functional performance test mimicking

activities of daily living, but there was a signifi cant decline

in physical function in the placebo group (Merriam et al

1997, 2003; Cummings and Merriam 2003). This tantalizing

fi nding, suggesting that GHRH can stabilize if not improve

physical function, needs confi rmation. There is only one other

published study of chronic GHRH in normal aging, which

reported positive effects on exercise testing after 3 months

of treatment (Veldhuis et al 2005).

Sleep and cognition were also studied in the GHRH trial,

with surprising results. GHRH failed to improve and may

even have impaired deep sleep, despite the rise in IGF-I and

pulsatile GH. However, GHRH treatment was associated


Hersch and Merriam

with improved scores in several domains of fl uid (but not

crystallized) intelligence – those measures previously found

correlated with circulating IGF-I levels (Vitiello et al 2006).

This intriguing preliminary fi nding is now being studied

more systematically at the University of Washington in a

new NIH-funded study (the Somatotrophics, Memory, and

Aging Research Trial, or “SMART”).

Thus as with GH, there is a consensus on hormonal and

body composition effects but inconsistent functional effects

on function; and in addition there is a very encouraging but

still unconfi rmed positive effect on some domains of fl uid

intelligence.

Ghrelin, which is produced in the stomach and increases

during periods of fasting or under conditions associated with

negative energy balance (such as starvation or anorexia), acts

at both hypothalamic and pituitary levels via mechanisms

distinct from GHRH, and thus has different effects from

GHRH or GH; subjects often gain weight and do not lose,

or even gain body fat) (Merriam et al 2000, 2002; Liddle

2006). The effects of ghrelin on GH secretion depend in

part on the presence of GHRH; and thus if GHRH secretion

declines with aging, ghrelin’s effects may be blunted. While

the effects of these two GHS differ clinically, they have syn-

ergistic effects on GH release, and therefore supplementation

of both substances may be more effective than either alone

in aging (Merriam et al 2000, 2002). Additionally, there are

other substances which can enhance GH response to GHS by

suppressing somatostatin secretion, including arginine and

beta-adrenergic antagonists, which could potentially enhance

treatment effects (Merriam et al 1997).

Several studies have shown short-term effects of GHS on

GH secretion, but so far only three groups have conducted

studies of their chronic effects in normal aging. Bowers and

colleagues showed that chronic repeated injections or subcu-

taneous infusions of GH-releasing peptide-2 (GHRP-2) could

stimulate and maintain increases in episodic GH secretion

and IGF-I (Bowers et al 2004). Thorner and colleagues at the

University of Virginia have conducting a study of two years’

oral treatment with the non-peptidyl GHS MK-677. As with

previous studies, there was a sustained increase in IGF-I and

episodic GH secretion, and an increase in lean body mass

(Thorner et al 2006). Preliminary functional results over one

year of treatment, recently reported at an abstract presenta-

tion, however, did not show signifi cant improvements.

In cooperation with investigators at Duke University

and several other sites, we conducted a trial of the Pfi zer

investigational oral GH capromorelin in pre-frail older men

and women (Merriam et al 2006). This protocol recruited

over 300 subjects and was initially planned as a two-year

intervention. The study was unfortunately stopped, how-

ever, after all subjects had been treated for 6 and many for

12 months, due to failure to see an increase in per cent lean

body mass, which was a pre-set non-effi cacy termination

criterion. Absolute lean body mass did increase signifi cantly,

but due to the appetite-stimulating effect of this ghrelin

mimetic – unforeseen in early 1999 when the study was

designed and ghrelin was still unknown – subjects also gained

weight (about 1.5 Kg) and this washed out the effect on per

cent lean body mass. However, even this truncated study is

currently the largest clinical trial of chronic GHS treatment

in aging. It showed the expected increases in IGF-I levels

and (as noted) total lean body mass. There were also encour-

aging effects on physical functional performance. Of seven

functional tests, one improved signifi cantly after 6 months

of treatment, and another after 12 months. Two other mea-

sures showed non-signifi cant trends toward improvement,

and the three remaining measures showed no effect. Effects

on clinical endpoints such as falls could not be assessed

with this relatively brief duration of treatment. Side effects

were generally mild, including increases in fasting blood

sugar within the normal range. Interestingly, there was a

self-reported deterioration of sleep quality, though formal

sleep testing was not performed. Cognition was not studied

in this trial.

Thus as with GH and GHRH, reports of the hormonal

and body composition effects of ghrelin mimetic GHS in

normal aging are relatively consistent, but there is no con-

sensus on functional effects among these very few studies,

and of course none could assess clinical fi nal outcomes or

long-term risks.

ConclusionSarcopenia and subsequent frailty lead to loss of indepen-

dence. While aging is not a disease, it results in signifi cant

body composition and functional changes which affect the

individual and the community at large. Aging represents a

milder form of adult GHD, and GH replacement in GHD

has met with success. Since the aging pituitary remains

responsive to GH and GHS, it is reasonable to suggest that

GH replacement or stimulation might be indicated in aging.

However, elders are more sensitive to GH, and thus more

susceptible to the side effects of replacement. Stimulating

GH with GHS instead of GH replacement has the advantage

of a more physiological approach to increase endogenous GH

pulsatility with theoretically decreased risk for side effects

(Arvat et al 2000).



Reports of stabilization or even improvement of physical

function with GHRH or with oral GHS are extremely tan-

talizing, but they are hardly proof that Ponce de Leon’s

“Fountain of Youth” has been found. The failure to replicate

these fi ndings consistently across studies reminds us of the

origins of the word “tantalizing.” In mythology, Tantalus,

chained to a rock, bent down to drink from the pool of water

around him – and the water receded just out of reach. So far,

defi nitive conclusions regarding functional effects of GHRH

and GHS in normal aging have also been out of our reach;

and until we know whether the age-related decline in GH

secretion is pathological or adaptive, and until more studies

are undertaken to study this and the long term effects of GH

and GHS supplementation, conclusive statements about the

benefi ts of treatment cannot be made and we can only rec-

ommend their use in well-controlled clinical studies. Long

term studies on hard clinical endpoints, such as decreased

fractures and falls, increased function and quality of life, and

decreased morbidity and mortality from vascular disease need

to be performed in order to establish the role, if any, for GH

and GHS treatment in normal aging.

NotePresented in part at the Second Annual Meeting of the Society

for Applied Research in Aging (SARA), 11 November 2006.

AcknowledgmentsWe thank our colleagues at the National Institutes of Health,

the University of Virginia, the University of Washington,

and Duke University, especially Drs Marc Blackman, Cyril

Bowers, David Buchner, David Cummings, Marie Gelato,

S Mitchell Harman, Ken Ho, the late Lawrence Larsen, Saul

Malozowski, Karen Moe, the late Eugenia Pavlov, Robert

Schwartz, Michael Thorner, Mary Lee Vance, Johannes

Veldhuis, Michael Vitiello, and Heidi White, for their

inspiration and collaboration; and Pamela Asberry, Suzanne

Barsness, Colleen Carney, and Monica Kletke for expert

professional assistance; and Dr Sharon Falzgraf for support

and critical review of the manuscript.

ReferencesAleman A, Verhaar HJ, DeHaan EH, et al. 1999. Insulin-like growth factor-I

and cognitive function in healthy older men. J Clin Endocrinol Metab, 84:471–5.

Anawalt BD, Merriam GR. 2001, Neuroendocrine aging in men: andropause and somatopause. Endocrinology and Metabolism Clinics of North America, 30:647–69.

Arvat E, Giordano R, Broglio F, et al. 2000. GH secretagogues in aging. J Anti-Aging Medicine, 3:149–58.

Blackman MR, Sorkin JD, Munzer T, et al. 2002. Growth hormone and sex steroid administration in healthy aged women and men: a randomized controlled trial. JAMA, 288:2282–92.

Bowers CY, Granda R, Mohan S, et al. 2004. Sustained elevation of pulsatile growth hormone (GH) secretion and insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and IGFBP-5 concentrations during 30-day continuous subcutaneous infusion of GH-releasing peptide-2 in older men and women. J Clin Endocrinol Metab, 89:2290–300.

Cummings DE, Merriam GR. 2003. Growth hormone therapy in adults. Annu Rev Med, 54:513.

Ho KY, Evans WS, Blizzard RM, et al. 1987. Effects of sex and age on the 24 hour profi le of growth hormone secretion in man: Importance of endogenous estradiol concentrations. J Clin Endocrinol Metab, 64:51–8.

Liddle, RA. Up to Date. 2006. Ghrelin [online]. Accessed 8 Sep 2006. URL: http://www.uptodate.com.

Melmed S. Up to Date. 2006. Physiology of growth hormone [online]. Accessed 8 Sep 2006. URL: http://www.uptodate.com

Merriam GR, Blackman M, Hoffman A, et al. 2006. Effects of chronic treatment with an oral growth hormone (GH) secretagogue on nocturnal GH and insulin-like growth factor-I (IGF-I) in older men and women. Frontiers in Neuroendocrinology, 27:36 (published abstract).

Merriam GR, Schwarz RS, Vitiello MV. 2003. Growth hormone-releasing hormone and growth hormone secretagogues in normal aging. Endocrine, 22:1–7.

Merriam GR, Barsness S, Buchner D, et al. 2002. Growth hormone-releasing hormone treatment in normal aging. J Anti Aging Med, 4:1–13.

Merriam GR, Cummings DE. 2003. Growth hormone and growth hormone secretagogues in adults. In Meikle W (ed). Endocrine replacement therapy in clinical practice. Totowa, NJ: Humana Press. p 63–94.

Merriam GR. 2002. Growth hormone as anti-aging therapy, and other emerg-ing (and submerging) indications. Clinical Endocrinology Update, The Endocrine Society, Chevy Chase, MD.

Merriam GR, Kletke M, Barsness S, et al. 2000. Growth hormone-releasing hormone in normal aging: An Update. Today’s Therapeutic Trends, 18:335–54.

Merriam GR, Buchner DM, Prinz PN, et al. 1997. Potential applications of GH secretagogs in the evaluation and treatment of the age-related decline in growth hormone secretion. Endocrine, 7:1–3.

Merriam GR, Wyatt FG. 2006. Diagnosis and treatment of growth hormone defi ciency in adults: current perspectives. Current Opinion in Endocri-nology and Diabetes, 13:362–8.

Molitch M, Clemmons D, Malozowski S, et al. 2006. Evaluation and treatment of adult growth hormone defi ciency: An Endocrine Society Clinical Practice Guideline. J. Clin. Endocrinol. Metab, 91:1621–34.

Papadakis MA, Grady D, Black D, et al. 1996. Growth hormone replacement in healthy older men improves body composition but not functional ability. Ann Intern Med, 124:708–16.

Pavlov EP, Harman SM, Merriam GR, et al. 1986. Responses of growth hormone and somatomedin-C to growth hormone-releasing hormone in healthy aging men. J Clin Endocrinol Metab, 62:595–600.

Rudman D, Feller AG, Nagraj HS, et al. 1990. Effects of human growth hormone in men over 60 years old. NEJM, 323:1–6.

Thorner MO, Nass R, Pezzoli SS, et al. 2006. Orally active ghrelin mimetic (MK-677) prevents and partially reverses sarcopenia in healthy older men and women: a double-blind, placebo controlled, crossover study. Endocrine Society Annual Meeting, Boston, 24 June 2006, abstract OR5–5.

Veldhuis JD, Patri JM, Frick K, et al. 2005. Administration of recombinant human GHRH-1,44-amide for 3 months reduces abdominal visceral fat mass and increases physical performance measures in postmenopausal women. Eur J Endocrinol, 153:669–77.

Vitiello MV, Moe KE, Merriam GR, et al. 2006. Chronic growth hormone releasing hormone treatment improves cognition of healthy older adults. Neurobiology of Aging, 27:318–23.

Vitiello MV, Schwartz RS, Buchner KE, et al. 2001. Treating age-related changes in somatotrophic hormones, sleep, and cognition. Dialogs in Clinical Neuroscience, 3:229–36.

Vitiello MV, Wilkinson CW, Merriam GR, et al. 1997. Successful six-month endurance training does not alter insulin-like growth factor-I in healthy older men and women. J Gerontol Med Sci, 52A:149–54.

Clinical Interventions in Aging 2006:1(4) 307–308© 2006 Dove Medical Press Limited. All rights reserved

307

EDITORIAL FOREWORDVolume 1 • Number 4 • 2006

Richard F Walker

International Society for Applied Research in Aging (SARA)

Sermorelin: A better approach to management of adult-onset growth hormone insuffi ciency?

Growth hormone replacement therapy (GHRT) using recombinant human growth

hormone (rhGH) has been embraced by many age management practitioners as one

of the most effective methods for opposing somatic senescence currently available.

However, its routine use has been controversial because few clinical studies have been

performed to determine the potential risks of long-term therapy. Also, certain medical

and legal issues have not been resolved causing some practitioners to restrict their use

of the product. Some of these issues include the fact that:

• Improper dosing can lead to side effects that may be serious in some patients,

• Injection of hGH creates unnatural conditions of exposure to the hormone that

may erode normal physiology,

• The Code of Federal Regulations specifi cally forbids the use of rhGH in adults

except for treatment of AIDS or human growth hormone defi ciency (GHD)

diagnosed pursuant to regularly accepted guidelines.

While there is a wealth of information showing that long-term administration

of rhGH reduces intrinsic disease and extends life in adults suffering pathogenic

GHD, consensus on whether extrapolation of those data to the aging condition

is justifi ed has not been reached (Perls et al 2005). Most of the major concerns

derive from the fact that rhGH is mitogenic and may awaken latent cancers, that

improper dose selection may promote metabolic disorders such as diabetes, and

perhaps that pharmacological presentation may exacerbate decline of endocrine

function by distorting essential hormonal interactions. Of course, all these concerns

are speculative and will not be resolved until suffi cient scientifi c evidence for or

against GHRT eventually accumulate. In the interim, the value of rhGH in GHRT

will continue to be debated; unfortunately based more upon personal prejudice than

objective information.

Despite the eventual outcome to the “Great Hormone Debate” as it has been

titled in media articles (Landsmann 2006), certain negative aspects of GHRT using

rhGH cannot be disputed and justify searching for a better alternative. For example,

“square wave” or pharmacological presentation of the exogenous hormone cannot

be avoided since it is administered as a bolus, subcutaneous injection. Since the

amount of rhGH entering the general circulation is not controlled by normal feedback

mechanisms, tissue exposure to elevated concentrations is persistent and eventually

may lead to tachyphylaxis and reduced effi cacy. Also, because the body cannot

modulate tissue exposure to rhGH, the practitioner is required to “best guess” the

appropriate dosage based upon little other than serum measurements of insulin-like

growth factor-1 (IGF-1) and subjective comments from the patient about perceived

responses to the hormone. Thus, it would seem that an alternative method(s) of GHRT

that circumvented these problems would be of great value so long as it retained the

positive attributes of rhGH.

One possibility that is receiving growing attention is the use of GH secretagogues

to promote pituitary health and function during aging. An example of such molecules

is growth hormone releasing factor 1-29 NH2-acetate, or sermorelin, that recently

became available to practitioners for use in longevity medicine (Merriam et al

2001). Other alternatives include orally active growth hormone-releasing peptides


Walker

that are currently being developed by pharmaceutical

companies. Some of these have been reported to be effective

at improving physical performance in the elderly (Fahy

2006). However, it is unlikely that they will be marketed

for several years. On the other hand, sermorelin, an analog

of naturally occurring growth hormone-releasing hormone

(GHRH) whose activity declines during aging, may

presently offer a more immediate and better alternative to

rhGH for GHRT in aging (Russell-Aulet et al 2001). The

molecule was commercially produced and marketed for

many years as an alternative to rhGH for use in children

with growth retardation, but it could not compete with

rhGH and was withdrawn as a therapeutic entity by the

manufacturer. Paradoxically sermorelin failed as a growth-

promoting agent in children for the very reason that it is

a better alternative for GHRT in aging adults. Growth-

defi cient children need higher doses of growth hormone

than can be achieved by stimulating production of their

own hormone, whereas the benefi cial effects of sermorelin

on pituitary function and simulation of youthful growth

hormone secretory dynamics in aging adults have little

effect on growth rate in children. Unlike exogenous rhGH

that causes production of the bioactive hormone IGF-1 from

the liver, sermorelin simulates the patients own pituitary

gland by binding to specifi c receptors to increase production

and secretion of endogenous hGH. Because sermorelin

increases endogenous hGH by stimulating the pituitary

gland, it has certain physiological and clinical advantages

over hGH that include:

• Effects are regulated by negative feedback involving

the inhibitory neurohormone, somatostatin, so that

unlike administration of exogenous rhGH, overdoses

of endogenous hGH are diffi cult if not impossible to

achieve,

• Because of the interactive effects of sermorelin and

somatostain, release of hGH by the pituitary is episodic or

intermittent rather than constant as with injected rhGH.

• Tachphylaxis is avoided because sermorelin-induced

release of pituitary hGH is not “square wave”, but instead

simulates more normal physiology,

• Sermorelin stimulates pituitary gene transcription of

hGH messenger RNA, increasing pituitary reserve

and thereby preserving more of the growth hormone

neuroendocrine axis, which is the fi rst to fail during

aging (Walker et al 1994).

• Pituitary recrudescence resulting from sermorelin helps

slow the cascade of hypophyseal hormone failure that

occurs during aging thereby preserving not only youthful

anatomy but also youthful physiology (Villalobos et al

1997).

Finally, there is the question of lawful practice. Unlike

rhGH which has legal restrictions on its clinical use, the

off-label prescribing of sermorelin is not prohibited by

federal law. Thus, it can be carefully employed and evaluated

by the practitioner to objectively determine whether it

provides greater benefi ts with less risk to his/her patients.

In support of this effort, the Society for Applied Research

in Aging will be providing sermorelin free of cost on a

competitive basis to practitioners willing to study its effects

under protocol conditions and to report the outcomes in

a peer-reviewed journal such as Clinical Interventions in

Aging. Hopefully, through such efforts we can contribute

to development of a paradigm for evidence-based GHRT in

clinical age management.

For more information on this effort and to participate in

the protocol, please contact [email protected].

ReferencesFahy J. 2006. Drug could fi ght effects of aging [online]. Accessed on 22

June 2006. Pittsburgh Post-Gazette. URL: http://www.post-gazette.com/pg/06173/700274-114.stm.

Landsmann MA. 2006. Forever young? What role does human growth hormone play in the aging process? The question is rife with controversy. ADVANCE for Healthy Aging, 2:54-61.

Merriam GR, Barness S, Buchner D, et al. 2001. Growth hormone releasing hormone treatment in normal aging. J Anti-Aging Med, 4:331-43.

Perls TT, Reisman NR, Olshansky SJ. 2005. Provision and distribution of growth hormone for “antiaging”: clinical and legal issues. JAMA, 294:2086-90.

Russell-Aulet M, Dimaraki EV, Jaffe CA, et al. 2001. Aging-related growth hormone (GH) decrease is a selective hypothalamic GH-releasing hormone pulse amplitude mediated phenomenon. J Gerontol A Biol Sci Med Sci, 56:M124-9.

Villalobos C, Núñez L, Frawley LS, et al. 1997. Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion. Proc Natl Acad Sci U S A, 94:14132-7.

Walker RF, Eichler DC, Bercu BB. 1994. Inadequate pituitary stimulation: a possible cause of growth hormone insuffi ciency and hyperprolactinemia in aged rat. Endocrine, 2:633-8.

Clinical Science (2006) 110, 563–573 (Printed in Great Britain) doi:10.1042/CS20050374 563

Use of growth-hormone-releasing peptide-6(GHRP-6) for the prevention of multiple

organ failure

Danay CIBRIAN∗, Hussam AJAMIEH†, Jorge BERLANGA∗, Olga S. LEON∗,Jose S. ALBA∗, Micheal J.-T. KIM‡, Tania MARCHBANK§, Joseph J. BOYLE‡,Freya FREYRE∗, Diana GARCIA DEL BARCO∗, Pedro LOPEZ-SAURA∗,Gerardo GUILLEN∗, Subrata GHOSH‡, Robert A. GOODLAD‖and Raymond J. PLAYFORD§∗Center for Genetic Engineering and Biotechnology, Ave 31 e/158 & 190 Playa 10600, Havana, Cuba, †Center for BiologicalStudies, Food and Drug Institute, University of Havana, Ave 23 e/44 & 222 La Coronela, La Lisa, Havana, Cuba,‡Department of Gastroenterology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road,London W12 0NN, U.K., §Imperial College School of Medicine, Barts & The London, Queen Mary’s School of Medicine andDentistry, Turner Street, London E1 2AD, U.K., and ‖Cancer Research UK, Lincoln’s Inn Fields, London WC2A 3PX, U.K.

A B S T R A C T

Novel therapies for the treatment of MOF (multiple organ failure) are required. In the presentstudy, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) oncell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer(HT29) cells as in vitro models of injury. In addition, we examined its efficacy when givenalone and in combination with the potent protective factor EGF (epidermal growth factor)in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 minof ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion).In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its additioncaused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P < 0.01),but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that,compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage(histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipidperoxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 µg/kgof body weight, intraperitoneally) alone truncated these effects by 50–85% (all P < 0.05) and anadditional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of bodyweight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments.In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhanceits effects, may provide a novel simple approach for the prevention and treatment of MOFand other injuries of the gastrointestinal tract. In view of these findings, further studies appearjustified.

Key words: epidermal growth factor (EGF), growth-hormone-releasing peptide (GHRP), gut injury, ischaemia/reperfusion, multipleorgan failure, repair, recombinant peptide.Abbreviations: ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; DMEM, Dulbecco’s modified Eagle’s medium;EGF, epidermal growth factor; FCS, foetal calf serum; GH, growth hormone; GHRP, GH-releasing peptide; i.p., intraperitoneally;I/R, ischaemia/reperfusion; 45 min/45 min I/R, 45 min of ischaemia, followed by 45 min of reperfusion; 90 min/120 min I/R, 90 minof ischaemia, followed by 120 min of reperfusion; MDA, malondialdehyde; MOF, multiple organ failure; MPO, myeloperoxidase;rhEGF, recombinant human EGF; SOD, superoxide dismutase; TGF, transforming growth factor; THP, total hydroperoxides.Correspondence: Professor Raymond J. Playford (email [email protected]).

C© 2006 The Biochemical Society

564 D. Cibrian and others

INTRODUCTIONMOF (multiple organ failure) is a severe life-threateningcondition that usually occurs as a result of major trauma,burns or fulminant infections. Whatever the initiatingevent, once established, MOF has a high mortality (up to80 %) [1]. The pathophysiological mechanisms underly-ing this condition are unclear, although important contri-butory factors probably include hypoxia, increased intes-tinal permeability, bacterial translocation, endotoxaemiaand uncontrolled systemic inflammatory responses [2].

Several studies suggest that the splanchnic circulationis particularly vulnerable to hypoperfusion, as occursin low-flow states, such as haemorrhagic shock, andthat this hypoperfusion is out of proportion with theoverall reduction in cardiac output [3]. Although it isobvious that tissue ischaemia initiates a series of eventsthat can ultimately lead to cellular dysfunction andnecrosis, resumption of blood flow can paradoxicallycreate more tissue injury, possibly because of productionof oxygen-derived cytotoxic products [4]. The use ofI/R (ischaemia/reperfusion) models of injury, therefore,not only have relevance to acute vascular disruption(thrombosis and embolism) and major hepatic surgery,including transplantation, but also to the pathogenesis ofdevelopment of MOF.

Synthetic and recombinant peptides are being usedincreasingly for clinical purposes (e.g. human insulin fordiabetes and erythropoietin for anaemia of renal failure),but assessment of their value for the treatment of luminalgastroenterological problems is at a much earlier stage [5].

GH (growth hormone) secretagogues compose agroup of heterogeneous synthetic peptides and non-peptides that, as well as inducing pituitary GH secretion,also bind to GH secretagogue receptors on peripheraltissues, such as the myocardium, pancreas and bonemarrow [6,7]. The physiological role of these peripheralreceptors is, however, unclear and the potential valueof GHRP (GH-releasing peptide)-6 administration onhepatic and gastrointestinal mucosal integrity is untested.

In this series of studies, we therefore initially examinedwhether GHRP-6 had potentially useful ‘pro-healing’ ac-tivity using various in vitro models of gut injury. Havingfound positive results, we progressed to test the effectof systemic administration of GHRP-6 in a rat liver I/Rmodel of hepatic injury and MOF. In addition, as we havefound previously a beneficial effect of the potent growthfactor EGF (epidermal growth factor) when using a re-lated mesenteric I/R model [8], we also examined the res-ults of giving EGF alone and in combination with GHRP-6 (to begin to examine additive/synergistic effects).

MATERIALS AND METHODSSynthetic and recombinant peptidesGHRP-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2) waspurchased from BCN Peptides. The product in a lyo-

philized form, certified as pyrogen- and contaminant-free, was stored at −20 ◦C and diluted in sterile salinejust prior to its administration.

rhEGF1−52 (recombinant human EGF1−52), expressedin Saccharomyces cerevisiae, was obtained from Heber-Biotec in a lyophilized form. This product consists ofa 60:40 mixture of EGF1−52 and EGF1−51 and is as bio-logically active as the full length EGF1−53 form [9]. Priorto administration, EGF was diluted in 0.9 % saline understerile conditions.

EthicsExperiments were conducted according to current Localand National regulatory and ethical guidelines.

Study series 1: in vitro models

Effect of exogenous GHRP-6 on an in vitro cellmigration modelOne of the earliest biological repair responses followinginjury to tissue cells is the migration of surviving cellsover the denuded area caused by the injury to re-establishepithelial integrity. Since it is extremely difficult to studythis effect upon tissue inside a human or animal, cellculture models are commonly used as surrogate markersof this pro-migratory response. This method also allowsdirect actions of the test peptide on the cells to bedetermined.

Cell migration assays were performed using ourmethods published previously [10]. Briefly, human colo-nic carcinoma (HT29) cells or rat intestinal epithelial(IEC6) cells were grown to confluence in six-wellplates in DMEM (Dulbecco’s modified Eagle’s medium)containing 10 % (v/v) FCS (foetal calf serum) at 37 ◦Cin 5 % CO2. The monolayers were then wounded byscraping a disposable pipette tip across the dishes, washedwith fresh serum-free medium and cultured in serum-free medium in the presence of various test factors.The rate of movement of the anterior edges of thewounded monolayers was then determined by takingserial photomicrographs at various times after wounding[10]. Twenty measurements per field were performedby placing a transparent grid over the photograph andmeasuring the distance moved from the original woundline. All results are expressed as means +− S.E.M. of threeseparate experiments.

The various test factors used were GHRP-6 (10–400 µg/ml) and EGF (10 µg/ml; used as a positivecontrol). This dose of EGF was used as we have shownpreviously [10a] that this stimulates maximal restitutionresponses in this system. The importance of TGF (trans-forming growth factor) β in any response seen wasanalysed by using additional wells which containedGHRP-6 (40 µg/ml) and a TGFβ-neutralizing antibody(100 µg/ml; R&D Systems).


Use of GHRP-6 for multiple organ failure 565

Effect of exogenous GHRP-6 on an in vitro cellproliferation modelCell proliferation assays were performed using ourmethods published previously [10]. Briefly, HT29 andIEC6 cells were grown in DMEM containing 4 mmol/lglutamine, 10 % (v/v) FCS and various test factors. Effectsof addition of various doses of GHRP-6 and EGF(10 µg/ml; used as a positive control) were subsequentlytested under serum-starved conditions.

To assess the degree of proliferation, [3H]thymidine(2 µCi/well) was included 24 h after the addition of thetest factors, and cells were left for a further 24 h. Foreach condition, the stimulatory or inhibitory effect of thesolutions was measured in quadruplicate in six separatewells. Cell viability, determined by the ability to exclude0.2 % Trypan Blue, was always greater than 90 %.

Study series 2: in vivo model of I/R

Introduction to methodHaving shown that GHRP-6 possesses potentially usefulbiological activity in the in vitro systems, we proceededto examine its effects when used in an in vivo hepaticI/R model. Two different timed protocols were usedto examine if any effects seen were applicable to bothrelatively short and more prolonged periods of ischaemia.GHRP-6 was tested alone and also in combination withEGF, as we have shown previously a beneficial effectof EGF in a related mesenteric I/R model [8] and wewanted to determine if any additive/synergistic responseswere apparent. The dose of EGF used in the present study(1 mg/kg of body weight) was similar to that used in ourstudy reported previously [8].

AnimalsAdult male Wistar rats (200–250 g) were purchased fromthe National Center for Laboratory Animals and wereallowed access to food and water ad libitum.

Induction of I/R injuryAnimals were anaesthetized with urethane [10 mg/kg ofbody weight, i.p. (intraperitoneally)] and placed in asupine position on a heating pad in order to maintainbody temperature between 36 and 37 ◦C. To induce hep-atic ischaemia, a midline laparatomy was used and theblood supply of the right lobe of the liver was interruptedby placing a bulldog clamp (Fine Science Tools) at thelevel of the hepatic artery and the portal vein branches.Upon completion of the ischaemia time, reperfusion wasinitiated by removing the clamp. Reflow was confirmedby the macroscopic inspection of the target lobe. Noanimals were discarded due to non-reflow states. Animalsremained anaesthetized throughout the experiment.

Two different I/R time protocols were used: (i) 45 min/45 min I/R (45 min of ischaemia, followed by 45 min of

reperfusion; n = 6 per group), and (ii) 90 min/120 min I/R(90 min of ischaemia, followed by 120 min of reperfusion;n = 10–12 per group).

Experimental designFor both I/R protocols, rats were randomly assignedto five experimental groups as follows: group 1 (shamischaemia), animals received saline (placebo; 1 ml, i.p.)and 40 min later underwent all procedures, includinglaparotomy, liver exposure and manipulation, but thehepatic artery and the portal vein branches were notclamped; group 2 (I/R group), animals received saline(placebo, 1 ml, i.p.) and 40 min later underwent I/R;group 3 (I/R with GHRP-6), animals received GHRP-6 (120 µg/kg of body weight, i.p.) and 40 min laterunderwent I/R; group 4 (I/R with EGF), animals receivedrhEGF (1 mg/kg of body weight, i.p.) and 40 min laterunderwent I/R; group 5 (I/R with GHRP-6 + EGF),animals received GHRP-6 (120 µg/kg of body weight,i.p.) and rhEGF (1 mg/kg of body weight, i.p.) and 40 minlater underwent I/R.

Autopsy and sample processingAt the end of the study periods, blood samples wereobtained from the abdominal aorta for biochemical deter-minations. Serum was obtained, aliquoted and stored at−20 ◦C until processing. Rats were subjected to autopsy,and samples of different regions from the right ischaemiclobe were collected for subsequent histopathologicalexamination and tissue homogenization. In addition, re-presentative samples were collected from lungs, kidneys,jejunum and ileum. Samples to be processed for histo-logical study were immediately placed in 10 % bufferedformalin and subsequently paraffin-embedded andstained with haematoxylin/eosin.

Blood analysesSerum levels of ALAT (alanine aminotransferase) andASAT (aspartate aminotransferase), used as markers ofhepatocyte injury, were determined using a commercialkit according to the manufacturer’s instruction (Sigma).Serum creatinine levels, used as a marker of renal function,were determined using standard colorimetric methods.

Tissue biochemical analysesThe oxidative state of the liver was analysed by meas-urement of both enzyme activities [SOD (superoxidedismutase) and catalase] and chemical components [THP(total hydroperoxides) and MDA (malondialdehyde)levels]. MDA levels are a commonly used marker oflipid peroxidation. In addition, liver and intestinal MPO(myeloperoxidase) activities were measured as a markerof neutrophilic infiltration.

For liver tissue biochemical studies of MDA, THP andSOD, tissue was homogenized [1:10 (w/v)] in 50 mmol/l



KCl/5 mmol/l histidine buffer (pH 7.4), followed bycentrifugation at 5000 g for 20 min at 4 ◦C. The super-natants were collected, aliquoted and stored at −20 ◦Cuntil assay. All the biochemical parameters were deter-mined by spectrophotometric methods. MDA contentwas assessed using the Bioxytech LPO-586 kit (Bio-Rad Laboratories), and THP were determined usingthe Bioxytech H2O2-560 kit (Bio-Rad Laboratories).SOD activity was determined by following changes inautoxidation of pyrogallol in response to adding thehomogenate [11]. MPO activity was determined usinga modification of the method described by Krawiszet al. [12], and 1 unit of MPO activity was defined as thequantity of enzyme that degrades 1 µmol of H2O2/minat 25 ◦C. Biochemical data were adjusted to reflect totalprotein concentration using a commercial spectrophoto-metric protein dye kit (Bio-Rad Laboratories).

Histological assessmentAll tissues were assessed in a blinded manner.

Small intestine The total lengths of the small intestinewere measured and then split longitudinally to allow amacroscopic assessment of the percentage injured area.The percentage of damage was calculated by measuring(cm) all the regions showing gross macroscopic changes,such as petechiae and haemorrhagic areas, and con-sidering the whole length of the small intestine (in cm) as100 %. Eight equal-spaced 2 cm segments from the lengthof the small bowel were then collected for histologicalassessment.

For the microscopic assessment, mucosal damage ofthe small intestine was quantitatively assessed accordingto the grading system of Chiu et al. [13]. This system usesa scale of 0–5, where 0 is normal mucosa; 1 is developmentof subepithelial (Gruenhagen’s) spaces; 2 is extension ofthe subepithelial space with moderate epithelial liftingfrom the lamina propria; 3 is extensive epithelial lift-ing with occasional denuded villi tips; 4 is denuded villiwith exposed lamina propria and dilated capillaries, and 5is disintegration of the lamina propria, haemorrhage andulceration. The mean scores of 30–40 villi from each of theeight segments for each animal were pooled to provide anaverage score for the intestine of that animal.

Liver For each animal, the degree of liver damage wasdetermined in at least five different lobar regions andgraded using the modified Suzuki scoring system [14].Briefly, the various changes noted are sinusoidal conges-tion, hepatocyte necrosis and ballooning degeneration.The specimen was then graded from 0–4, where no nec-rosis or congestion/centrilobular ballooning was given ascore of 0, and severe congestion/ballooning degenerationas well as >60 % lobular necrosis was given a value of 4.

Kidney Each sample was classified in a blinded fashioninto one of three groups: 0, essentially normal histology;

1, moderate, probably reversible, changes (hydropic cyto-plasmic changes); and 2, severe changes (nuclear break-down or cellular detachment from the tubule basementmembrane).

Lungs Lung interstitial damage ranged from normalto showing varying degrees of septal thickening, hyper-cellularity, neutrophilic recruitment, interstitial adhesionand alveolar luminal reduction. Each sample was classi-fied in a blinded fashion into one of three groups: 0, essen-tially normal histology; 1, abnormal showing some ofthe changes described above, and 2, grossly abnormalshowing all of the changes described above.

Data analysisData were analysed using ANOVA with treatmentas factor. Where significant effects were seen on theANOVA (P < 0.05), individual comparisons based onthe group mean square error and residual were per-formed, a method equivalent to multiple comparisonsanalyses.

RESULTS

Study series 1: in vitro studies

Restitution assaysGHRP-6 caused pro-migratory activity of woundedmonolayers in both HT29 and IEC6 cells in a dose-dependent manner. Maximal effects were observed at40 µg/ml for HT29 cells (Figure 1A) and 160 µg/ml forIEC6 cells (Figure 1B).

The addition of a neutralizing anti-TGFβ antibody didnot affect the cell migration response caused by GHRP-6(Figure 1C), suggesting that cell migration in response toGHRP-6 is independent of TGF-β production.

Proliferation assayGHRP-6 did not induce increased thymidine uptake inHT29 or IEC6 cells at any of the doses tested (Figure 1D).

Study series 2: in vivo model of I/RFor both of the short (45 min/45 min I/R)- and longer(90 min/120 min I/R)-timed protocols, the results wereessentially the same. The results from the 90 min/120 minI/R protocol are therefore discussed in detail and shownin the Figures and Table 1. The main results from the45 min/45 min I/R protocol are shown in Table 2 and,in the few instances where the results differ from the90 min/120 min protocol, these are mentioned in the text.

LiverBiochemical analyses I/R caused an approx. 10-foldincrease in serum ASAT and ALAT. Pre-administrationof either GHRP-6 or EGF alone reduced this rise by



Figure 1 Effect of GHRP-6 on the rate of migration (restitution) or proliferation of various gastrointestinal cell linesThe addition of GHRP-6 to wounded monolayers of (A) HT29 cells or (B) IEC6 cells caused a dose-dependent increase in the rate of migration compared with thenegative control (�, no GHRP-6 added). The various doses tested were 1 µg/ml (+), 20 µg/ml (�), 40 µg/ml (∗) and 160 µg/ml (�). Maximum effects wereseen at 40 µg/ml in HT29 cells and 160 µg/ml in IEC6 cells. Cells were treated with 10 % (v/v) FCS as positive control (�). P < 0.01 compared with thenegative control at all doses above 1 µg/ml at each time point after 4 h. (C) The pro-migratory effect of GHRP-6 on HT29 cells was not affected by co-incubatingwith a neutralizing anti-TGFβ antibody. �, Negative control (no GHRP-6); ∗ , cells incubated with 40 µg/ml GHRP-6; and X, cells incubated with 40 µg/mlGHRP-6 and a neutralizing anti-TGFβ antibody. Similar results were seen using IEC6 cells (results not shown). (D) HT29 cells incubated in DMEM alone (negativecontrol; − ve) had a [3H]thymidine uptake of approx. 400 000 c.p.m. Addition of EGF (10 µg/ml, positive control; + ve) caused an approximate doubling of[3H]thymidine uptake, whereas GHRP-6 (50–400 µg/ml) did not increase [3H]thymidine uptake above baseline. Similar results were seen with IEC6 cells (resultsnot shown).

approx. 50 % and combination treatment resulted in afurther reduction in enzyme levels (Figure 2 and Table 1).

I/R caused the MDA levels (marker of lipid per-oxidation) to increase by approx. 4-5 fold (Figure 2).In the 90 min/120 min protocol, this rise was truncatedby approx. 50 % in animals that had received GHRP-6 or EGF alone and virtually completely prevented bypre-treatment with GHRP-6 + EGF together (Figure 2).Similar results were seen in animals undergoing the45 min/45 min I/R protocol, although the rise in MDAwas slightly less marked and either peptide givenalone was sufficient to prevent an increase in MDAlevels (Table 2). Similarly, animals that received placeboand underwent the 90 min/120 min I/R protocol had a3–4-fold increase in THP (Table 1). GHRP-6 or EGF

alone truncated this response by approx. 75 % withcombination treatment preventing the rise completely(Table 1). Similar results were seen in animals that under-went the 45 min/45 min I/R protocol, although theamount of THP produced was less (I/R + saline-treatedanimals having an approx. 2-fold increase above sham-operated animals; Table 2).

I/R caused an approx. 60 % fall in hepatic SOD levels,and this change was partially reversed by pre-treatmentwith either GHRP-6 or EGF alone. A further improve-ment was seen in animals that had received the com-bination treatment (Figure 2 and Table 2).

Catalase activity was increased by approx. 30-fold inresponse to I/R. This increase was markedly truncatedin animals that had received either GHRP-6 or EGF



Figure 2 Influence of pre-administration of GHRP-6 and EGF alone or in combination on injury sustained in various organsRats (10–12 per group) were pre-treated with GHRP-6 (120 µg/kg of body weight, i.p.) and EGF (1 mg/ml, i.p.) and then underwent organ injury induced by 90 minof hepatic vessel clamping, followed by 120 min of reperfusion. Animals were then killed and blood and tissue collected for various assays of tissue injury. ALAT is amarker of hepatic injury, MDA is a marker of lipid peroxidation and, along with SOD, allows assessment of the oxidative state of the liver. Liver and intestinal MPOactivity was measured as a marker of neutrophilic infiltration. Serum creatinine was used as a marker of renal function. Values are means+− S.E.M. ∗P < 0.05 and∗∗P < 0.01 compared with the equivalent value in sham-operated animals. +P < 0.05 and ++P < 0.01 compared with the equivalent value in animals treatedwith I/R + saline. $P < 0.05 and $$P < 0.01 when the values in animals given combination therapy (GHRP-6+ EGF) are compared with the values in animalsgiven the same dose of either GHRP-6 or EGF alone.

Table 1 Effect of GHRP-6 and EGF on injury induced by 90 min/120 min of hepatic I/RValues are means+− S.E.M., n = 10–12 per group. Also see Figure 1. ∗P < 0.05 and ∗∗P < 0.01 compared with the equivalent value in sham-operated animals.++P < 0.01 compared with the equivalent value in I/R animals. $P < 0.05 and $$P < 0.01 when the values in animals given combination therapy (GHRP-6+ EGF)are compared with those in animals given the same dose of either GHRP-6 or EGF alone. IU, international units.

Sham operation (laparotomy) I/R I/R + GHRP-6 I/R + EGF I/R + GHRP-6 + EGF

ASAT (IU/l) 34+− 4 1452+− 308∗∗ 543+− 123∗++ 404+− 82++ 115+− 33++

Catalase (units · min−1 · mg−1 of protein) 16+− 4 581+− 57∗∗ 31+− 4++ 58 +− 13++ 20 +− 4++

THP (µmol/mg of protein) 27+− 3 109+− 16∗∗ 51+− 2∗++$$ 43 +− 2++$ 21 +− 2++



Table 2 Effect of GHRP-6 and EGF on injury induced by 45 min/45 min of hepatic I/RValues are means+− S.E.M., n = 6 for each group. ∗P < 0.05 and ∗∗P < 0.01 compared with the equivalent value in sham-operated animals. ++P < 0.01compared with the equivalent value in I/R animals. $P < 0.05 and $$P < 0.01 when the values seen in animals given combination therapy (GHRP-6+ EGF)are compared with those in animals given the same dose of either GHRP-6 or EGF alone. IU, international units.

Sham operation(laparotomy) I/R I/R + GHRP-6 I/R + EGF I/R + GHRP-6 + EGF

MPO intestine (units · min−1 · mg−1 of protein) 26+− 3 148+− 13∗∗ 60+− 8∗∗++$$ 40 +− 5++$ 16 +− 2++

MDA liver (nmol/mg of protein) 0.31+− 0.01 1.17+− 0.11∗∗ 0.37+− 0.01++$ 0.29 +− 0.01++ 0.19 +− 0.01++

MPO liver (units · min−1 · mg−1 of protein) 19+− 3 105+− 9∗∗ 38+− 6++$ 34 +− 10++$ 8 +− 1++

ASAT (IU/l) 13+− 2 116+− 11∗∗ 62+− 7∗∗++$ 57 +− 2∗∗++$ 33 +− 4∗++

ALAT (IU/l) 21+− 3 157+− 19∗∗ 69+− 16∗∗++ 82 +− 13∗∗++ 61 +− 8∗++

Catalase (units · min−1 · mg−1 of protein) 9+− 2 288+− 28∗∗ 106+− 7∗∗++$$ 63 +− 2∗∗++ 47 +− 5∗++

THP (µmol/mg of protein) 182+− 16 295+− 13∗∗ 168+− 5++$ 123+− 20∗++ 108+− 8∗∗++

10−3 × SOD (units · min−1 · mg−1 of protein) 32.5+− 1.3 14.2+− 1.8∗∗ 22.6+− 1.0∗∗++$ 20.2 +− 0.5∗∗++$$ 26.5 +− 0.9∗∗++

Creatinine (µmol/l) 43+− 5 84+− 8∗∗ 67+− 16 70 +− 5* 79 +− 11∗∗

alone (causing a 60–70 % reduction), with GHRP-6 + EGF combination treatment truncating this responseby approx. 90 % (Tables 1 and 2).

Histology Sham-operated animals had an essentiallynormal liver histology (Figure 3). Animals that hadundergone I/R with placebo (saline) injection had severechanges, consisting of areas of necrosis, haemorrhage,cytoplasmic ballooning and sinusoidal distension. Ani-mals that had been pre-treated with GHRP-6 alone, EGFalone or the GHRP-6 + EGF combination therapy allshowed improvements compared with the I/R group,with the combination therapy appearing to have the mostprotective effect (Figure 3). Assessment using the micro-scopic scoring system confirmed these results; all animalsthat underwent I/R and received placebo had scores of3 or 4, whereas six out of ten animals that had receivedcombination therapy had scores of 0 (Figure 4).

IntestineI/R alone resulted in macroscopically obvious injuryaffecting 73 +− 4 % of the intestinal length. Pre-treatmentwith either peptide alone significantly decreased (P <

0.01) the degree of macroscopic injury (27 +− 3 and30 +− 2 % for GHRP-6- and EGF-treated animals res-pectively), with the most beneficial effect being seenin animals that had received both GHRP-6 and EGF(19 +− 2 %; P < 0.01 compared with I/R alone or I/R pluseither peptide given alone).

Histological assessment showed I/R caused severemucosal damage, with most animals showing completeloss of villous architecture and extensive areas ofmucosal infarction (Figure 3). These changes were muchless prominent in animals that had received GHRP-6alone, EGF alone or the GHRP-6 + EGF combinationtreatment (Figure 3). Quantitative assessment showedsimilar effects; all animals that underwent I/R andreceived placebo had scores of 4 or 5, whereas six out

of ten animals that had received combination therapy hadscores of 0 (Figure 4).

KidneyBiochemical analysis In the animals undergoing the90 min/120 min I/R protocol, serum creatinine levels rosefrom 45 to 70 µmol/l in response to I/R. Pre-treatmentwith GHRP-6 was associated with a 30 % (non-sig-nificant) fall in creatinine levels, whereas pre-treatmentwith EGF either alone or in combination with GHRP-6resulted in the creatinine levels remaining in the normal(sham-operated) range (Figure 2). A similar trend wasseen in animals that underwent the 45 min/45 min I/Rprotocol, although the beneficial effects were less markedand non-significant (Table 2).

Histology Animals that had undergone I/R but not re-ceived GHRP-6 or EGF all showed moderate or severerenal injury comprising nuclear breakdown or cellulardetachment from the tubule basement membrane. Ad-ministration of GHRP-6 or EGF given alone, or incombination, tended to reduce the degree of injury;GHRP-6 + EGF combination treatment had the mostbeneficial effect with nine out of ten animals havingessentially normal renal histology by semi-quantitativescoring (Figure 4).

LungHistology Animals that had received I/R withoutGHRP-6 or EGF had severe changes comprising septalthickening, hypercellularity, neutrophilic recruitment,interstitial adhesion and alveolar luminal reduction (Fig-ure 3). None of the animals that had received I/R withoutGHRP-6 or EGF had normal lung histology, whereasnine out of ten animals that had received both peptideshad normal histology. Animals that had received eitherpeptide alone occupied intermediate positions (Figures 3and 4).



Figure 3 Histopathology of rats given placebo (saline), or GHRP-6 and EGF alone or in combination prior to 90 min hepaticvessel clamping followed by 120 min reperfusionCompared with sham-operated animals, rats that underwent I/R, but did not receive GHRP-6 or EGF, had severe changes. Administration of either peptide aloneimproved histological appearances with the most improvement being seen in animals that received the combination of GHRP-6 and EGF. Original magnification ofintestine, lungs and liver was ×10, ×10 and ×40 respectively.

DISCUSSION

Using in vitro models of injury and repair, we haveshown that GHRP-6 stimulates gut epithelial restitution,but not proliferation. In vivo studies have shown thatpre-administration of GHRP-6 reduced the amount ofintestinal and extra-intestinal injury caused by hepaticvessel I/R and that added benefit was observed if EGFwas co-administered with GHRP-6.

The control of release of endogenous GH from thepituitary gland is thought to be partially mediated bythe presence of GHRP receptors acting via a specificG-protein-coupled receptor pathway, the natural ligandof which is probably the 28-amino-acid peptide ghrelin[15,16]. During the course of research into the control ofGH release, several peptides that induce GH secretionwere developed and one of the most potent was thehexapeptide GHRP-6 [15,16] used in the present study.Using a variety of GH secretagogue molecules, it isnow known that, in addition to being present withinthe pituitary gland, GHRP receptors are also present inseveral peripheral tissues, including bone marrow, spleen,pancreas, thyroid and myocardium [6,7], suggesting

additional roles for GHRP ligands that extend beyondGH release.

GHRP-6 stimulated cell migration of the humancolonic cell line HT29 and the rat intestinal cell line IEC6,showing that these effects were not species specific andthat GHRP-6 was able to influence gut epithelial functionby acting directly on the cells. The pro-migratory effectsof some of the well established pro-migratory ‘growthfactors’, such as IFNγ (interferon γ ), TGFα and EGF,are dependent upon their ability to induce TGFβ releaseinto the medium [17]. It is, therefore, of interest thatwe found that the pro-migratory activity of GHRP-6was not blocked by adding a neutralizing anti-TGFβ

antibody. Caution always has to be shown, however, inextrapolating from the in vitro situation (utilizing cancercell lines) to the in vivo situation.

GHRP-6 has been reported previously to stimulateproliferation of the hepatoma cell line HepG2, humanpancreatic and prostate cancer cell lines and rat pitui-tary somatotrophs, possibly acting through the MAPK(mitogen-activated protein kinase) and ERK (extra-cellular-signal-regulating kinase) pathways [18,19]. Incontrast, GHRP-6 possessed anti-proliferative activity



Figure 4 Histomorphometric assessment of histological injury in various organsQuantitative assessment, using well-validated histological scoring systems, was performed on the livers (modified Suzuki scoring scheme [14]) and intestines (Chiuscoring scheme [13]). In addition, semi-quantitative assessments of lungs and kidneys (scale: 0, normal; and 2, grossly abnormal) were also performed. See text fordetails of the parameters of assessment.

when added to the human lung cancer cell line CALU-1[20]. To the best of our knowledge, studies on the effectof GHRP-6 on luminal gut epithelial cells have not beenassessed previously. We found that GHRP-6 had no effecton proliferation using either HT29 or IEC6 cells, eventhough GHRP-6 receptors are presumably present (basedon the pro-restitutive activity in the same cells).

The use of arterial occlusion followed by reperfusionis a well-established model of injury resulting from acutevascular occlusion as occurs following embolism orthrombosis. In addition, it is used as a model for loss of theintestinal barrier function associated with haemorrhagicshock, major burns and multiple traumas, which canresult in MOF [21]. Several models have been used tomimic the early stages of MOF. I/R has the advantage ofbeing more physiologically relevant than administrationof toxic agents, such as thioacetamide [22], as the majorfactors causing injury are probably internally generatedpro-inflammatory cytokines and free radical production[4,23,24], rather than resulting from metabolism of anexternal damaging agent. Mesenteric artery occlusionis one of the most popular models used (for example,[8]), but suffers from the drawback that much of theintestinal injury is induced directly. The mesenteric I/Rmodel, therefore, although of direct relevance if studies

are being performed in relation to therapies of mesentericthrombosis, has limitations if therapeutic interventionsare being studied in relation to gut changes in MOF, wherecomplete occlusion of the mesenteric vessels usually doesnot occur. It was because of these issues that we decidedto use the liver vessel clamping technique.

I/R caused marked hepatic necrosis as demonstrated byhistology and elevated ALAT and ASAT plasma levels.Addition of GHRP-6 markedly truncated the degreeof damage determined using all of these parameters.The mechanisms underlying I/R-induced injury and theprotective effects of GHRP-6 are likely to be complexand multi-factorial. During hypoxic conditions, there isup-regulation of cell adhesion molecules [25], facilitatingrecruitment of inflammatory cells to ischaemic areas.Our studies confirmed a marked influx of inflammatoryinfiltrate within the liver, along with a rise in its associatedmarker, MPO. Although GHRP-6 has not been directlyassessed, administration of ghrelin, the natural receptorligand homologue of GHRP-6, has been shown to reducethe adhesion of mononuclear cells to endothelial cellsactivated with TNFα (tumour necrosis factor α) [26]. Itis important to note, however, that the influx of inflam-matory cells was not restricted to the intestine, but alsoaffected distant organs such as the lungs. This must either



be due to an alteration in circulating factor(s), such as pro-inflammatory cytokines, or to the priming and activationof inflammatory cells (mainly neutrophils) at the hepaticsite that subsequently migrate to distant organs.

Production of highly reactive oxygen species andother free-radical-damaging metabolites is known tooccur during I/R [23,24,27]. Uncontrolled production ofsuch factors results in cellular damage, including lipidperoxidation, as well as induction of both apoptosisand necrosis [28,29]. We found excessive free radicalproduction in I/R-treated animals, measured indirectly asmarkedly raised hepatic MDA levels (indicating increasedlipid peroxidation) and a general shift in the redox state, asdemonstrated by changes in both the enzyme constituents(SOD and catalase) and chemical components (TPHand MDA). The molecular mechanisms underlying thereduction in MDA levels may be due to several factors,including immune modulation. In support of this ideais the finding that ghrelin can directly reduce the pro-inflammatory response of stressed endothelial cells [26],which normally results in a pro-inflammatory cascadeand increased free radical production. In addition,GHRP-6 may also have up-regulated the production ofcellular antioxidant enzymes. Further studies in this areacould potentially measure changes in antioxidant enzymelevels in various hepatic and gastrointestinal cell lines.

GHRP-6 has been shown to reduce the amount ofapoptosis in the cerebellar cells of aged rats [30]. Thechanges seen in our present studies may have been par-tially mediated by alteration in apoptosis within the liverand other tissues, although the predominant histologicalfeature seen in the liver and intestine was of necrosis.Further investigation into these mechanisms is complex,however, as single cell-culture model systems do notcontain inflammatory cells and these are likely to be ofmajor importance in the damaging process in vivo (asdemonstrated in the present study by raised MPO levelsand histology). Similarly, there are major difficulties inattempting to measure the degree of apoptosis withintissues containing large amounts of necrotic tissue. Lessdamaging models will probably have to be developed toaddress this question.

Over the last few years, recombinant peptides havebeen introduced increasingly into the clinical arena(e.g. colony-derived growth factor for bone marrowsupport and interferon therapy for viral hepatitis). Wehave examined the effects of EGF in rats undergoingmesenteric I/R previously [8] and also in a clinical trialwhen administered via enema to patients with colitis [31].In view of the positive nature of these studies, we alsoexamined and compared the effect of EGF given alone andin combination with GHRP-6 in the present model. Wefound that EGF given alone was approximately similarin its beneficial effects to those seen with GHRP-6 givenalone (although the dose used was 8 times that of GHRP-6). Administration of both peptides together gave additive

or synergistic responses, suggesting that, in the clinicalarena, use of multiple therapies may have advantages anddeserve further research.

In conclusion, our present studies provide preliminaryevidence that the synthetic hexamer GHRP-6 which,because of its small size, is relatively simple and cheap tomake may be of benefit for injury associated with visceralvascular hypoperfusion. If patients at high risk of MOFcan be identified at an early stage of their admission tohospital, rapid intervention with GHRP-6 may maintainorgan viability. Further studies of GHRP-6 given alone,or possibly in combination with EGF to enhance effects,in additional models that allow administration of thepeptides after MOF has been induced therefore appearjustified.

ACKNOWLEDGMENTS

This work was partially funded by the WellcomeTrust (grant number 054787/B/98/Z), Wexham ParkGastrointestinal Trust (grant number 2004/6772), anda DDF/Belmont Trust Award. The EGF used in thisstudy was produced by Heber-Biotec (Havana, Cuba),which is the commercial arm of the Center for GeneticEngineering and Biotechnology.

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Received 20 December 2005; accepted 17 January 2006Published as Immediate Publication 17 January 2006, doi:10.1042/CS20050374


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