Download Effects of plasmid-mediated growth hormone releasing hormone

Effects of plasmid-mediated growth hormone releasing hormone
supplementation in young, healthy Beagle dogs1
R. Draghia-Akli2, K. K. Cummings, A. S. Khan, P. A. Brown, and R. H. Carpenter
ADViSYS, Inc., The Woodlands, TX 77381
ABSTRACT: Our study focused on the evaluation of
the pharmacological and toxicological effects of plasmid-mediated GHRH supplementation with electroporation in normal adult dogs over a 180-d period. Twentyeight dogs (<2 yr of age) were randomized to four groups.
Three groups (four dogs/sex for each group) were
treated with ascending doses of GHRH-expressing plasmid: 0.2, 0.6, and 1 mg. One group (two dogs of each
sex) served as the control. Clinical observations and
body weights were recorded. Hematological, serum biochemical, and urine analyses were performed. Serum
IGF-I, ACTH, and insulin were determined. Necropsies
were performed on d 93 and 180; organs were weighed
and tissues were fixed and processed for light microscopy. Selected tissues were used to assess plasmid
biodistribution on d 93. At all doses, plasmid GHRH
caused increased weight gain (P < 0.001), without organomegaly. Serum glucose and insulin in fasted dogs
remained within normal ranges at all time points. Adrenocorticotropic hormone was normal in all groups. Significant increases in number of red blood cells, hematocrit, and hemoglobin (P < 0.01) were observed. In conclusion, our study shows that plasmid-mediated GHRH
supplementation is safe in electroporated doses up to
1.0 mg in young healthy dogs.
Key Words: Dogs, Electroportation, Insulin-Like Growth Factor, Plasmids, Somatoliberin, Somatotropin
2003 American Society of Animal Science. All rights reserved.
Introduction
Regulated GH secretion is essential for optimal linear
growth, homeostasis of carbohydrate, protein, and fat
metabolism, and for the promotion of a positive nitrogen
balance (Murray and Shalet, 2000). These effects are
both direct and mediated by IGF-I, the downstream
effector of GH. The GH synthesis and secretion from
the anterior pituitary are stimulated by GHRH, a hypothalamic peptide hormone (Muller et al., 1999). Although administration of exogenous recombinant GH
(rGH) produces anabolic effects in a variety of situations, rGH therapy has disadvantages. An alternate
1
The authors would like to particularly thank D. Kern for his
support of this work, C. Tone for the editorial correction of this manuscript, and to the members of our research team for their input during
this study: M. Pope, L. A. Hill, and B. Malone. Our greatest appreciation for the staff at Stillmeadow, Inc., for their professionalism, and
especially to A. Perez de Leon. We would also like to thank D. Hildebrandt at Pathco, Inc., for the histological analysis of the tissues. We
are grateful to T. Spencer at Texas A&M University for his PCR tissue
analysis. We acknowledge support for this study from ADViSYS, Inc.
(The Woodlands, TX).
2
Correspondence: 2700 Research Forest Dr., Suite 180 (phone: 281296-7300, ext. 107, fax: 281-296-7333, E-mail: ruxandradraghia@
advisys.net).
Received March 24, 2003.
Accepted May 21, 2003.
J. Anim. Sci. 2003. 81:2301–2310
method to increase GH production and release would
be to administer GHRH. In a 56-d proof-of-concept
study, we observed anabolic effects, correction of anemia and weight loss, and improved quality of life in dogs
with cancer following injection of a GHRH-expressing
plasmid (Draghia-Akli et al., 2002a). The objective of
the present experiment was to determine whether a
plasmid-mediated GHRH therapy would be nontoxic
and would produce long-term beneficial effects at different dosages. This pilot study was conducted in young
healthy male and female Beagle dogs. Follow-up evaluations demonstrated increased weight and serum IGFI concentrations as an indicator of GHRH activity, together with significantly increased red blood cell production. Necropsy and histopathology evaluation
showed no adverse effects associated with plasmid delivery and GHRH expression over a period of 180 d.
These results suggest that plasmid-mediated GHRH
therapy can be a viable alternative to injectable recombinant protein therapies.
Materials and Methods
Deoxyribonucleic Acid Constructs
The plasmid pSPc5-12 contains a 360-bp SacI/BamHI
fragment of the SPc5-12 synthetic promoter (Li et al.,
1999) in the SacI/BamHI sites of a pSK-GHRH back-
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Draghia-Akli et al.
bone (Draghia-Akli et al., 1997). The synthetic GHRH
complementary DNA (cDNA), HV-GHRH, was obtained by site-directed mutagenesis (Altered Sites II in
vitro Mutagenesis System, Promega, Madison, WI), and
cloned into the BamHI/HindIII sites of pSK-GHRH.
The GHRH cDNA is followed by the 3′ untranslated
region of GH. Characterization of the vector and the
long half-life analog HV-GHRH has been previously
described (Draghia-Akli et al., 1999).
Animals
Twenty-eight dogs (Beagles; Harlan Sprague-Dawley, Inc., Houston, TX), approximately 2 yr of age,
weighing 10 to 18 kg for males and 7 to 13 kg for females,
were divided into one group of four animals (Group I)
and three groups of eight animals each (Groups II to
IV). Each group had an equal number of males and
females. Normal growth, appearance, and behavior
were factors used to select healthy animals for testing.
Each animal received a pretest physical examination
by a laboratory animal veterinarian. Animals were
identified by tattoos and cage cards. Necropsies were
performed on half the animals from each group on d 93
(two control animals—one male and one female, and
four treated animals from each treatment group—two
males and two females per group). At approximately d
180, necropsies were performed on the other half of
the animals from each group (two control animals—one
male and one female, and four treated animals from
each treatment group—two males and two females
per group).
Intramuscular Injection of Plasmid DNA
The endotoxin-free plasmid (Qiagen Inc., Chatsworth, CA) preparation of pSPc5-12-HV-GHRH was diluted in water to 5 mg/mL. Animals in Groups II to
IV were treated per FDA regulations in target animal
safety studies with 1× (a total of 0.2 mg of plasmid), 3×
(a total of 0.6 mg of plasmid), and 5× (a total of 1 mg
of plasmid) the projected therapeutic dosage range of
HV-GHRH plasmid by intramuscular injection followed
by electroporation on d 0. Group I animals underwent
the electroporation procedure but were not injected
with the test article and served as untreated controls.
The dogs were anesthetized with 2 mL of a 50:50 mixture of xylazine (Phoenix Scientific, Inc., St. Joseph,
MO) and ketamine (Abbott Laboratories, Inc., North
Chicago, IL) delivered intramuscularly. All animals received a tattoo of the projected injection area in order
to properly identify and isolate the site. The plasmid
was injected directly into the semitendinosus muscle
with a 1-cc insulin syringe and a 21-gauge, 1.5-in needle
(Becton-Dickinson, Franklin Lakes, NJ). Two minutes
after injection, the injected muscle was electroporated
(three pulses in one orientation, three pulses in an orientation perpendicular to the first one, 100 V/cm, 60
ms/pulse) with a two-needle electrode device, and BTX-
830 electroporator (Genetronics, San Diego, CA). Animals were observed during recovery from anesthesia
and then returned to their cages.
Weight, and Serum and Urine Endpoints
Animal Weight. Dogs were weighed and numbered
before the injection/electroporation procedure. Two preinjection blood draws (d −6 and 0), and six post-treatment draws (d 28, 56, 93, 120, 157, and 180) were performed. Average complete blood counts (CBC), biochemistry, and hormone values for d −6 and 0 served
as baseline reference for each dog. Whole blood was
collected in Monoject Lavender Stopper blood collection
tubes with 3.0 mg of EDTA (Sherwood Medical, St.
Louis, MO) and submitted for CBC analysis (Antech
Diagnostics, Irvine, CA). Serum was aliquoted for radioimmunoassay and biochemical analysis (Antech Diagnostics). Serum was stored at −80°C before analysis.
Biochemical analysis occurred within 48 h after serum
collection. The IGF-I assay was performed within 90 d
after serum collection.
Insulin-like Growth Factor-I Radioimmunoassay. Dog
IGF-I was measured by a heterologous human assay
(Diagnostic System Lab., Webster, Texas). The sensitivity limit of the assay was 0.8 ng/mL; the intra- and
interassay variations were 3.4 and 4.5%, respectively.
Biochemistry, CBC, Urinalysis, and Hormone Levels.
Biochemical and blood chemistry, insulin, and ACTH
levels were assayed at Antech Diagnostic at the previously stated time points. During anesthesia prior to
injection and on d 93 and 180 at necropsy, a urine
sample was drawn from each animal and a urine analysis was performed. A laboratory animal veterinarian
reviewed all values. The following parameters were
evaluated: serum chemistry, hematology, and urine
analysis.
Serum chemistry tested for amylase, globulin, γ-glutamyltransferase, total bilirubin, blood urea nitrogen,
creatinine, total protein, albumin, direct bilirubin, serum alanine aminotransferase, serum aspartate aminotransferase, alkaline phosphatase, glucose, inorganic
phosphorus, chloride, calcium, sodium, potassium, triglyceride, and cholesterol.
Hematology looked at erythrocyte counts, hematocrit, hemoglobin, total leukocyte count, and differential
leukocyte counts (neutrophils, lymphocytes, monocytes,
eosinophils, and basophils), platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean
corpuscular hemoglobin concentration, and partial prothrombin time.
Urine analysis analyzed color, consistency, volume,
glucose, bilirubin, ketone (acetoacetic acid), specific
gravity, blood, pH, protein, urobilinogen, nitrite, leukocytes, and microscopic examination of formed elements.
Necropsy
Gross Necropsy. All the dogs were killed with an overdose of sodium pentobarbital (Fatal Plus, Vortech Phar-
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maceuticals, Dearborn, MI). The gross necropsy included the following: gross observations of external surfaces, all orifices, and cranial, thoracic, abdominal, and
pelvic cavities as well as accompanying organs in situ.
Histopathology Analysis of Tissues. Samples of selected organs (heart, lung, liver, spleen, stomach, intestine, pancreas, gall bladder, kidney, gonads, brain, pituitary, adrenals, thyroid, parathyroid, retina and optic
nerve, injected muscle, and the injection/electroporation site (skin and muscle) were examined and then
assessed histologically by a licensed, independent veterinary pathologist.
Polymerase Chain Reaction Analysis
for Presence of Plasmid
The DNA extraction from injected muscle, liver, and
first lymph node was performed according to standard
procedures (Sambrook et al., 1989). The PCR was performed for presence of β-actin as a control (forward
primer 5′-ATGTACGTGGCCATCCA-3′ and reverse
primer 5′-AGGAGGAGGGACCTCTT-3′) and origin of
replication (ori) for presence of the injected HV-GHRH
plasmid (forward primer 5′-CGAGCGGTATCAGCTCA3′ and reverse primer 5′-ATCGTCTCGCTCCATACAT3′). Primers for PCR amplification were synthesized by
Sigma Genosys (The Woodlands, TX). The sensitivity
of the conventional PCR reaction is routinely one copy
of plasmid per microgram of genomic DNA (Ledwith et
al., 2000). Amplification using ori primers was conducted in a GeneAmp 9700 Thermocycler (Pekin Elmer,
Norwalk, CT). Polymerase chain reaction was performed in 50-␮L reactions with 2× PCR mix #1 (Sigma),
0.2 ␮M forward and reverse primers, and 40 ng of dog
tissue DNA or 1 ng of control plasmid DNA. Water
was substituted for template DNA in negative controls.
Polymerase chain reaction conditions were as follows:
1) 94°C for 4 min; 2) five cycles of 94°C for 30 s, 57°C
for 30 s, 72°C for 30 s; 3) 30 cycles of 94°C for 30 s,
53°C for 30 s, 72°C for 30 s; and 4) 72°C for 5 min.
Products were detected by agarose electrophoresis of
an aliquot (60%) of each reaction vs. a 100-bp ladder
marker (Invitrogen, Carlsbad, CA).
Statistical Analysis
Data consisted of repeated measures in seven different time points with an unbalanced study design
(Group I, n = 4; Groups II to IV, n = 8). After d 93, the
sample size was reduced to half (Group I, n = 2; Groups
II to IV, n = 4). Considering all these facts, we chose
the MIXED model of SAS (SAS Inst., Inc., Cary, NC;
analysis of simple main effects) to examine if there were
any significant differences among the groups of each
variable at different time points. Multiple comparisons
were adjusted by the Tukey-Krammer method. Mean
values were compared with Student’s t-test, ANOVA,
or linear regression, with <0.05 taken as the level of
statistical significance.
Results
Electroporation and Injection Site
Erythema was the most common observation at the
injection/electroporation site on d 1 and resolved in a
few days. The in-life observations showed one male dog
in Group II that developed an abscess on or near the
injection site 3 d after injection. Healing occurred
around d 19. All other dogs were normal and presented
no abnormal findings throughout the study that might
be test-article or procedure related.
Weights
During the study, weights increased in plasmidtreated dogs (Group II by 11.3%, Group III by 6.7%, and
Group IV by 14.1%), and slightly decreased in control
animals (Group I decreased by 0.8%; Table 1). When
data were compared by analysis of covariance using
the baseline weight as the regression variable in the
analysis, Groups II and IV had a significant weight
increase during the study (r2 = 0.59, P < 0.02 and r2 =
0.65, P < 0.001, respectively), whereas Group III had a
weight increase that did not reach statistical significance due to high interanimal variability (r2 = 0.20, P
< 0.20) (Figure 1).
Insulin-Like Growth Factor-I Levels
An increase in serum IGF-I concentration over the
baseline value was taken as a measure of plasmid
GHRH activity (de Boer et al., 1996; Aimaretti et al.,
1998). During the study, IGF-I levels were increased
in plasmid-treated dogs, and values were statistically
significant for days postinjection (sum of average IGFI levels from d 0 to d 180: Group I = 121.9, Group II =
142.6, Group III = 153.4, and Group IV = 153.2, P <
0.001). A dose-related trend in IGF-I levels was observed among the treated groups, but the effect of different treatment doses was secondary (P < 0.09) once days
postinjection had been considered. In this case, the variability between groups in the mean values of the various
assessed parameters was not greater than the variability within each group. The data from all treated groups
of animals, therefore, were combined for comparison
with Group I (P < 0.001). When each dog was compared
to its own baseline (IGF-I postinjection − IGF-I preinjection/IGF-I preinjection), the group treated with 1 mg
of plasmid had a significant increase at all time points
analyzed (Figure 2 and Table 2). Insulin-like growth
factor-I levels were within normal range for all dogs.
Analysis of Blood, Chemical Parameters,
and Urinalysis
Several variables were significantly different between controls and treated groups at different time
points after treatment. Hemoglobin and hematocrit
were significantly increased in all treated dogs, with
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Table 1. Weights in dogs treated with plasmid-mediated GHRH therapya
Day
Baseline
Day 28
Day 42
Day 56
Day 93
Day 120
Day 147
Day 180
Group I
Control
Group II
0.2-mg dose
Group III
0.6-mg dose
Group IV
1-mg dose
11.59 ± 0.70b
11.95 ± 0.47
P = 0.06
12.23 ± 0.72
P = 0.17
12.05 ± 0.66
P = 0.16
11.58 ± 0.63
P = 0.04
11.05 ± 0.11
P = 0.33
11.35 ± 0.11
P = 0.0001
11.50 ± 0.07
P = 0.33
11.36 ± 0.97
11.80 ± 1.02
P = 0.05
12.14 ± 1.19
P = 0.03
12.13 ± 1.24
P = 0.08
11.93 ± 1.15
P = 0.09
11.75 ± 1.34
P = 0.006
12.35 ± 1.56
P = 0.02
12.66 ± 1.37
P = 0.0004
11.43 ± 0.86
12.21 ± 0.79
P = 0.007
12.30 ± 0.85
P = 0.01
12.46 ± 0.82
P = 0.002
12.04 ± 0.75
P = 0.08
12.00 ± 0.63
P = 0.27
12.13 ± 0.78
P = 0.21
12.20 ± 0.69
P = 0.18
11.46 ± 0.72
12.28 ± 0.76
P = 0.0002
12.64 ± 0.81
P = 0.0003
12.53 ± 0.83
P = 0.0008
12.09 ± 0.77
P = 0.001
12.73 ± 0.63
P = 0.008
13.10 ± 0.72
P = 0.01
13.08 ± 0.79
P = 0.006
a
The results are presented as means ± SEM. P-values vs. baseline weights are included for each group
and time point.
b
Weights were recorded at least eight different times throughout the study for each individual animal on
a precision balance. Group 1, n = 4; Groups II through IV, n = 8.
least squares means <0.05 (Figure 3), but within normal
values for dogs. Calcium:phosphorous ratio, an indication of bone remodeling (Anderson, 1996; Yilmaz et al.,
1999), was significantly increased in dogs in Group IV
vs. controls at d 180 postinjection (2.75 ± 0.2 vs. 2.28
± 0.04, P < 0.045). All other values were within the
normal reference laboratory range and not indicative
of any abnormality. No toxicological significance can be
attributed to any of these changes.
Fasting glucose and insulin levels were normal for
all groups throughout the study. The ACTH levels were
assayed on d 120 postinjection. There were no significant differences between treated and control groups.
Polymerase Chain Reaction Analysis
for Presence of Plasmid
Polymerase chain reactions were performed on tissues harvested at the interim necropsy on d 93. The
entire injected muscle, first lymph node, and liver of
all dogs, treated or control, were assayed. One dog in
Group III and one dog in Group IV had a strongly positive PCR reaction on the injected muscle. One dog in
Group III had a positive PCR on the injected muscle
and liver. One control dog had a false positive reaction
in the first lymph node, probably due to a contamination
of the tissue harvest that was not reconfirmed when
the assay was repeated. All other PCR reactions with
dog tissues were negative. Both the positive and negative controls consistently yielded the respective predicted reactions.
Histopathological Evaluation of Tissues
Figure 1. Weights in dogs treated with plasmid-mediated GHRH therapy. The linear regression curves are
presented. For Group I (control), n = 4; for Groups II (0.2
mg of plasmid GHRH), III (0.6 mg of plasmid GHRH),
and IV (1 mg of plasmid GHRH), n = 8 per group to d
93. For Group I, n = 2, and for Groups II to IV, n = 4 per
group from d 93 to the end of the study. Group II, *P <
0.02, Group IV, **P < 0.001.
Samples of the selected organs listed in the Materials
and Methods section were examined and then histologically assessed by a board-certified veterinary pathologist. Not all samples could be collected from all dogs
due to time constraints during the necropsy process.
For some organ/tissue samples, extensive dissection
procedures were necessary, and the quality/quantity of
tissue obtained was inadequate for complete histological evaluation. As presented in Table 3, there were no
test article-related lesions identified by histopathologic
examination. Occasional observations, such as pitu-
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Effects of plasmid GHRH in dogs
Figure 2. Serum IGF-I levels in dogs treated with plasmid-mediated GHRH therapy. The linear regression
curves are presented. For Group I (control), n = 4; for
Groups II (0.2 mg of plasmid GHRH), III (0.6 mg of plasmid GHRH), and IV (1 mg of plasmid GHRH), n = 8 per
group to d 93. For Group I, n = 2, and for Groups II to
IV, n = 4 per group from d 93 to the end of the study.
Two-way ANOVA, *P < 0.001 for days after treatment
for treated vs. control animals.
itary cysts, are often seen in young dogs, and the frequency was not different among groups.
Discussion
Preclinical studies have suggested that anabolic hormones, such as GH, IGF-I, and IGFBP-3, may reverse
the catabolic state associated with cachexia, and thus
result in increases in weight, lean body mass, and work
output in cancer patients (Colao et al., 1999; Kotler,
2000) or patients with kidney failure (Ramirez et al.,
1990; Pasqualini et al., 1996). For instance, malnutrition and wasting are important determinants of morbidity and mortality in companion animals with chronic
renal failure on dialysis. Even pets with a relatively
modest degree of chronic renal insufficiency are characterized by reduced lean body mass, bone mineral content, and basal energy expenditure (O’Sullivan et al.,
2002).
Growth hormone synthesis and secretion from the
anterior pituitary are stimulated by GHRH, a hypothalamic peptide hormone (Muller et al., 1999). Low levels
of GHRH (about 100 pg/mL) are sufficient to induce GH
secretion (Esch et al., 1982; Corpas et al., 1993). Hence,
an alternate method to increase GH production and
release would be to administer GHRH. Unfortunately,
the short half-life of the GHRH peptide in plasma (<12
min) mandates frequent i.v. or s.c. injections of the
hormone to sustain its activity (Evans et al., 1985;
Thorner et al., 1986b), making recombinant GHRH
therapy impractical for chronic conditions.
Both GH and GHRH are currently administered therapeutically as recombinant proteins. Current knowledge about the interaction between GH and its receptor
suggests that the molecular heterogeneity of circulating
GH may have important implications in growth and
homeostasis. Although administration of exogenous
rGH produces anabolic effects in a variety of situations
(Lieberman et al., 1994; Molon-Noblot et al., 1998; Pichard et al., 1999), rGH therapy has disadvantages.
Growth hormone must be administered s.c. or i.m. as
frequently as once a day over the entire treatment period, and the nonphysiological hormonal peaks and
Table 2. Serum IGF-I concentrations in dogs treated
with plasmid-mediated GHRH therapya
Day
Baseline
Day 28
Day 56
Day 93
Day 120
Day 157
Day 180
Median
Mean
Group I
Control
Group II
0.2-mg dose
Group III
0.6-mg dose
Group IV
1-mg dose
13.51 ± 4.81b
16.30 ± 4.03
P = 0.35
14.86 ± 3.47
P = 0.49
14.20 ± 1.50
P = 0.38
17.33 ± 4.02
P = 0.44
22.31 ± 8.82
P = 0.08
23.41 ± 3.06
P = 0.24
16.3
17.42 ± 1.49
14.81 ± 2.28
17.21 ± 3.26
P = 0.26
21.83 ± 4.83
P = 0.04
17.18 ± 5.40
P = 0.33
26.38 ± 4.51
P = 0.02
22.11 ± 4.28
P = 0.06
23.03 ± 4.72
P = 0.07
21.83
20.37 ± 1.54
16.55 ± 4.58
26.22 ± 6.56
P = 0.08
18.77 ± 5.09
P = 0.11
19.34 ± 6.05
P = 0.33
25.03 ± 4.84
P = 0.04
26.09 ± 8.57
P = 0.12
21.38 ± 3.76
P = 0.06
21.38
21.91 ± 1.47
12.31 ± 3.04
17.98 ± 2.90
P = 0.0004
14.34 ± 1.30
P = 0.01
19.37 ± 3.80
P = 0.006
34.47 ± 4.52
P = 0.01
29.91 ± 6.58
P = 0.06
24.77 ± 6.50
P = 0.04
19.37
21.88 ± 3.1
a
The results are presented as means ± SEM. P-values vs. baseline weights are included for each group
and time point.
b
The IGF-I concentrations were measured at least eight different times throughout the study for each
individual animal; each sample was assayed in duplicate; assays were twice repeated. Group 1, n = 4; for
Groups II through IV, n = 8.
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Figure 3. Hemoglobin and PCV in dogs treated with
plasmid-mediated GHRH therapy. The results are presented as means ± SEM. A) Least squares means analysis
of hemoglobin for the dogs treated with 0.2 mg, Group
II, **P < 0.002; dogs treated with 0.6 mg, Group III, ***P
< 0.0001; and dogs treated with 1 mg, Group IV, vs. Group
I, controls, *P < 0.05. B) Least squares means analysis of
PCV for the dogs treated in Group II, **P < 0.01, dogs
treated in Group III, ***P < 0.0001, and dogs treated in
Group IV, vs. Group I, *P < 0.025. For Group I (control),
n = 4; for Groups II (0.2 mg of plasmid GHRH), III (0.6
mg of plasmid GHRH), and IV (1 mg of plasmid GHRH),
n = 8 per group to d 93. For Group I, n = 2, and for Groups
II to IV, n = 4 per group from d 93 to the end of the study.
troughs that follow such injections often result in impaired glucose tolerance and insulin resistance (Angelopoulos et al., 1998). Moreover, it is clear that biological
responses to exogenous rGH are not similar to physiological responses to the naturally occurring isoforms of
this protein through rapid negative feedback regulation
on pituitary release (Satozawa et al., 2000; Leung et
al., 2002). Numerous studies in humans, sheep, and
pigs showed that continuous infusion with recombinant
GHRH protein restores the normal GH pattern without
desensitizing GHRH receptors or depleting GH supplies
since this system is capable of feed-back regulation,
which is abolished in the GH therapies (Vance et al.,
1985; Dubreuil et al., 1990; Vance, 1990), thus avoiding
adverse effects (Thorner et al., 1986a; Abribat et al.,
1989).
Preliminary studies in small and large animal models
suggested that a single injection of a GHRH plasmid
into skeletal muscle will ensure physiologic GHRH expression for many months (Draghia-Akli et al., 1999;
Draghia-Akli et al., 2002b). In a phase I trial in dogs
with spontaneous malignancies, we administered a
GHRH-expressing plasmid once intramuscularly to severely debilitated, anemic dogs with naturally occurring tumors (Draghia-Akli et al., 2002a). Most (75%)
of the animals showed a physiological increase in serum
IGF-I concentration, which was associated with the correction of anemia and a significant increase in circulating lymphocytes. There were no discernible adverse effects associated with this therapy. Taken together,
these observations demonstrated the feasibility of
GHRH-plasmid mediated therapy to stimulate GH synthesis and release in large animals, leading to the correction of anemia and other catabolic processes associated with cachexia. Similar benefits may be achievable
in renal failure or geriatric companion animals. Nevertheless, because dogs from the previous study had numerous preexisting conditions and spontaneous malignancies, a clear conclusion about the possible adverse
effects of the therapy cannot be made. The present
study was designed to address the specific issue of longterm effects of GHRH plasmid-mediated therapy on
healthy dogs.
This new treatment modality is now practical in vivo
because of the significant enhancement of plasmid delivery obtained using electroporation. Electroporation
has been used successfully to enhance plasmid uptake
by tumor cells after injection (Matsubara et al., 2001;
Lucas et al., 2002) and in various organs to deliver
therapeutic genes that encode for a variety of hormones,
cytokines, or enzymes in mice (Lucas et al., 2001; Vilquin et al., 2001; Lesbordes et al., 2002), rats (Terada
et al., 2001; Yasui et al., 2001), and dogs (Fewell et
al., 2001) without major adverse effects linked to the
procedure itself. Intramuscular injection of plasmid followed by electroporation has been used successfully in
ruminants for vaccination purposes (Babiuk et al.,
2003; Tollefsen et al., 2003). The dogs that received
the plasmid injection followed by electroporation had
minimal and transitory erythema at the injection point,
but no permanent damage to skin or muscle.
By PCR analysis, we were unable to demonstrate
the presence of plasmid in the muscles of all treated
animals, even when its biological activity was obvious.
The entire injected muscle was collected and processed,
and the plasmid dose was relatively low. The limit of
detection of the assay is such that we could detect the
presence of approximately 5,000 molecules or 0.2 pg of
plasmid per gram of tissue (Manam et al., 2000). It is
thus possible we were not able to detect it under the
assay parameters. Nevertheless, previous biodistribution studies using PCR showed similar results. In mini
pigs, the plasmid was detected in the treatment sites
and also in the inguinal lymph nodes on d 2. At d 57,
plasmid was present in the treatment sites only, and by
d 141, it seemed to have cleared, even if the transgene
product was still present (Haworth and Pilling, 2000;
Pilling et al., 2002).
Effects of plasmid GHRH in dogs
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Table 3. The combined results (from both d 93 and 180), from males (M) and females
(F) from the microscopic examination of all available samples
Brain
Peripheral nerve
Hypothalamus
Pituitary
Cyst
Retina
Optic nerve
Adrenal cortex
Adrenal medulla
Kidney
Pelvis-lymphocytic infiltrate
Lymphocytic infiltrate
Chronic inflammation
Mineralization
Testis
Prostate
Chronic inflammation
Uterus/cervix
Ovaries
Lung
Chronic inflammation
Stomach
Small intestine
Cystic mucosal gland
Large intestine
Spleen
Pancreas
Liver
Central cytoplasmid vacuolization
Acute inflammation
Gall bladder
Bone
Bone marrow
Heart
Skin
Chronic inflammation
First lymph node
Pigmented
Hemorrhage
Skeletal muscle
Injection site
The weight gain associated with increased production
of GHRH is in agreement with other studies that utilized recombinant porcine GH in dogs. In one of these
studies, recombinant porcine GH was administered for
14 wk in dogs (Prahalada et al., 1998). Porcine GH
caused increased BW gain in mid- and high-dose groups
(2.8 and 4.7 kg, respectively) compared with 0.4 and
0.8 kg in the control and low-dose groups, respectively.
During our 6-mo study, GHRH-treated animal weights
increased by up to 1.6 kg (controls had a slight weight
loss). At necropsy, the body composition was similar
among groups.
Insulin-like growth factor-I levels were significantly
increased in treated dogs, but the increase was not
dose dependent, and levels were identical between the
groups that received 0.6 and 1 mg of plasmid. As the
system maintains feedback and regulation, this is not
an unexpected finding. In a study on male Beagles that
received GHRH[1-29]NH2 (25 ␮g/kg, subcutaneously,
twice daily) for 57 d, it was shown that GHRH injections
Normal (M/F)
Normal (M/F)
Normal (M/F)
1 M in Groups I, II, III and IV
1 F in Group I, 2 F in Groups III and IV
Normal (M/F)
Normal (M/F)
Normal (M/F)
Normal (M/F)
2 M in Group III, 1 F in Group II
1 F in Group III
1 F in Group II
1 F in Group IV
Normal (M)
1 M in Groups I, II, and IV
Normal (F)
Normal (F)
1 F in Group III
Normal (M/F)
1 M in Group II
Normal (M/F)
Normal (M/F)
Normal (M/F)
1M in Group II and IV, 1 F in Group III
1 F in Group III
Normal (M/F)
Normal (M/F)
Normal (M/F)
Normal (M/F)
1F in Group III
3 M in Group III, 1 M in Group IV, 1 F in Group II
1 F in Group IV
Normal (M/F)
Normal (M/F)
produced a significant increase in GH release following
each injection, and an increase in GH response over
time. The concentration of IGF-I increased for 5 wk and
then reached a plateau (Dubreuil et al., 1996) and was
constant for the remainder of the study. The short stature of some dog breeds is associated with low serum
levels of IGF-I (Guler et al., 1989). When these animals
are given recombinant IGF-I, serum levels of IGF-I increase to a certain value and stay constant thereafter.
Also, radial length and BW are not increased to a
greater extent in the IGF-I-infused dogs than in controls. These facts are indicators of a tight regulation in
the GHRH axis in dogs.
In our current study, glucose and insulin levels were
monitored and found to be normal in all circumstances.
Dogs were healthy and playful and did not show signs
of lameness. It has been suggested that adverse effects,
including insulin resistance, may result from the fact
that the basal GH levels are raised and the natural
GH episodic pulses are abolished with exogenous GH.
2308
Draghia-Akli et al.
Numerous studies have shown that continuous infusion
with GHRH restores normal GH pulsatile pattern, with
no desensitization of GHRH receptors or depletion of
GH supplies. At the same time, this system is capable
of feedback. Virtually no side effects have been reported
for GHRH therapies (Thorner et al., 1986b). Thus,
GHRH therapy may be more physiological than GH
therapy.
An entire class of GH-releasing peptides is also used
in clinics to stimulate GH and IGF-I in humans and
dogs. Hexarelin, a potent and well-studied GH-releasing peptide, is capable of causing profound GH release
in normal individuals. The GH response to hexarelin
in humans becomes appreciably attenuated following
longterm administration (attenuation that is partial
and reversible) and could seriously limit the potential
longterm therapeutic use of hexarelin and similar
agents (Rahim and Shalet, 1998). Growth hormonereleasing peptides similar to corticotropin-releasing
hormone also possess acute ACTH- and cortisol-releasing activity, although the mechanisms underlying the
stimulatory effect of GH-releasing peptides on the hypothalamo-pituitary-adrenal axis are still unclear (Rigamonti et al., 2002). More recent studies in humans have
demonstrated acute increases in ACTH (Ghigo et al.,
1999), cortisol, and prolactin (Svensson and Bengtsson,
1999) after GH-releasing peptide administration (hexarelin, MK-0677) (Jacks et al., 1996; Schleim et al.,
1999). The potential adverse effects of repeated episodes of transient (even minor) hyperprolactinemia and
hypercortisolemia during long-term therapy with GHreleasing peptides and similar agents have raised concern and require further study (Rahim et al., 1999). In
our study, ACTH levels were normal throughout and
not significantly different among groups.
The beneficial effect on hemoglobin level and red cell
number was not anticipated, because GH administration to normal Beagle dogs over 14 wk of age produced
a dose-related normochromic, normocytic, and nonregenerative anemia (Prahalada et al., 1998). Growth
hormone-releasing hormone stimulates IGF-I, and
there are data to suggest that IGF-I, rather than erythropoietin, is the primary mediator of erythropoiesis during catabolic states and in uremic patients (Urena et
al., 1992) and can induce a proportional increase in
body mass and oxygen transport capacity (Kurtz et al.,
1990). The increase in hemoglobin and hematocrit confirms the in vivo erythropoietic growth-promoting effects of GH that are also observed during GH treatment
in GH-deficient children or adults (Christ et al., 1997;
Valerio et al., 1997). No dogs showed evidence of polycythemia following plasmid administration, indicating
that this approach to stimulating the GHRH/GH/IGF-I
axis respects normal physiological levels of hemoglobin
and red blood cell synthesis.
Implications
The ability of plasmid-mediated growth hormone-releasing hormone supplementation to induce growth
hormone production and release through natural physiological mechanisms may be a useful single-dose alternative to multiple growth hormone injections. The absence of adverse effects as shown in this study indicates
that this treatment strategy can produce increased levels of growth hormone and associated hormones without adversely affecting homeostasis. This treatment
methodology represents a significant advance in the
ability of veterinarians to treat different conditions in
companion or production animals.
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