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

Sermorelin Growth hormone-releasing hormone (GHRH) Losing muscle mass and tone? Gaining weight? Feeling fatigued? Low sex drive? SERMORELIN IS A ...
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Sermorelin Growth hormone-releasing hormone (GHRH)

Losing muscle mass and tone?

Gaining weight?

Feeling fatigued?

Low sex drive?

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

STATEMENTS NOT REVIEWED BY FDA Pricing: $650 per month, including all supplies New Age Medical Clinic, PA. www.GrowthHormoneNJ.com

(908) 598-0509

Contents: 1. Overview: What is Sermorelin? a. Benefits b. Side effects 2. Administration and Dosing a. Instructions for Administration 3. Studies

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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:

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Increase development of lean body 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

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Improves sleep quality Increase calcium retention Increases protein synthesis and stimulates the growth of all internal 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.

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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

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

New Age Medical Clinic, PA.

www.GrowthHormoneNJ.com

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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.

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

New Age Medical Clinic, PA.

www.GrowthHormoneNJ.com

(908) 598-0509

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 §

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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.

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New Age Medical Clinic, PA.

www.GrowthHormoneNJ.com

(908) 598-0509

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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.

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

New Age Medical Clinic, PA.

www.GrowthHormoneNJ.com

(908) 598-0509

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

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REVIEW

Growth hormone (GH)–releasing hormone and GH secretagogues in normal aging: Fountain of Youth or Pool of Tantalus? Elizabeth C Hersch George R Merriam VA Puget Sound Health Care System and University of Washington School of Medicine, Tacoma and Seattle, Washington USA

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 deficiency (AGHD) is a distinct clinical entity, and GH replacement in AGHD can improve body composition, strength, aerobic capacity, and mood, and may reduce vascular disease risk. While there are some hormone-related side effects, the balance of benefits and risks is generally favorable, and several countries have approved GH for clinical use in AGHD. GH secretion declines progressively 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 benefits 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 studies 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 benefits and risks of GH supplementation in normal aging remains uncertain. Keywords: growth hormone, growth hormone-releasing hormone, growth hormone secretagogues, aging, sarcopenia, frailty

Introduction

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

Frailty in the elderly is a syndrome of progressive loss of strength and aerobic capacity 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 support 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 findings in frail elders are similar in many ways to those signs and symptoms found in younger adults with GH deficiency (AGHD). Replacement of GH or stimulation of GH secretion with GHreleasing 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 axis GH 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

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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 modulates 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 colleagues (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 receptor (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 physiological pattern of pulsatile GH secretion (Anawalt and Merriam 2001). There are approximately 10 pulses of GH secretion

? Ghrelin ? HYPOTHALAMUS

?

GHRH

SRIF

? ? PITUITARY Ghrelin ? STOMACH GH

LIVER

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.

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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 modified 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 sodiumpotassium pumps and facilitating the renin-angiotensinaldosterone system (Merriam and Cummings 2003).

GH influences 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 synthesis of DNA and procollagen mRNA in bone (Merriam and Cummings 2003).

Normal aging vs adult growth hormone deficiency GH 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 reduction 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.

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in body composition, hormonal status, and functional capacity that mimic the changes seen in AGHD or partial hypogonadism (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 profiles (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 classifying different cognitive domains, often they are grouped as “crystallized” vs “fluid” intelligence. The former includes vocabulary and long-term memory; the latter includes shortterm memory and active problem-solving and declines more markedly with aging. A number of studies have shown that in older adults there is a significant correlation between performance on tests of fluid intelligence and circulating levels of IGF-I (Aleman et al 1999), suggesting that GH may play a role in maintenance of fluid 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 first 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 sensitivity to IGF-I, and that there may be some relative deficiency 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 deficiency. 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 normal 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

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the likelihood of GHD is the clinical context (Merriam and Cummings 2003). Among adults with AGHD, 85% acquire the deficiency 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 difficult 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 deficiencies 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 cardiovascular 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 significant abnormalities in hormone profiles, 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 difficulties 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. Hypertension is more common, thrombogenic blood components are increased, and an atherogenic lipid profile 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 replacement and its side effects While 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 qualities, clinical trials in AGHD were begun soon after recombinant GH was approved for pediatric use in 1985, and results of these studies began to appear in the late 1980’s. In 1996

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GH secretagogues in normal aging

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 significant decrease with aging, and pituitary GH responses are well preserved even in the oldest subjects. From Pavlov et al 1986.

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 benefits 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

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to the current adult practice of beginning with a low fixed 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-deficient state than AGHD, GH replacement seems a potentially reasonable approach to prevention or even reversal of the frailty symptoms of aging. The first studies in non-GHD older adults took place soon after its effects in AGHD were published. In a widely cited

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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 density Atherogenic lipid profile Thin, dry skin; poor venous access Impaired sweating Psychosocial 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.

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 improvement was especially remarkable. Although the Rudman study did not include any functional measures, given these results, it 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 profile 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.

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was postulated that GH treatment might also improve muscle strength and functional performance. Studies of physical functional effects, however, have been generally disappointing 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 fit 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 efficacy, 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 Institute 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 conditioning, and one study of GHRH were funded and have now been completed. While a comprehensive review of the findings 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 reproduce 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 confirmed its beneficial 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 definitive 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 definitively 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 reflects more a lack of information than a demonstration of safety.

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GH secretagogues in normal aging

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 fluid 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 fixed 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 specific 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 beneficial 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 specific dosing targets and in the specific population for which the use is being proposed can adequately assess those benefits and risks.

Growth hormone-releasing hormone and growth hormone secretagogues Growth 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

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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 physiologic 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 influenced 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 fitness associated with GHRH injections. Testing again confirmed the benefits 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 significant decline in physical function in the placebo group (Merriam et al 1997, 2003; Cummings and Merriam 2003). This tantalizing finding, suggesting that GHRH can stabilize if not improve physical function, needs confirmation. 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

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with improved scores in several domains of fluid (but not crystallized) intelligence – those measures previously found correlated with circulating IGF-I levels (Vitiello et al 2006). This intriguing preliminary finding 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 unconfirmed positive effect on some domains of fluid 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 synergistic 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 subcutaneous 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 presentation, however, did not show significant improvements. In cooperation with investigators at Duke University and several other sites, we conducted a trial of the Pfizer investigational oral GH capromorelin in pre-frail older men and women (Merriam et al 2006). This protocol recruited

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over 300 subjects and was initially planned as a two-year intervention. The study was unfortunately stopped, however, 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-efficacy termination criterion. Absolute lean body mass did increase significantly, 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 encouraging effects on physical functional performance. Of seven functional tests, one improved significantly after 6 months of treatment, and another after 12 months. Two other measures showed non-significant 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 consensus on functional effects among these very few studies, and of course none could assess clinical final outcomes or long-term risks.

Conclusion Sarcopenia and subsequent frailty lead to loss of independence. While aging is not a disease, it results in significant 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).

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GH secretagogues in normal aging

Reports of stabilization or even improvement of physical function with GHRH or with oral GHS are extremely tantalizing, but they are hardly proof that Ponce de Leon’s “Fountain of Youth” has been found. The failure to replicate these findings 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, definitive 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 benefits of treatment cannot be made and we can only recommend 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.

Note Presented in part at the Second Annual Meeting of the Society for Applied Research in Aging (SARA), 11 November 2006.

Acknowledgments We 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.

References Aleman 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.

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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 profile 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 emerging (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 deficiency in adults: current perspectives. Current Opinion in Endocrinology and Diabetes, 13:362–8. Molitch M, Clemmons D, Malozowski S, et al. 2006. Evaluation and treatment of adult growth hormone deficiency: 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.

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EDITORIAL FOREWORD Volume 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 insufficiency? 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 specifically forbids the use of rhGH in adults except for treatment of AIDS or human growth hormone deficiency (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 justified 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 sufficient scientific 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 efficacy. 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 Clinical Interventions in Aging 2006:1(4) 307–308 © 2006 Dove Medical Press Limited. All rights reserved

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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 growthpromoting agent in children for the very reason that it is a better alternative for GHRT in aging adults. Growthdeficient children need higher doses of growth hormone than can be achieved by stimulating production of their own hormone, whereas the beneficial 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 specific 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 difficult 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 first 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

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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 benefits 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].

References Fahy J. 2006. Drug could fight 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 insufficiency and hyperprolactinemia in aged rat. Endocrine, 2:633-8.

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

Use of growth-hormone-releasing peptide-6 (GHRP-6) for the prevention of multiple organ failure ´ ∗ , Hussam AJAMIEH†, Jorge BERLANGA∗ , Olga S. LEON ´ ∗, Danay CIBRIAN ∗ 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 Biological Studies, 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 and Dentistry, Turner Street, London E1 2AD, U.K., and Cancer Research UK, Lincoln’s Inn Fields, London WC2A 3PX, U.K.

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Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell 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 given alone 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 min of 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 addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P < 0.01), but did not increase proliferation ([3 H]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 lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 µg/kg of body weight, intraperitoneally) alone truncated these effects by 50–85 % (all P < 0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, 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 enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.

Key words: epidermal growth factor (EGF), growth-hormone-releasing peptide (GHRP), gut injury, ischaemia/reperfusion, multiple organ 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 min of 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]).

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INTRODUCTION MOF (multiple organ failure) is a severe life-threatening condition that usually occurs as a result of major trauma, burns or fulminant infections. Whatever the initiating event, once established, MOF has a high mortality (up to 80 %) [1]. The pathophysiological mechanisms underlying this condition are unclear, although important contributory factors probably include hypoxia, increased intestinal permeability, bacterial translocation, endotoxaemia and uncontrolled systemic inflammatory responses [2]. Several studies suggest that the splanchnic circulation is particularly vulnerable to hypoperfusion, as occurs in low-flow states, such as haemorrhagic shock, and that this hypoperfusion is out of proportion with the overall reduction in cardiac output [3]. Although it is obvious that tissue ischaemia initiates a series of events that can ultimately lead to cellular dysfunction and necrosis, resumption of blood flow can paradoxically create more tissue injury, possibly because of production of oxygen-derived cytotoxic products [4]. The use of I/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 of development of MOF. Synthetic and recombinant peptides are being used increasingly for clinical purposes (e.g. human insulin for diabetes and erythropoietin for anaemia of renal failure), but assessment of their value for the treatment of luminal gastroenterological problems is at a much earlier stage [5]. GH (growth hormone) secretagogues compose a group of heterogeneous synthetic peptides and nonpeptides that, as well as inducing pituitary GH secretion, also bind to GH secretagogue receptors on peripheral tissues, such as the myocardium, pancreas and bone marrow [6,7]. The physiological role of these peripheral receptors is, however, unclear and the potential value of GHRP (GH-releasing peptide)-6 administration on hepatic and gastrointestinal mucosal integrity is untested. In this series of studies, we therefore initially examined whether GHRP-6 had potentially useful ‘pro-healing’ activity using various in vitro models of gut injury. Having found positive results, we progressed to test the effect of systemic administration of GHRP-6 in a rat liver I/R model of hepatic injury and MOF. In addition, as we have found previously a beneficial effect of the potent growth factor EGF (epidermal growth factor) when using a related mesenteric I/R model [8], we also examined the results of giving EGF alone and in combination with GHRP6 (to begin to examine additive/synergistic effects).

MATERIALS AND METHODS Synthetic and recombinant peptides GHRP-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2 ) was purchased from BCN Peptides. The product in a lyo C

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philized form, certified as pyrogen- and contaminantfree, was stored at − 20 ◦ C and diluted in sterile saline just prior to its administration. rhEGF1−52 (recombinant human EGF1−52 ), expressed in Saccharomyces cerevisiae, was obtained from HeberBiotec in a lyophilized form. This product consists of a 60:40 mixture of EGF1−52 and EGF1−51 and is as biologically active as the full length EGF1−53 form [9]. Prior to administration, EGF was diluted in 0.9 % saline under sterile conditions.

Ethics Experiments were conducted according to current Local and National regulatory and ethical guidelines.

Study series 1: in vitro models Effect of exogenous GHRP-6 on an in vitro cell migration model One of the earliest biological repair responses following injury to tissue cells is the migration of surviving cells over the denuded area caused by the injury to re-establish epithelial integrity. Since it is extremely difficult to study this effect upon tissue inside a human or animal, cell culture models are commonly used as surrogate markers of this pro-migratory response. This method also allows direct actions of the test peptide on the cells to be determined. Cell migration assays were performed using our methods published previously [10]. Briefly, human colonic carcinoma (HT29) cells or rat intestinal epithelial (IEC6) cells were grown to confluence in six-well plates in DMEM (Dulbecco’s modified Eagle’s medium) containing 10 % (v/v) FCS (foetal calf serum) at 37 ◦ C in 5 % CO2 . The monolayers were then wounded by scraping a disposable pipette tip across the dishes, washed with fresh serum-free medium and cultured in serumfree medium in the presence of various test factors. The rate of movement of the anterior edges of the wounded monolayers was then determined by taking serial photomicrographs at various times after wounding [10]. Twenty measurements per field were performed by placing a transparent grid over the photograph and measuring the distance moved from the original wound line. All results are expressed as means + − S.E.M. of three separate experiments. The various test factors used were GHRP-6 (10– 400 µg/ml) and EGF (10 µg/ml; used as a positive control). This dose of EGF was used as we have shown previously [10a] that this stimulates maximal restitution responses in this system. The importance of TGF (transforming growth factor) β in any response seen was analysed by using additional wells which contained GHRP-6 (40 µg/ml) and a TGFβ-neutralizing antibody (100 µg/ml; R&D Systems).

Use of GHRP-6 for multiple organ failure

Effect of exogenous GHRP-6 on an in vitro cell proliferation model Cell proliferation assays were performed using our methods published previously [10]. Briefly, HT29 and IEC6 cells were grown in DMEM containing 4 mmol/l glutamine, 10 % (v/v) FCS and various test factors. Effects of addition of various doses of GHRP-6 and EGF (10 µg/ml; used as a positive control) were subsequently tested under serum-starved conditions. To assess the degree of proliferation, [3 H]thymidine (2 µCi/well) was included 24 h after the addition of the test factors, and cells were left for a further 24 h. For each condition, the stimulatory or inhibitory effect of the solutions was measured in quadruplicate in six separate wells. Cell viability, determined by the ability to exclude 0.2 % Trypan Blue, was always greater than 90 %.

Study series 2: in vivo model of I/R Introduction to method Having shown that GHRP-6 possesses potentially useful biological activity in the in vitro systems, we proceeded to examine its effects when used in an in vivo hepatic I/R model. Two different timed protocols were used to examine if any effects seen were applicable to both relatively short and more prolonged periods of ischaemia. GHRP-6 was tested alone and also in combination with EGF, as we have shown previously a beneficial effect of EGF in a related mesenteric I/R model [8] and we wanted to determine if any additive/synergistic responses were apparent. The dose of EGF used in the present study (1 mg/kg of body weight) was similar to that used in our study reported previously [8].

Animals Adult male Wistar rats (200–250 g) were purchased from the National Center for Laboratory Animals and were allowed access to food and water ad libitum.

Induction of I/R injury Animals were anaesthetized with urethane [10 mg/kg of body weight, i.p. (intraperitoneally)] and placed in a supine position on a heating pad in order to maintain body temperature between 36 and 37 ◦ C. To induce hepatic ischaemia, a midline laparatomy was used and the blood supply of the right lobe of the liver was interrupted by placing a bulldog clamp (Fine Science Tools) at the level of the hepatic artery and the portal vein branches. Upon completion of the ischaemia time, reperfusion was initiated by removing the clamp. Reflow was confirmed by the macroscopic inspection of the target lobe. No animals were discarded due to non-reflow states. Animals remained 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 design For both I/R protocols, rats were randomly assigned to five experimental groups as follows: group 1 (sham ischaemia), animals received saline (placebo; 1 ml, i.p.) and 40 min later underwent all procedures, including laparotomy, liver exposure and manipulation, but the hepatic artery and the portal vein branches were not clamped; 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 GHRP6 (120 µg/kg of body weight, i.p.) and 40 min later underwent I/R; group 4 (I/R with EGF), animals received rhEGF (1 mg/kg of body weight, i.p.) and 40 min later underwent 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 min later underwent I/R.

Autopsy and sample processing At the end of the study periods, blood samples were obtained from the abdominal aorta for biochemical determinations. Serum was obtained, aliquoted and stored at − 20 ◦ C until processing. Rats were subjected to autopsy, and samples of different regions from the right ischaemic lobe were collected for subsequent histopathological examination and tissue homogenization. In addition, representative samples were collected from lungs, kidneys, jejunum and ileum. Samples to be processed for histological study were immediately placed in 10 % buffered formalin and subsequently paraffin-embedded and stained with haematoxylin/eosin.

Blood analyses Serum levels of ALAT (alanine aminotransferase) and ASAT (aspartate aminotransferase), used as markers of hepatocyte injury, were determined using a commercial kit 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 analyses The oxidative state of the liver was analysed by measurement of both enzyme activities [SOD (superoxide dismutase) and catalase] and chemical components [THP (total hydroperoxides) and MDA (malondialdehyde) levels]. MDA levels are a commonly used marker of lipid peroxidation. In addition, liver and intestinal MPO (myeloperoxidase) activities were measured as a marker of neutrophilic infiltration. For liver tissue biochemical studies of MDA, THP and SOD, tissue was homogenized [1:10 (w/v)] in 50 mmol/l  C

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KCl/5 mmol/l histidine buffer (pH 7.4), followed by centrifugation at 5000 g for 20 min at 4 ◦ C. The supernatants were collected, aliquoted and stored at − 20 ◦ C until assay. All the biochemical parameters were determined by spectrophotometric methods. MDA content was assessed using the Bioxytech LPO-586 kit (BioRad Laboratories), and THP were determined using the Bioxytech H2 O2 -560 kit (Bio-Rad Laboratories). SOD activity was determined by following changes in autoxidation of pyrogallol in response to adding the homogenate [11]. MPO activity was determined using a modification of the method described by Krawisz et al. [12], and 1 unit of MPO activity was defined as the quantity of enzyme that degrades 1 µmol of H2 O2 /min at 25 ◦ C. Biochemical data were adjusted to reflect total protein concentration using a commercial spectrophotometric protein dye kit (Bio-Rad Laboratories).

Histological assessment All tissues were assessed in a blinded manner. Small intestine The total lengths of the small intestine were measured and then split longitudinally to allow a macroscopic 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 considering the whole length of the small intestine (in cm) as 100 %. Eight equal-spaced 2 cm segments from the length of the small bowel were then collected for histological assessment. For the microscopic assessment, mucosal damage of the small intestine was quantitatively assessed according to the grading system of Chiu et al. [13]. This system uses a scale of 0–5, where 0 is normal mucosa; 1 is development of subepithelial (Gruenhagen’s) spaces; 2 is extension of the subepithelial space with moderate epithelial lifting from the lamina propria; 3 is extensive epithelial lifting with occasional denuded villi tips; 4 is denuded villi with exposed lamina propria and dilated capillaries, and 5 is disintegration of the lamina propria, haemorrhage and ulceration. The mean scores of 30–40 villi from each of the eight segments for each animal were pooled to provide an average score for the intestine of that animal. Liver For each animal, the degree of liver damage was determined in at least five different lobar regions and graded using the modified Suzuki scoring system [14]. Briefly, the various changes noted are sinusoidal congestion, hepatocyte necrosis and ballooning degeneration. The specimen was then graded from 0–4, where no necrosis or congestion/centrilobular ballooning was given a score of 0, and severe congestion/ballooning degeneration as well as > 60 % lobular necrosis was given a value of 4. Kidney Each sample was classified in a blinded fashion into one of three groups: 0, essentially normal histology;  C

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1, moderate, probably reversible, changes (hydropic cytoplasmic changes); and 2, severe changes (nuclear breakdown or cellular detachment from the tubule basement membrane). Lungs Lung interstitial damage ranged from normal to showing varying degrees of septal thickening, hypercellularity, neutrophilic recruitment, interstitial adhesion and alveolar luminal reduction. Each sample was classified in a blinded fashion into one of three groups: 0, essentially normal histology; 1, abnormal showing some of the changes described above, and 2, grossly abnormal showing all of the changes described above.

Data analysis Data were analysed using ANOVA with treatment as factor. Where significant effects were seen on the ANOVA (P < 0.05), individual comparisons based on the group mean square error and residual were performed, a method equivalent to multiple comparisons analyses.

RESULTS Study series 1: in vitro studies Restitution assays GHRP-6 caused pro-migratory activity of wounded monolayers in both HT29 and IEC6 cells in a dosedependent manner. Maximal effects were observed at 40 µg/ml for HT29 cells (Figure 1A) and 160 µg/ml for IEC6 cells (Figure 1B). The addition of a neutralizing anti-TGFβ antibody did not affect the cell migration response caused by GHRP-6 (Figure 1C), suggesting that cell migration in response to GHRP-6 is independent of TGF-β production.

Proliferation assay GHRP-6 did not induce increased thymidine uptake in HT29 or IEC6 cells at any of the doses tested (Figure 1D).

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

Liver Biochemical analyses I/R caused an approx. 10-fold increase in serum ASAT and ALAT. Pre-administration of either GHRP-6 or EGF alone reduced this rise by

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Figure 1 Effect of GHRP-6 on the rate of migration (restitution) or proliferation of various gastrointestinal cell lines

The 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 the negative control (䊊, no GHRP-6 added). The various doses tested were 1 µg/ml (+), 20 µg/ml (䉭), 40 µg/ml (∗ ) and 160 µg/ml (䉮). Maximum effects were seen 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 (䊐). P < 0.01 compared with the negative 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-incubating with a neutralizing anti-TGFβ antibody. 䊊, Negative control (no GHRP-6); ∗ , cells incubated with 40 µg/ml GHRP-6; and X, cells incubated with 40 µg/ml GHRP-6 and a neutralizing anti-TGFβ antibody. Similar results were seen using IEC6 cells (results not shown). (D) HT29 cells incubated in DMEM alone (negative control; − ve) had a [3 H]thymidine uptake of approx. 400 000 c.p.m. Addition of EGF (10 µg/ml, positive control; + ve) caused an approximate doubling of [3 H]thymidine uptake, whereas GHRP-6 (50–400 µg/ml) did not increase [3 H]thymidine uptake above baseline. Similar results were seen with IEC6 cells (results not shown).

approx. 50 % and combination treatment resulted in a further reduction in enzyme levels (Figure 2 and Table 1). I/R caused the MDA levels (marker of lipid peroxidation) to increase by approx. 4-5 fold (Figure 2). In the 90 min/120 min protocol, this rise was truncated by approx. 50 % in animals that had received GHRP6 or EGF alone and virtually completely prevented by pre-treatment with GHRP-6 + EGF together (Figure 2). Similar results were seen in animals undergoing the 45 min/45 min I/R protocol, although the rise in MDA was slightly less marked and either peptide given alone was sufficient to prevent an increase in MDA levels (Table 2). Similarly, animals that received placebo and underwent the 90 min/120 min I/R protocol had a 3–4-fold increase in THP (Table 1). GHRP-6 or EGF

alone truncated this response by approx. 75 % with combination treatment preventing the rise completely (Table 1). Similar results were seen in animals that underwent the 45 min/45 min I/R protocol, although the amount of THP produced was less (I/R + saline-treated animals having an approx. 2-fold increase above shamoperated animals; Table 2). I/R caused an approx. 60 % fall in hepatic SOD levels, and this change was partially reversed by pre-treatment with either GHRP-6 or EGF alone. A further improvement was seen in animals that had received the combination treatment (Figure 2 and Table 2). Catalase activity was increased by approx. 30-fold in response to I/R. This increase was markedly truncated in animals that had received either GHRP-6 or EGF  C

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Figure 2 Influence of pre-administration of GHRP-6 and EGF alone or in combination on injury sustained in various organs

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

ASAT (IU/l) Catalase (units · min−1 · mg−1 of protein) THP (µmol/mg of protein)

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Sham operation (laparotomy)

I/R

I/R + GHRP-6

I/R + EGF

I/R + GHRP-6 + EGF

34 + −4 16 + −4 27 + −3

∗∗ 1452 + − 308 ∗∗ 581 + − 57 ∗∗ 109 + − 16

∗++ 543 + − 123 ++ 31 + −4 ∗++$$ 51 + −2

++ 404 + − 82 ++ 58 + − 13 ++$ 43 + −2

++ 115 + − 33 ++ 20 + −4 ++ 21 + −2

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Table 2 Effect of GHRP-6 and EGF on injury induced by 45 min/45 min of hepatic I/R ∗ ∗∗ ++ Values are means + P < 0.01 − S.E.M., n = 6 for each group. P < 0.05 and P < 0.01 compared with the equivalent value in sham-operated animals. $ $$ compared 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.

MPO intestine (units · min−1 · mg−1 of protein) MDA liver (nmol/mg of protein) MPO liver (units · min−1 · mg−1 of protein) ASAT (IU/l) ALAT (IU/l) Catalase (units · min−1 · mg−1 of protein) THP (µmol/mg of protein) 10−3 × SOD (units · min−1 · mg−1 of protein) Creatinine (µmol/l)

Sham operation (laparotomy)

I/R

I/R + GHRP-6

I/R + EGF

I/R + GHRP-6 + EGF

26 + −3 0.31 + − 0.01 19 + −3 13 + −2 21 + −3 9+ −2 182 + − 16 32.5 + − 1.3 43 + −5

∗∗ 148 + − 13 ∗∗ 1.17 + − 0.11 ∗∗ 105 + −9 ∗∗ 116 + − 11 ∗∗ 157 + − 19 ∗∗ 288 + − 28 ∗∗ 295 + − 13 ∗∗ 14.2 + − 1.8 ∗∗ 84 + −8

∗∗++$$ 60 + −8 ++$ 0.37 + − 0.01 ++$ 38 + −6 ∗∗++$ 62 + −7 ∗∗++ 69 + − 16 ∗∗++$$ 106 + −7 ++$ 168 + −5 ∗∗++$ 22.6 + − 1.0 67 + − 16

++$ 40 + −5 ++ 0.29 + − 0.01 ++$ 34 + − 10 ∗∗++$ 57 + −2 ∗∗++ 82 + − 13 ∗∗++ 63 + −2 ∗++ 123 + − 20 ∗∗++$$ 20.2 + − 0.5 70 + − 5*

++ 16 + −2 ++ 0.19 + − 0.01 ++ 8+ −1 ∗++ 33 + −4 ∗++ 61 + −8 ∗++ 47 + −5 ∗∗++ 108 + −8 ∗∗++ 26.5 + − 0.9 ∗∗ 79 + − 11

alone (causing a 60–70 % reduction), with GHRP6 + EGF combination treatment truncating this response by approx. 90 % (Tables 1 and 2). Histology Sham-operated animals had an essentially normal liver histology (Figure 3). Animals that had undergone I/R with placebo (saline) injection had severe changes, consisting of areas of necrosis, haemorrhage, cytoplasmic ballooning and sinusoidal distension. Animals that had been pre-treated with GHRP-6 alone, EGF alone or the GHRP-6 + EGF combination therapy all showed improvements compared with the I/R group, with the combination therapy appearing to have the most protective effect (Figure 3). Assessment using the microscopic scoring system confirmed these results; all animals that underwent I/R and received placebo had scores of 3 or 4, whereas six out of ten animals that had received combination therapy had scores of 0 (Figure 4).

Intestine I/R alone resulted in macroscopically obvious injury affecting 73 + − 4 % of the intestinal length. Pre-treatment with either peptide alone significantly decreased (P < 0.01) the degree of macroscopic injury (27 + − 3 and 30 + − 2 % for GHRP-6- and EGF-treated animals respectively), with the most beneficial effect being seen in animals that had received both GHRP-6 and EGF (19 + − 2 %; P < 0.01 compared with I/R alone or I/R plus either peptide given alone). Histological assessment showed I/R caused severe mucosal damage, with most animals showing complete loss of villous architecture and extensive areas of mucosal infarction (Figure 3). These changes were much less prominent in animals that had received GHRP-6 alone, EGF alone or the GHRP-6 + EGF combination treatment (Figure 3). Quantitative assessment showed similar effects; all animals that underwent I/R and received placebo had scores of 4 or 5, whereas six out

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

Kidney Biochemical analysis In the animals undergoing the 90 min/120 min I/R protocol, serum creatinine levels rose from 45 to 70 µmol/l in response to I/R. Pre-treatment with GHRP-6 was associated with a 30 % (non-significant) fall in creatinine levels, whereas pre-treatment with EGF either alone or in combination with GHRP-6 resulted in the creatinine levels remaining in the normal (sham-operated) range (Figure 2). A similar trend was seen in animals that underwent the 45 min/45 min I/R protocol, although the beneficial effects were less marked and non-significant (Table 2). Histology Animals that had undergone I/R but not received GHRP-6 or EGF all showed moderate or severe renal injury comprising nuclear breakdown or cellular detachment from the tubule basement membrane. Administration of GHRP-6 or EGF given alone, or in combination, tended to reduce the degree of injury; GHRP-6 + EGF combination treatment had the most beneficial effect with nine out of ten animals having essentially normal renal histology by semi-quantitative scoring (Figure 4).

Lung Histology Animals that had received I/R without GHRP-6 or EGF had severe changes comprising septal thickening, hypercellularity, neutrophilic recruitment, interstitial adhesion and alveolar luminal reduction (Figure 3). None of the animals that had received I/R without GHRP-6 or EGF had normal lung histology, whereas nine out of ten animals that had received both peptides had normal histology. Animals that had received either peptide alone occupied intermediate positions (Figures 3 and 4).  C

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Figure 3 Histopathology of rats given placebo (saline), or GHRP-6 and EGF alone or in combination prior to 90 min hepatic vessel clamping followed by 120 min reperfusion

Compared with sham-operated animals, rats that underwent I/R, but did not receive GHRP-6 or EGF, had severe changes. Administration of either peptide alone improved histological appearances with the most improvement being seen in animals that received the combination of GHRP-6 and EGF. Original magnification of intestine, lungs and liver was ×10, ×10 and ×40 respectively.

DISCUSSION Using in vitro models of injury and repair, we have shown that GHRP-6 stimulates gut epithelial restitution, but not proliferation. In vivo studies have shown that pre-administration of GHRP-6 reduced the amount of intestinal and extra-intestinal injury caused by hepatic vessel I/R and that added benefit was observed if EGF was co-administered with GHRP-6. The control of release of endogenous GH from the pituitary gland is thought to be partially mediated by the presence of GHRP receptors acting via a specific G-protein-coupled receptor pathway, the natural ligand of which is probably the 28-amino-acid peptide ghrelin [15,16]. During the course of research into the control of GH release, several peptides that induce GH secretion were developed and one of the most potent was the hexapeptide GHRP-6 [15,16] used in the present study. Using a variety of GH secretagogue molecules, it is now known that, in addition to being present within the pituitary gland, GHRP receptors are also present in several peripheral tissues, including bone marrow, spleen, pancreas, thyroid and myocardium [6,7], suggesting  C

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additional roles for GHRP ligands that extend beyond GH release. GHRP-6 stimulated cell migration of the human colonic cell line HT29 and the rat intestinal cell line IEC6, showing that these effects were not species specific and that GHRP-6 was able to influence gut epithelial function by acting directly on the cells. The pro-migratory effects of some of the well established pro-migratory ‘growth factors’, such as IFNγ (interferon γ ), TGFα and EGF, are dependent upon their ability to induce TGFβ release into the medium [17]. It is, therefore, of interest that we found that the pro-migratory activity of GHRP-6 was not blocked by adding a neutralizing anti-TGFβ antibody. Caution always has to be shown, however, in extrapolating from the in vitro situation (utilizing cancer cell lines) to the in vivo situation. GHRP-6 has been reported previously to stimulate proliferation of the hepatoma cell line HepG2, human pancreatic and prostate cancer cell lines and rat pituitary somatotrophs, possibly acting through the MAPK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulating kinase) pathways [18,19]. In contrast, GHRP-6 possessed anti-proliferative activity

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Figure 4 Histomorphometric assessment of histological injury in various organs

Quantitative assessment, using well-validated histological scoring systems, was performed on the livers (modified Suzuki scoring scheme [14]) and intestines (Chiu scoring scheme [13]). In addition, semi-quantitative assessments of lungs and kidneys (scale: 0, normal; and 2, grossly abnormal) were also performed. See text for details 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 effect of GHRP-6 on luminal gut epithelial cells have not been assessed previously. We found that GHRP-6 had no effect on proliferation using either HT29 or IEC6 cells, even though GHRP-6 receptors are presumably present (based on the pro-restitutive activity in the same cells). The use of arterial occlusion followed by reperfusion is a well-established model of injury resulting from acute vascular occlusion as occurs following embolism or thrombosis. In addition, it is used as a model for loss of the intestinal barrier function associated with haemorrhagic shock, major burns and multiple traumas, which can result in MOF [21]. Several models have been used to mimic the early stages of MOF. I/R has the advantage of being more physiologically relevant than administration of toxic agents, such as thioacetamide [22], as the major factors causing injury are probably internally generated pro-inflammatory cytokines and free radical production [4,23,24], rather than resulting from metabolism of an external damaging agent. Mesenteric artery occlusion is one of the most popular models used (for example, [8]), but suffers from the drawback that much of the intestinal injury is induced directly. The mesenteric I/R model, therefore, although of direct relevance if studies

are being performed in relation to therapies of mesenteric thrombosis, has limitations if therapeutic interventions are being studied in relation to gut changes in MOF, where complete occlusion of the mesenteric vessels usually does not occur. It was because of these issues that we decided to use the liver vessel clamping technique. I/R caused marked hepatic necrosis as demonstrated by histology and elevated ALAT and ASAT plasma levels. Addition of GHRP-6 markedly truncated the degree of damage determined using all of these parameters. The mechanisms underlying I/R-induced injury and the protective effects of GHRP-6 are likely to be complex and multi-factorial. During hypoxic conditions, there is up-regulation of cell adhesion molecules [25], facilitating recruitment of inflammatory cells to ischaemic areas. Our studies confirmed a marked influx of inflammatory infiltrate within the liver, along with a rise in its associated marker, MPO. Although GHRP-6 has not been directly assessed, administration of ghrelin, the natural receptor ligand homologue of GHRP-6, has been shown to reduce the adhesion of mononuclear cells to endothelial cells activated with TNFα (tumour necrosis factor α) [26]. It is important to note, however, that the influx of inflammatory cells was not restricted to the intestine, but also affected distant organs such as the lungs. This must either  C

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be due to an alteration in circulating factor(s), such as proinflammatory cytokines, or to the priming and activation of inflammatory cells (mainly neutrophils) at the hepatic site that subsequently migrate to distant organs. Production of highly reactive oxygen species and other free-radical-damaging metabolites is known to occur during I/R [23,24,27]. Uncontrolled production of such factors results in cellular damage, including lipid peroxidation, as well as induction of both apoptosis and necrosis [28,29]. We found excessive free radical production in I/R-treated animals, measured indirectly as markedly raised hepatic MDA levels (indicating increased lipid peroxidation) and a general shift in the redox state, as demonstrated by changes in both the enzyme constituents (SOD and catalase) and chemical components (TPH and MDA). The molecular mechanisms underlying the reduction in MDA levels may be due to several factors, including immune modulation. In support of this idea is the finding that ghrelin can directly reduce the proinflammatory response of stressed endothelial cells [26], which normally results in a pro-inflammatory cascade and increased free radical production. In addition, GHRP-6 may also have up-regulated the production of cellular antioxidant enzymes. Further studies in this area could potentially measure changes in antioxidant enzyme levels in various hepatic and gastrointestinal cell lines. GHRP-6 has been shown to reduce the amount of apoptosis in the cerebellar cells of aged rats [30]. The changes seen in our present studies may have been partially mediated by alteration in apoptosis within the liver and other tissues, although the predominant histological feature seen in the liver and intestine was of necrosis. Further investigation into these mechanisms is complex, however, as single cell-culture model systems do not contain inflammatory cells and these are likely to be of major importance in the damaging process in vivo (as demonstrated in the present study by raised MPO levels and histology). Similarly, there are major difficulties in attempting to measure the degree of apoptosis within tissues containing large amounts of necrotic tissue. Less damaging models will probably have to be developed to address this question. Over the last few years, recombinant peptides have been introduced increasingly into the clinical arena (e.g. colony-derived growth factor for bone marrow support and interferon therapy for viral hepatitis). We have examined the effects of EGF in rats undergoing mesenteric I/R previously [8] and also in a clinical trial when administered via enema to patients with colitis [31]. In view of the positive nature of these studies, we also examined and compared the effect of EGF given alone and in combination with GHRP-6 in the present model. We found that EGF given alone was approximately similar in its beneficial effects to those seen with GHRP-6 given alone (although the dose used was 8 times that of GHRP6). Administration of both peptides together gave additive  C

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or synergistic responses, suggesting that, in the clinical arena, use of multiple therapies may have advantages and deserve further research. In conclusion, our present studies provide preliminary evidence that the synthetic hexamer GHRP-6 which, because of its small size, is relatively simple and cheap to make may be of benefit for injury associated with visceral vascular hypoperfusion. If patients at high risk of MOF can be identified at an early stage of their admission to hospital, rapid intervention with GHRP-6 may maintain organ viability. Further studies of GHRP-6 given alone, or possibly in combination with EGF to enhance effects, in additional models that allow administration of the peptides after MOF has been induced therefore appear justified.

ACKNOWLEDGMENTS This work was partially funded by the Wellcome Trust (grant number 054787/B/98/Z), Wexham Park Gastrointestinal Trust (grant number 2004/6772), and a DDF/Belmont Trust Award. The EGF used in this study was produced by Heber-Biotec (Havana, Cuba), which is the commercial arm of the Center for Genetic Engineering and Biotechnology.

REFERENCES 1 Zimmerman, J. E., Knuas, W. A., Sun, X. et al. (1996) Severity stratification and outcome prediction for multisystem organ failure and dysfunction. World J. Surg. 20, 401–405 2 Nguyen, T. T., Gilpin, D. A., Meyer, N. A. et al. (1996) Current treatment of severely burned patients. Ann. Surgery 1, 14–25 3 McNeill, J. R., Stark, R. D. and Greenway, C. V. (1970) Intestinal vasoconstriction after haemorrhage: role of vasopressin and angiotensin. Am. J. Physiol. 219, 1342–1347 4 Parks, D. A. and Granger, D. N. (1986) Contributions of ischaemia and reperfusion to mucosal lesion formation. Am. J. Physiol. 250, G749–G753 5 Playford, R. J. and Ghosh, S. (2005) Cytokines and growth factor modulators in intestinal inflammation and repair. J. Pathol. 205, 417–425 6 Bodart, V., Bouchard, J. F., McNicoll, N. et al. (1999) Identification and characterization of a new growth hormone-releasing peptide receptor in the heart. Circ. Res. 85, 796–802 7 Papotti, M., Ghe, C., Cassoni, P. et al. (2000) Growth hormone secretagogue binding sites in peripheral human tissues. J. Clin. Endocrinol. Metab. 85, 3803–3807 8 Berlanga, J., Prats, P., Remirez, D. et al. (2002) Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage. Am. J. Path. 161, 373–379 9 Calnan, D. P., Fagbemi, A., Berlanga-Acosta, J. et al. (2000) Potency and stability of C-terminal truncated human epidermal growth factor. Gut 47, 622–627 10 Playford, R. J., Marchbank, T., Chinery, R. et al. (1995) Human spasmolytic polypeptide is a cytoprotective agent that stimulates cell migration. Gastroenterology 108, 108–116

Use of GHRP-6 for multiple organ failure

10a Chinery, R. and Playford, R. J. (1995) Combined intestinal trefoil factor and epidermal growth factor is prophylactic against indomethacin-induced gastric damage in the rat. Clin. Sci. 88, 401–403 11 Keesey, J. (1987) Enzymes for routine quantitative analysis. In Biochemica Information: a Revised Biochemical Reference Source, pp. 80–81, Boehringer-Mannheim GmbH Biochemica, Mannheim 12 Krawisz, J. E., Sharon, P. and Stenson, W. F. (1984) Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. Gastroenterology 87, 1344–1350 13 Chiu, C. J., McArdle, A. H., Brown, R. et al. (1970) Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch. Surg. 101, 478–483 14 Shen, X. D., Ke, B., Zhai, Y. et al. (2003) Stat4 and Stat6 signaling in hepatic ischemia/reperfusion injury in mice: HO-1 dependence of Stat4 disruption-mediated cytoprotection. Hepatology 37, 296–303 15 Anderson, L. L., Jeftinija, S., Scanes, C. G. et al. (2005) A physiology of ghrelin and related peptides. Domest. Anim. Endocrinol. 29, 111–144 16 Bowers, C. Y., Momany, F. A., Reynolds, G. A. et al. (1984) On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114, 1537–1545 17 Dignass, A. U. and Podolsky, D. K. (1993) Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor β. Gastroenterology 105, 1323–1332 18 Jeffery, P. L., Herington, A. C. and Chopin, L. K. (2002) Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines. J. Endocrinol. 172, R7–R11 19 Nanzer, A. M., Khalaf, S., Mozid, A. M. et al. (2004) Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. Eur. J. Endocrinol. 151, 233–240

20 Ghe, C., Cassoni, P., Catapano, F. et al. (2002) The antiproliferative effect of synthetic peptidyl GH secretagogues in human CALU-1 lung carcinoma cells. Endocrinology 143, 484–491 21 Biffl, W. L. and Moore, E. E. (1996) Splanchnic ischaemia/reperfusion and multiple organ failure. Br. J. Anaesthetics 77, 59–70 22 Caballero, M. E., Berlanga, J., Ramirez, D. et al. (2001) Epidermal growth factor reduces multiorgan failure induced by thioacetamide. Gut 48, 34–40 23 Granger, D. N. and Korthuis, R. J. (1995) Physiologic mechanisms of postischemic tissue injury. Annu. Rev. Physiol. 57, 311–332 24 Kong, S. E., Blennerhassett, L. R., Heel, K. A. et al. (1998) Ischaemic-reperfusion injury to the intestine. Aust. N.Z. J. Surg. 68, 554–561 25 Meyer, K., Brown, M. F., Zibari, G. et al. (1998) ICAM-1 upregulation in distant tissues after hepatic ischemia/ reperfusion: a clue to the mechanism of multiple organ failure. J. Pediatr. Surg. 33, 350–353 26 Li, W. G., Gavrila, D., Liu, X. et al. (2004) Ghrelin inhibits proinflammatory responses and nuclear factor-κB activation in human endothelial cells. Circulation 109, 2221–2226 27 Carden, D. L. and Granger, D. N. (2000) Pathophysiology of ischaemia-reperfusion injury. J. Pathol. 190, 255–266 28 Crompton, M. (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341, 233–249 29 Sandoval, M., Zhang, X. J., Liu, X. et al. (1997) Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells: attenuation by L-ascorbic acid. Free Radical Biol. Med. 22, 489–495 30 Paneda, C., Arroba, A. I., Frago, L. M. et al. (2003) Growth hormone-releasing peptide-6 inhibits cerebellar cell death in aged rats. NeuroReport 14, 1633–1635 31 Sinha, A., Nightingale, J., West, K. P. et al. (2003) Epidermal growth factor enemas with oral mesalamine for mild-to-moderate left-sided ulcerative colitis or proctitis. N. Engl. J. Med. 349, 350–357

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

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