Double-blinded, Placebo-controlled Plasmid GHRH Trial for Cancer-associated Anemia in Dogs

Abstract

The use of growth hormone releasing hormone (GHRH) plasmid-based therapy to treat companion dogs with spontaneous malignancies and anemia receiving a cancer-specific treatment was examined in a double-blinded, placebo-controlled trial. The dogs (age 10.5 +/- 2.5 years, weight 24.9 +/- 12.9 kg) received a single 0.35 mg dose of plasmid or placebo intramuscularly (i.m.), followed by electroporation (EP), and were analyzed for up to 120 days. The response rate was defined as > or = 5% increase above the nadir in the red blood cell (RBC), hemoglobin (Hb), and hematocrit (Ht) levels. Plasmid-treated dogs had at least a 7% increase in all three parameters. The initial response rates for the plasmid-treated dogs were 40.6 and 35.5%, respectively on days 40 and 60, which increased to 54.2% on day 90. Although the response rate reduced to 47.1% by day 120, it was still 22.1% higher than in the control dogs. Post-hoc analysis of the GHRH-treated group showed that responder dogs survived 84% longer, 178 +/- 26 days post-treatment, while nonresponders and controls survived for 95 +/- 16 and 97 +/- 31 days post-treatment, respectively. The quality of life, defined by 10 different parameters, dramatically improved with treatment. Overall, the possibility of a GHRH plasmid-based therapy for anemia in cancer-afflicted subjects is important enough to deserve further investigation.

Full text

© The American Society of Gene Therapy
original article
862 www.moleculartherapy.org vol. 16 no. 5, 862–870 may 2008
Double-blinded, Placebo-controlled Plasmid
GHRH Trial for Cancer-associated Anemia in Dogs
Angela M Bodles-Brakhop
1
, Patricia A Brown
2
, Melissa A Pope
1
and Ruxandra Draghia-Akli
1
1
VGX Pharmaceuticals, The Woodlands, Texas, USA;
2
VGX Animal Health, The Woodlands, Texas, USA
The use of growth hormone releasing hormone (GHRH)
plasmid–based therapy to treat companion dogs with
spontaneous malignancies and anemia receiving a cancer-
specific treatment was examined in a double-blinded,
placebo-controlled trial. The dogs (age 10.5 ± 2.5 years,
weight 24.9 ± 12.9 kg) received a single 0.35 mg dose of
plasmid or placebo intramuscularly (IM), followed by elec-
troporation (EP), and were analyzed for up to 120 days.
The response rate was defined as ≥5% increase above the
nadir in the red blood cell (RBC), hemoglobin (Hb), and
hematocrit (Ht) levels. Plasmid-treated dogs had at least
a 7% increase in all three parameters. The initial response
rates for the plasmid-treated dogs were 40.6 and 35.5%,
respectively on days 40 and 60, which increased to 54.2%
on day 90. Although the response rate reduced to 47.1% by
day 120, it was still 22.1% higher than in the control dogs.
Post-hoc analysis of the GHRH-treated group showed that
responder dogs survived 84% longer, 178 ± 26 days post-
treatment, while nonresponders and controls survived for
95 ± 16 and 97 ± 31 days post-treatment, respectively.
The quality of life, defined by 10 different parameters,
dramatically improved with treatment. Overall, the pos-
sibility of a GHRH plasmid–based therapy for anemia in
cancer-afflicted subjects is important enough to deserve
further investigation.
Received 10 October 2007; accepted 31 January 2008; published online
18 March 2008. doi:10.1038/mt.2008.31
INTRODUCTION
Cachexia, a common illness aecting up to 5 million people in
the United States alone, and its associated disorders are common
complications of cancer, aging, acquired immunodeciency syn-
drome, chronic kidney, or heart failure. is disease can oen lead
to premature death.
1
Prevalent cancer-associated conditions such
as fatigue may be induced by the cancer itself or by cancer treat-
ments (chemotherapy), with anemia being one of its leading causes
in patients.
2
e majority of cancer patients experience at least a
mild form of anemia especially aer chemotherapy treatment, and
erythropoietin (EPO) is the treatment of choice. However, the use
of EPO is currently under question as several reports suggest that
it may actually exert a growth-promoting eect on cancer cells.
3,4
is information, taken together with the fact that most therapies
are eective only in a minority of patients, warrants a dierent
approach and further studies.
Growth hormone releasing hormone (GHRH), GH, and
insulin-like growth factor-I (IGF-I) are molecules required for
the normal growth and development of animals and humans that
decline with aging and due to certain pathologic circumstances,
such as cachexia. It has also been suggested that overexpression of
GHRH/GH/IGF-I may be correlated with tumorigenesis,
5
while
other conicting results suggest that there is no direct cause–eect
connection.
6,7
Studies have shown that some tumors may secrete
GHRH,
8
and therefore an overproduction of GHRH may not be a
contributing factor but a consequential one. Endocrine or exog-
enous supplemented GH has been shown not to be involved in
tumorigenesis, while autocrine GH produced by malignant cells
induces proliferation.
9
Recent research shows that complications
of cancer may be reversed or prevented as demonstrated in the
treatment of mice with plasmid-based GHRH delivered by elec-
troporation (EP) using implanted Lewis lung adenocarcinoma
cells to create tumor-bearing immunocompetent mice and nude
mice.
10,11
In both cases, tumor growth decreased and serum IGF-I
levels (a measure of GHRH activity) increased in the GHRH
plasmid–treated animals compared to controls; cachexia was pre-
vented and a nonspecic immune stimulation was noted.
Previously, we have reported that optimization of the EP
method can result in signicant overall improvement in health
in large mammals such as pigs, dogs, or cows with a compara-
tively small one-time dose of plasmid-GHRH.
12
We examined a
group of companion dogs that had been diagnosed with naturally
occurring tumors (in order to determine the eectiveness of plas-
mid-based GHRH therapy delivered by EP), in treating anemia
and cachexia and their related disorders in cancer and aging,
13
as
well as the length of expression aer a single plasmid administra-
tion in the disease model;
14
in one of these studies, encouraging
eects of the therapy were seen for up to 440 days aer one single
plasmid administration. A toxicology study on young healthy
laboratory dogs
15
was also performed. In normal healthy dogs,
plasmid-GHRH treatment resulted in increased serum IGF-I,
as well as signicant increases in the number of red blood cells
(RBCs), hematocrit (Ht) and hemoglobin (Hb) levels, and weight
gain compared to control and baseline measurements.
To further these ndings we carried out a randomized, double
blind placebo-controlled study to evaluate the ecacy of plas-
mid-GHRH as being potentially therapeutic in the management
Correspondence: Ruxandra Draghia-Akli, VGX Pharmaceuticals, Inc., 2700 Research Forest Drive, Suite 180, The Woodlands, Texas 77381, USA.
E-mail: rdraghia@vgxp.com

Molecular erapy vol. 16 no. 5 may 2008 863
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
of cancer-related anemia in dogs. Based on the preliminary
data collected from pilot studies discussed above
13,14
successful
treatment was dened as a ≥5% increase above baseline levels
of all RBC, Ht, and Hb aer day 60 on the study. is study
with 55 dogs shows that GHRH therapy might be an eective
and ecient method for reversing cancer-related anemia in
patients, while increasing their chance of survival during specic
cancer therapy.
RESULTS
Study population
All dogs enrolled were companion dogs diagnosed with sponta-
neously occurring malignancies. e 55 dogs enrolled were ran-
domized in a 4:1 ratio for GHRH plasmid (GHRH-T) (Figure 1)
to placebo treatment (P-T) (Table 1). e high plasmid-to-pla-
cebo ratio was decided in consultation with the attending vet-
erinarians, in order to give more dogs the possibility of a better
quality of life based on preliminary studies.
13,14
Table 2 shows
the number and percentage of dogs with the various histopatho-
logical types that were included in the study, the evaluated cases,
and the number and percentage of dogs that were not included
in the study analysis or could not be evaluated due to various
reasons such as death, loss to follow-up, or noncompliance.
e principal explanation for study discontinuation was death
from natural causes, or more oen due to euthanasia upon the
owner’s request. Of the 55 dogs, 45 received chemotherapy only,
9 radiotherapy only, 5 a combination regimen of chemotherapy/
radiotherapy; 1 dog did not receive cancer-specic therapy.
Analysis of treatment success
All dogs that could be evaluated were assessed for treatment success
or treatment failure. Animals that died prior to the minimum evalu-
ation period were not evaluated. Analysis of treatment success was
determined on days 60, 90, and 120 for dogs treated with GHRH-T
compared to P-T dogs (Figure 2) and categorized as either a
responder or nonresponder. More of the P-T dogs were catego-
rized as treatment failures than the GHRH-T dogs (see Table 3).
A post-hoc analysis following the un-blinding of the study groups
compared responders (GHRH-R) and nonresponders (GHRH-
NR) in the GHRH plasmid–treated group to assess dierences
in survival, hematological, and hormonal parameters. A total of
15 dogs were found to be GHRH-R, with a 5% increase in all RBC,
Ht, and Hb levels determined at least at two consecutive time
points of days 60, 90, and 120.
Analysis of survival
Of the 43 dogs that received GHRH-T, 23.26% died (n = 10)
before day 40, whereas in the case of the P-T dogs (n = 12) >40%
died (41.67%, n = 5) before day 40 (P < 0.001). Seventeen of the
thirty-two evaluable dogs that were enrolled in the study and
received GHRH plasmid survived until at least day 120 with four
of these dogs surviving beyond day 300 postinjection, and two
dogs surviving to day 390 post-treatment. Only four of the P-T
dogs survived until day 120, with two of them surviving until
day 210. On average, GHRH-T dogs survived for 130 ± 15 days
aer treatment, while controls survived for 97 ± 31 days (P =
0.06, due to the high intra-group variability for placebo controls)
(Figure 3). GHRH-T dogs that came with a hemangiosarcoma
diagnosis survived on average for 105 ± 22.5 days (P = 0.31 ver-
sus P-T), while dogs diagnosed with any of the other cancer types
survived for 141.8 ± 19 days (
P < 0.05 versus P-T). Most impor-
tant, GHRH-R survived on average 84% longer than did controls,
i.e., for 178 ± 26 days, while GHRH-NR survived for 95 ± 16 days
post-treatment (
P < 0.0026 R versus NR), similarly to P-T.
Hematopoietic analysis
Analysis of the individual results for RBC count (normal for dogs:
5.5–8.5 × 10
6
/mm
3
, study population 4.6 ± 0.15 × 10
6
/mm
3
at
nadir), Hb (normal for dogs: 12–18 g/dl, study population 10.8 ±
0.35 g/dl at nadir), and Ht (normal for dogs: 37–55%, study popula
-
tion 32.7 ± 0.14% at nadir) were also carried out. All hematological
parameters in GHRH-T dogs showed a distinct increasing trend, in
particular aer day 60, which was not seen in P-T. All three param-
eters were analyzed against their nadir (the lowest value on day 0 or
day 20) and tabulated as the percentage change in
Table 3 (P < 0.05,
Wilcoxon signed-rank test). Nevertheless, GHRH-R showed sig-
nicant increases over both P-T and GHRH-NR, while GHRH-NR
were not dierent from P-T (Figure 4); these increases occurred
earlier (as early as day 20), and were maintained throughout the
120-day study. All other hematological parameters analyzed, such
as white blood cells, neutrophils, lymphocytes, monocytes, and
basophils, fell within the normal ranges, with no signicant dier-
ences detected for either GHRH-T or P-T dogs.
Sac I (1)
HindIII (653)
XhoI (1,299)
KpnI (1,318)
LacZ
pUC ori
NEO (R)
PNEO promoter
pAV0125
3,534 bp
NEO
c5-12 synthetic promoter
Xbal (343)
BamHI (355)
GH 5′ UTR
hGH polyA
Modified porcine GHRH (HV)
Figure 1 Plasmid map of pAV0125 HV-GHRH. The plasmid pSPc5-
12-GHRH contains a muscle-specific SPc5-12 synthetic promoter. The
synthetic HV-GHRH complementary DNA (cDNA) encoding for a GHRH
analog with an extended half-life is depicted. bp, base pair GHRH,
growth hormone releasing hormone; UTR, untranslated region.
Table 1 Summary of gender and treatment assignment for evaluated
and nonevaluated dogs
Gender
Evaluated cases Nonevaluated cases
TotalGHRH-T
Placebo
treatment (P-T) GHRH-T P-T
N % N % N % N % N %
Female 12 37.5 2 28.57 3 27.27 3 60 20 36.36
Male 20 62.5 5 71.43 8 72.73 2 40 35 63.64
Total 32 100 7 100 11 100 5 100 55 100
Abbreviations: GHRH-T, growth hormone releasing hormone treatment; P-T,
placebo treatment.

864 www.moleculartherapy.org vol. 16 no. 5 may 2008
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
Biochemical analysis
Chlorine, potassium, and sodium levels, indicators of hydra-
tion, were all within normal ranges and statistically were not sig-
nicantly changed from predosing levels. Total circulating protein
levels decreased in the GHRH-T dogs (minus hemangiosarcomas)
compared to baseline, reaching signicance on day 120 (6.60 ±
0.10 versus 6.16 ± 0.17 respectively, P = 0.03); no change was seen
in P-T dogs. Glucose levels were within the normal ranges, and
decreased in GHRH-T dogs on day 60 and day 90 compared to
predosing (84.31 ± 4.16 versus 94.76 ± 3.20, P = 0.04; 84.96 ± 4.58
versus 94.76 ± 3.20, P = 0.04, respectively).
Weight and quality of life assessment
ere were no reports of adverse eects from the administration
of the plasmid-mediated GHRH-T or P-T from either the investi-
gator or the owners of the dogs. Weight increased during the study
Table 2 Summary of the histopathological tumor type, overall and by treatment assignment
Histopathological type
Evaluated cases Nonevaluated cases
GHRH-T P-T Total GHRH-T P-T Total
N
%
N
%
N
%
N
%
N
%
N
%
Hemangiosarcomas 13 40.63 1 14.29 14 35.90 3 27.27 1 20 4 25
Carcinoma 6 18.75 2 28.57 8 20.51 2 18.18 1 20 3 18.75
Sarcoma 4 12.5 2 28.57 5 12.82 2 18.18 1 20 3 18.75
Adenocarcinoma 4 12.5 1 14.29 5 12.82 1 9.09 0 0 1 6.25
Mast cell tumor 2 6.25 1 14.29 3 7.69 3 27.27 0 0 3 18.75
Melanoma 2 6.25 1 14.29 3 7.69 0 0 1 20 1 6.25
Cutaneous extramedullary
plasmacytoma
1 3.13 0 0 1 2.56 0 0 0 0 0 0
Malignant brous histocytoma 0 0 0 0 0 0 0 0 1 20 1 6.25
Total 32 100 7 7 39 100 11 100 5 100 16 100
Abbreviations: GHRH-T, growth hormone releasing hormone treatment; P-T, placebo treatment.
100
80
GHRH-T
P-T
60
n = 11
35.5%
n = 11
42.9%
n = 13
54.2%
n = 1
20.0%
n = 8
47.1%
n = 1
25.0%
40
Response rate (%)
20
10
Day 60 Day 90 Day 120
Figure 2 Analysis of treatment success. Percentage response rate for
GHRH-T and P-T dogs at day 60, 90, and 120 where there was at least a
5% increase in red blood cell count, hemoglobin levels, and hematocrit
levels. The percentage response rate and the number of dogs at each
time point for GHRH-T and P-T dogs are shown. GHRH-T, growth hor-
mone releasing hormone treatment; P-T, placebo treatment.
Table 3 Summary of percentage change from nadir in red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (Ht)
% Change in RBC % Change in Hb % Change in Ht
Visit Treatment Mean ± SD
P value
Mean ± SD
P value
Mean ± SD
P value
Day 40 GHRH-T (n = 32) 7.3 ± 18.7 0.0178* 8.0 ± 15.0 0.0016* 7.3 ± 18.9 0.0835
P-T (n = 7) 15.2 ± 26.3 0.1563 16.4 ± 27.7 0.2969 11.0 ± 21.5 0.2188
Day 60 GHRH-T (n = 31) 8.6 ± 19.1 0.0344* 9.0 ± 16.2 0.0045* 9.5 ± 17.3 0.0098*
P-T (n = 7) 19.4 ± 25.6 0.1563 15.8 ± 24.8 0.3125 10.1 ± 21.6 0.3125
Day 90 GHRH-T (n = 24) 13.5 ± 22.4 0.0028* 12.2 ± 16.4 0.0009* 8.2 ± 20.1 0.0156*
P-T (n = 5) 12.5 ± 29.0 0.4375 9.8 ± 30.8 0.8125 4.8 ± 20.4 1.000
Day 120 GHRH-T (n = 17) 8.2 ± 19.3 0.1454 11.0 ± 18.8 0.0174* 8.1 ± 13.8 0.0214*
P-T (n = 4) 23.0 ± 38.2 0.2500 21.0 ± 44.6 0.6250 11.5 ± 33.9 0.8750
Abbreviation: GHRH-T, growth hormone releasing hormone treatment; P-T, placebo treatment.
Mean percentage change from nadir for RBC, Hb, and Ht at days 40, 60, 90, and 120 for all evaluated dogs. Statistically significant changes were determined using
the Wilcoxon signed-rank test with a P value of < 0.05 (bold and*). P-T dogs experienced a decrease in RBC, Ht, and Hb from qualification to day 20, and a trend to
reversal to baseline thereafter, while values for the GHRH-T dogs represent increases versus baseline.
Figure 3 Percent survival of treated dogs. Analysis of survival was
determined using the Kaplan–Meier method. Percent survival for GHRH-T,
GHRH-R, GHRH-NR, and placebo treatment (P-T) dogs is shown over the
time points indicated. GHRH, growth hormone releasing hormone.
120
P-T
GHRH-T
GHRH-R
GHRH-NR
100
Percent survival (%)
80
60
40
20
0
Day 0 Day 20 Day 40 Day 60 Day 100 Day 120

Molecular erapy vol. 16 no. 5 may 2008 865
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
evaluation in GHRH-T dogs by 11%, while it decreased by 16.7%
in P-T, but the change was not statistically signicant due to intra-
group variability (P = 0.11).
All owners were requested to complete a questionnaire at each
visit to assess their dog’s quality of life. e investigator’s assess-
ment of performance status was determined using a modied
Karnofsky performance scale for veterinary use. Examination
of the change from predosing indicated that many parameters
assessed improved for the GHRH-T dogs while the P-T dogs
tended to worsen (Table 4). Due to the small number of P-T dogs,
no statistical signicance was noted within this group; nevertheless,
even with a reduced number of animals in the P-T group, some
parameters were signicant when comparing the GHRH-T with
the P-T group (Table 4).
IGF-I levels
An increase in IGF-I level compared to baseline is considered an
adequate measure of GHRH activity;
16
this surrogate assay is also
preferred as the GHRH assay involves a step of column purication
that introduces a large element of variability. In this study, we show
that IGF-I levels are increased in GHRH-T dogs. When compared
to baseline, IGF-I levels increased on day 20 by 34.15% ± 13.96 but
did not reach statistical signicance (48.34 ± 7.22 versus 64.84 ±
13.96 ng/ml,
P = 0.22). On day 40 the percentage change in IGF-I
was 51.15% ± 15.01 (48.34 ± 7.22 versus 73.07 ± 15.01 ng/ml,
P = 0.06), at day 60, 55.76% ± 12.06 (48.34±7.22 versus 75.30 ±
12.06 ng/ml,
P = 0.02) and on day 90, 52.50% ± 21.77 (48.34 ±
7.22 versus 73.72 ± 21.77 ng/ml,
P = 0.08). ere were no signi-
cant percentage changes in IGF-I levels in the P-T animals com-
pared to baseline. All values fell within the normal range for breed
and weight.
17
IGF-I levels in GHRH-T dogs positively correlated
with body weight at predosing (r = 0.39, P = 0.002), with a slightly
stronger correlation on day 20 (r = 0.45, P = 0.007), day 40 (r =
0.57, P = 0.001), day 60 (r = 0.49, P = 0.0008), and day 90 (r = 0.55,
P = 0.022). GHRH-NR had on average 25% higher IGF-I levels at
baseline compared to GHRH-R (54.7 ± 12.58 ng/ml versus 44.2 ±
9.37 ng/ml,
P = 0.25 due to intra-group variability), and no signi-
cant change in IGF-I values throughout the study.
DISCUSSION
e development of cancer cachexia is perhaps the most com-
mon manifestation of advanced malignant diseases.
18
e
abnormalities associated with the condition include progres-
sive weight loss, anorexia, asthenia, and anemia. e degree of
cachexia is inversely correlated with the survival time of the
patient and always implies a poor prognosis. Ideally, a therapeu-
tic regime targeting cancer-related complications would address
all these issues, while easily administered and with long-term
eects. e discovery of plasmid-based therapy in combination
with EP is such a method
19,20
and may be a treatment option
for cancer-associated cachexia and anemia. EP is a proven safe
and ecient method of delivering DNA in vivo, providing a
nonviral method and is an important alternative technique for
the gene therapy eld.
21–23
Intramuscular (IM) injection with
EP, compared to IM injection alone, results in higher plasmid
uptake and expression rate of the transgene product.
24
We have
successfully used optimized muscle-specic plasmids to obtain
long-term production of therapeutic proteins (in particular
GHRH) aer a single IM administration in conjunction with
EP in a multitude of large animal species (reviewed in ref. 25).
Previous reports show that EP has also been used to treat cuta-
neous and subcutaneous tumors with bleomycin in Phase I-III
human clinical trials.
26,27
Currently, several Phase I clinical trials
are underway to study the eect of plasmid injection and EP in
humans (as reported on http://www.clinicaltrials.gov).
e GHRH/GH/IGF-I axis has been implicated in reversing
the catabolic state associated with cachexia, resulting in increased
weight and general overall improvement in quality of life of
6
a
b
14
13
12
Hemoglobin (g/dL)
c
Hematocrit (%)
11
10
9
42
38
34
30
26
5
Red blood cells(10
6
/mm
3
)
4
3
Day 0 Day 20 Day 40 Day 60 Day 100 Day 120
Day 0 Day 20 Day 40 Day 60 Day 100 Day 120
Day 0 Day 20 Day 40 Day 60 Day 100 Day 120
*
*
*
P-T
GHRH-T
GHRH-R
GHRH-NR
P-T
GHRH-T
GHRH-R
GHRH-NR
P-T
GHRH-T
GHRH-R
GHRH-NR
Figure 4 Analysis of hematological parameters in GHRH-T, GHRH-R,
GHRH-NR, and placebo treatment (P-T) dogs. (a) Red blood cells,
(b) hemoglobin (Hb), and (c) hematocrit (Ht). GHRH-R had significantly
higher RBC, Ht, and Hb levels (*P < 0.01 and lower) at every time point
after qualification compared to P-T or GHRH-NR.
120
100
IGF-I (ng/ml)
80
60
40
20
0
Day 0
P-T
GHRH-T
GHRH-NR
GHRH-R
−27.0176
34.12726
6.538116
66.05894
−6.29042
34.44362
12.9354
56.30617
Percent change versus baseline
−10.1422
50.93663
18.38617
77.55961
0
0
−26.9423
18.57597
Day 20 Day 40 Day 60 Day 90
Day 20/0 Day 40/0 Day 60/0 Day 90/0
P-T
GHRH-T
GHRH-R
GHRH-NR
Figure 5 Serum insulin-like growth factor-I (IGF-I) levels in GHRH-T,
GHRH-R, GHRH-NR and placebo treatment dogs. Results are pre-
sented as the average ± SEM. All samples were assayed in duplicate.

866 www.moleculartherapy.org vol. 16 no. 5 may 2008
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
Table 4 Quality of life assessment
Day 0 Day 20 Day 40 Day 60 Day 90 Day 120
Overall score
P-T Avg. score 3.0 ± 0.4 2.9 ± 0.1 3.1 ± 0.2 3.1 ± 0.1 3.2 ± 0.2 3.5 ± 0.5
% Change –3.7% 4.2% 4.8% 6.7% 16.7%
P value versus day 0 0.40 0.37 0.37 0.31 0.25
GHRH-T
a
Avg. score 2.7 ± 0.1 3.3 ± 0.1 3.5 ± 0.1 3.2 ± 0.1 3.3 ± 0.2 3.2 ± 0.2
% Change 21.1% 28.9% 18.6% 20.7% 18.6%
P value versus day 0 0.0002 0.00004 0.004 0.02 0.05
Weight score
P-T Avg. score 3.0 ± 0.4 2.8 ± 0.3 3.3 ± 0.4 2.9 ± 0.3 2.8 ± 0.4 2.5 ± 0.3
% Change –8.3% 8.3% –4.8% –6.7% –16.7%
P value versus day 0 0.32 0.34 0.23 0.50 0.32
GHRH-T Avg. score 2.8 ± 0.2 3.0 ± 0.2 2.8 ± 0.2 3.1 ± 0.2 3.2 ± 0.2 3.1 ± 0.3
% Change 6.7% 0.0% 11.6% 14.4% 11.1%
P value versus day 0 0.22 0.50 0.16 0.15 0.11
Activity score
P-T Avg. score 2.6 ± 0.3 2.8 ± 0.3 2.9 ± 0.3 3.0 ± 0.3 3.4 ± 0.5 3.3 ± 0.8
% Change 6.9% 11.8% 16.7% 32.2% 26.4%
P value versus day 0 0.37 0.30 0.15 0.19 0.32
GHRH-T Avg. score 2.6 ± 0.1 3.3 ± 0.1 3.2 ± 0.2 3.3 ± 0.1 3.2 ± 0.2 3.3 ± 0.2
% Change 24.1% 21.8% 23.9% 21.7% 24.9%
P value versus day 0 0.0004 0.009 0.002 0.04 0.02
Exercise score
P-T Avg. score 2.4 ± 0.3 2.9 ± 0.3 3.0 ± 0.2 3 ± 0.3 3.2 ± 0.4 3.3 ± 0.5
% Change 21.6% 26.3% 26.3% 34.7% 36.8%
P value versus day 0 0.11 0.10 0.15 0.24 0.30
GHRH-T Avg. score 2.6 ± 0.2 3.2 ± 0.1 3.3 ± 0.1 3.3 ± 0.1 3.2 ± 0.2 3.4 ± 0.2
% Change 25.0% 28.5% 29.6% 26.0% 31.7%
P value versus day 0 0.001 0.0008 0.001 0.015 0.003
Alertness score
P-T Avg. score 2.9 ± 0.3 3.1 ± 0.1 3.1 ± 0.2 3.3 ± 0.2 3.2 ± 0.2 2.8 ± 0.3
% Change 9.4% 9.4% 15.0% 12.0% –3.8%
P value versus day 0 0.09 0.30 0.23 0.35 0.20
GHRH-T Avg. score 2.9 ± 0.1 3.2 ± 0.1 3.4 ± 0.1 3.3 ± 0.1 3.3 ± 0.1 3.4 ± 0.1
% Change 0.1 14.4% 13.4% 12.4% 12.4% 14.1%
P value versus day 0 0.009 0.0004 0.003 0.05 0.0008
Appetite score
P-T Avg. score 3.1 ± 0.5 2.8 ± 0.2 2.9 ± 0.3 2.8 ± 0.3 2.6 ± 0.2 3.0 ± 0.0
% Change –12.5% –9.1% –9.8% –17.3% –4.5%
P value versus day 0 0.24 0.33 0.24 0.24 0.50
GHRH-T Avg. score 2.6 ± 0.1 3.2 ± 0.1 3.5 ± 0.1 3.4 ± 0.1 3.3 ± 0.2 3.4 ± 0.2
% Change 21.1% 32.8% 27.2% 22.8% 27.5%
P value versus day 0 0.003 4.7 × 10
6
0.0004 0.001 0.0008
irst score
P-T Avg. score 3.1 ± 0.3 2.8 ± 0.2 3.1 ± 0.1 3.0 ± 0.0 2.8 ± 0.2 3.0 ± 0.0
% Change –12.5% 0.0% –4.6% –10.9% –4.5%
P value versus day 0 0.18 0.50 0.35 0.50 0.20
Table 4 continued on next page

Molecular erapy vol. 16 no. 5 may 2008 867
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
cancer patients.
28,29
GH production and secretion is stimulated by
low levels of GHRH.
30
erefore, the use of an expression plas-
mid encoding GHRH to restore normal levels of GH and IGF-I
and exert direct eects on certain cell types is a good alternative
method to the daily administration of recombinant peptides
which are not eective at reproducing natural physiological and
biological patterns.
31
Previous studies have shown that the single
IM injection of a muscle-specic GHRH-plasmid followed by EP
will result in long-term expression
32
and the treatment of severely
debilitated anemic dogs with naturally occurring tumors resulted
in a physiological increase in serum IGF-I;
13
it is of note that using
this therapeutic approach, the IGF-I levels are not increased above
the physiological level for normal healthy dogs. erefore, in this
study we aimed to show that IM treatment with GHRH-plasmid
followed by EP in dogs with cancer could improve cachexia-
associated anemia and improve the quality of life. Importantly, in
our previous studies we could not rigorously assess survival aer
therapy, nor compare GHRH-T dogs with P-T dogs, both issues
addressed in the current research study design. A post-hoc analy-
sis following the un-blinding of the study groups demonstrated
signicant dierences in survival, hematological parameters,
and serum IGF-I between responders and nonresponders in the
GHRH-T group. Of note, IGF-I levels in GHRH-NR were 25%
higher at baseline as compared to GHRH-R, and did not change
aer the treatment (data were not statistically signicant due to
animal-to-animal and breed-to-breed variability). Nevertheless,
GHRH-T Avg. score 2.9 ± 0.1 3.1 ± 0.1 3.2 ± 0.1 3.1 ± 0.1 3.1 ± 0.1 3.5 ± 0.2
% Change 8.6% 10.6% 8.8% 9.7% 23.9%
P value versus day 0 0.02 0.008 0.01 0.04 0.0008
Urination score
P-T Avg. score 3.0 ± 0.2 3.0 ± 0.0 3.1 ± 0.1 3.0 ± 0.0 3.0 ± 0.0 3.3 ± 0.3
% Change 0.0% 4.8% 0.0% 0.0% 8.3%
P value versus day 0 0.50 0.30 0.18 0.19 NS
GHRH-T Avg. score 3.1 ± 0.1 3.1 ± 0.1 3.1 ± 0.1 3.1 ± 0.1 3.1 ± 0.1 3.4 ± 0.1
% Change 0.1% 0.0% 0.3% 1.1% 10.4%
P value versus day 0 0.50 0.50 0.33 0.16 0.002
Bowel score
P-T Avg. score 3.0 ± 0.0 3.0 ± 0.0 2.97 ± 0.0 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0
% Change 0.0% 0.0% 0.0% 0.0% 0.0%
P value versus day 0 NS NS NS NS NS
GHRH-T Avg. score 3.0 ± 0.04 2.9 ± 0.04 3.0 ± 0.1 3.0 ± 0.05 3.0 ± 0.1 3.1 ± 0.1
% Change –2.1% –1.0% 0.0% 1.4% 3.9%
P value versus day 0 0.21 0.36 0.50 0.33 0.17
Diarrhea score
P-T Avg. score 3.0 ± 0.0 2.6 ± 0.2 3.0 ± 0.2 2.9 ± 0.1 3.0 ± 0.3 2.8 ± 0.3
% Change –13.3% 0.0% –4.8% 0.0% –6.7%
P value versus day 0 0.04 0.18 0.18 0.50 0.20
GHRH-T Avg. score 3.1 ± 0.1 2.8 ± 0.1 2.7 ± 0.1 2.7 ± 0.1 2.5 ± 0.1 2.7 ± 0.2
% Change –9.8% –11.8% –12.8% –16.9% –13.5%
P value versus day 0 0.01 0.01 0.01 0.002 0.04
Vomit score
P-T Avg. score 3.0 ± 0.0 2.8 ± 0.3 3.1 ± 0.2 2.9 ± 0.1 3.2 ± 0.2 3.0 ± 0.0
% Change –8.3% 4.2% –4.8% –6.7% 0.0%
P value versus day 0 0.18 0.09 0.18 0.19 NS
GHRH-T Avg. score 3.1 ± 0.1 2.7 ± 0.1 2.6 ± 0.1 2.7 ± 0.1 2.6 ± 0.1 2.6 ± 0.2
% Change –11.9% –13.7% –13.0% –14.3% –13.6%
P value versus day 0 0.008 0.0008 0.013 0.015 0.10
Abbreviations: Avg. score, average score; GHRH-T, growth hormone releasing hormone treatment; NS, not significant; P-T, placebo treatment.
QOL parameters from day 0 to day 120 of study where 1 = significantly decreased, 2 = decreased, 3 = no change, 4 = increased and 5 = significantly increased. For
each parameter, data are presented as average ± SEM. Percentage change over baseline values, as well as P value versus baseline values is included for each parameter
at each time point.
a
The following data was also significantly different between P-T and GHRH-T animals: overall assessment at day 20, P < 0.009; appetite levels at
days 20 and 40; thirst at day 120.
Table 4 Quality of life assessment (continued)
Day 0 Day 20 Day 40 Day 60 Day 90 Day 120

868 www.moleculartherapy.org vol. 16 no. 5 may 2008
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
the types of tumors, cancer-specic treatments, age, or breed
were not correlated to the GHRH-R status. While the number
of patients in each group is relatively small, and a denitive con-
clusion cannot be reached from this study, our current nding
seems to support this hypothesis; future studies will be designed
to address this issue, in particular the potential start of the therapy
versus cancer stage.
Our results indicate that the use of GHRH-plasmid com-
pared to P-T has the benecial eect of decreasing mortality. Only
23.26% of the GHRH-T dogs died by day 40 compared to 41.67%
of the P-T dogs. Of the dogs that could not be included in the
analysis due to the short survival time, 25% had hemangiosar-
coma (three GHRH-T and one P-T), more than any other type
of cancer, as shown in Table 2. However, 14 out of the 39 dogs
(~36%) diagnosed with hemangiosarcoma, survived at least to
day 60 and underwent evaluation. Hemangiosarcomas are rapidly
growing, highly invasive tumors typically lled with blood due to
extensive vascularization, oen rupturing and causing the victim
to rapidly bleed to death.
33
In fact when the data were analyzed
by malignancy type, the dogs with hemangiosarcomas appeared
not to perform as well as some of the other cancer types but the
number of cases per tumor type was too small to draw signicant
conclusions from among the groups.
Seventeen of the thirty-two dogs (that could be evaluated),
which were enrolled in the study and received GHRH plasmid,
survived until at least day 120 with four of these dogs surviv-
ing beyond day 300 postinjection. Only four of the P-T dogs sur-
vived until day 120, with two of them surviving until day 210.
Nine dogs (52.9%) were categorized as GHRH-NR at day 120.
Nevertheless, their hematopoietic parameters did remain stable
during the study, and they survived. Of the GHRH-T group,
61.54% survived from day 40 to at least day 120, whereas only
47.37% of GHRH-NR survived during the same period. Despite
the large number of hemangiosarcomas that have a poor general
prognosis, GHRH-T dogs survived on average 30% longer than
P-T ones. GHRH-T dogs with hemangiosarcomas survived on
average for 105 ± 22.5 days (this being not signicantly dierent
from P-T dogs, which survived for 97 ± 31 days), while all other
cancer types survived for 141.8 ± 19 days aer treatment, which
constitutes a 40% increase in survival (P < 0.05). GHRH-R sur-
vived 84% longer than both GHRH-NR and P-T (178 ± 26 days,
P
< 0.0026) groups. erefore, we can conclude that treatment
with the GHRH-plasmid appears to have a benecial eect on
survivability and extends the life span of dogs.
e quality of life of the plasmid GHRH-T dogs improved
dramatically based on a number of parameters assessed in the
study. e most signicant change occurred in the appetite level,
with the P-T dogs experiencing a decrease in appetite scores from
−4.5 to −17%, and GHRH-T dogs exhibiting an increase from
21 to 32.8% compared to their respective baseline levels (see
Table 4 for exact values at each study date). is nding is of par-
ticular importance for cancer patients with cachexia and anemia,
where lack of appetite is one of the most dicult complications
to treat.
34
Current therapies for the treatment of cancer cachexia
include the administration of appetite stimulants such as meges-
trol acetate or omega-3 fatty acids.
35,36
Supplementation with sh
oil decreased tumor growth and body weight loss while maintain-
ing food intake and increasing macrophage function and survival
in rats.
37
In this study, we have seen that GHRH treatment may
increase protein deposition in the tissues, as shown by modica-
tions in total circulating proteins, and a physiological increase
in IGF-I levels. Other parameters, related to complications of
the cancer-specic therapies, such as diarrhea and vomiting,
decreased signicantly in GHRH-T animals. ese ndings are
also of particular interest for cancer patients,
38,39
as many stud-
ies have shown that symptoms associated with shorter survival
included those of the anorexia–cachexia syndrome and impaired
physical well being.
Anemia is a frequent further complication of cancer, either
associated with cachexia or alone, that can also reduce the qual-
ity of life of cancer patients. Current therapies for anemia include
nutritional supplements, blood transfusions, treatment of infec-
tion/inammation, and EPO treatment.
40
In a separate study of
adult anemic cancer patients, EPO treatment once a week sub-
cutaneously for 16 weeks resulted in improvement in Hb levels
(9.82 ± 0.78 g/dl at baseline versus 12.56 ± 1.49 g/dl post-therapy;
P < 0.001) and a signicant improvement in quality of life in 76%
of patients.
41
Although administration of recombinant human
EPO appears to improve cancer-associated anemia, there are
drawbacks such as the required number of subcutaneous injec-
tions, which need to be administered weekly for at least 12 con-
secutive weeks. A single administration of GHRH IM injection
followed by EP into our study dogs has the ability to signicantly
increase the mean levels of RBC, Hb, and Ht over a 120-day
period (Figure 4). GHRH treatment also increased IGF-I levels,
which correlated with increases in the levels of RBCs, Hb, and Ht
at the next time point assayed. erefore, GHRH treatment has
the potential to improve the anemic condition of cancer-aicted
dogs. Importantly, these favorable changes were obtained aer
delivery of a low plasmid quantity, 0.35 mg, in conjunction with
EP, making this therapeutic approach attractive from a practical
point of view. e results in this study are highly encouraging and
also serve as ecacy models for the development of a therapy for
cancer-related anemia in humans.
GHRH administration is undergoing testing in two human
clinical trials, Phase II and III, respectively for aging or human
immunodeciency virus–associated lipodystrophy. e results
of the enabling studies showed that the therapy was well tol-
erated, and lacked severe adverse eects.
42–44
Nevertheless,
the very frequent administration (once a day to three times a
week) and the fact that almost all adverse eects were local at
the administration site, makes the peptide therapy less attrac-
tive than a plasmid-mediated therapy that would ensure thera-
peutic levels of GHRH for a longer period of time aer a single
administration.
Overall, the ability to control or reverse symptoms of cachexia
and/or anemia is of paramount importance for improving the
quality of life and aiding in the therapeutic treatment of cancer
patients. is dog study therefore addresses the important ques-
tion of cachexia-associated anemia in cancer-aicted patients.
e potential treatment by a single injection of GHRH-plasmid
followed by EP to reverse cancer-associated anemia can ben-
et not only man’s best friend but could be benecial to human
patients in the near future.

Molecular erapy vol. 16 no. 5 may 2008 869
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
MATERIALS AND METHODS
DNA constructs. e plasmid pSPc5-12-GHRH contains a 360-basepair
SacI/BamHI fragment of the SPc5-12 synthetic promoter
45
in the SacI/
BamHI sites of a pSK-GHRH backbone (Figure 1).
46
e synthetic HV-
GHRH complementary DNA encoding for a GHRH analog with an
extended half-life, obtained by site-directed mutagenesis (Altered Sites II
In vitro Mutagenesis System; Promega, Madison, WI) of wild-type GHRH
was cloned into the BamHI/HindIII sites of pSK-GHRH. Characterization
of the vector and the long half-life mutant HV-GHRH was previously
described.
32
Animal studies. To be eligible for this study, companion dogs diagnosed
with malignant cancer had to have an expected survival prognosis of at
least 60 days as determined by their attending veterinarian. e dogs were
of any age, breed, and sex, but weighed >4 kg. e dog’s owner had to agree
to his/her dog participating in the study, and to being available for blood
draws upon initial treatment (baseline) and on days 20, 40, 60, 90, and
120. Prior to the start of the study all dogs underwent a physical examina-
tion (including measurement of body weight), had body composite scor-
ing done and underwent a complete blood count analysis and urinalysis.
Dogs selected for the active drug treatment evaluation randomly received
one injection of 0.35 mg of GHRH-plasmid or placebo (saline) IM (time 0)
followed by EP (in a 4:1 ratio).
A total of 55 dogs were enrolled in the study. Out of this number 39 dogs
(14 female and 25 male; 32 of which received plasmid HV-GHRH) constituted
cases t for evaluation. e other 16 dogs were withdrawn from the study
due to death, because they had incomplete follow-ups, or were removed
due to noncompliance of study conditions by the owner as decided by the
investigator. Animals enrolled in the study had a mean age of 10.5 years,
and displayed a variety of cancers such as hemangiosarcoma, carcinoma,
sarcoma, adenocarcinoma, mast cell tumor, melanoma, and cutaneous
extramedullary plasmacytoma. Chemotherapy drugs given to treat the
cancer included, but were not limited to, doxorubicin (1 mg/kg intravenously
or <4.5 kg, 20 mg/m
2
; 4.5–9.0 kg, 25 mg/m
2
; 32–39 kg, 35 mg/m
2
; 39–50 kg,
40 mg/m
2
; >50 kg, 45 mg/m
2
, doses were given at 21-day intervals), vincris-
tine (0.5 mg/m
2
intravenously weekly), and cyclophosphamide (50 mg/m
2
on alternate days) at standard doses, per veterinarian recommendation.
Radiotherapy was also administered to some of the dogs in combination
with chemotherapy treatment: carcinoma (n = 2), hemangiosarcoma (n = 2),
adenocarcinoma (n = 2), melanoma (n = 1), mast cell tumor (n = 1), and
cutaneous extramedullary plasmacytoma (n = 1). ree out of the nine
dogs that were treated with both radiotherapy and chemotherapy were on
placebos and treatment was tailored to each individual dog’s needs by the
veterinary oncologist. Decisions about euthanasia were made by the owner,
in consultation with the attending veterinarian. Only if the general state of
the companion dog was deteriorating in spite of an acceptable standard of
care was the animal euthanized. e P-T animals were euthanized more
oen before the GHRH-T dogs because their general status was poor.
IM injection of plasmid DNA. e endotoxin-free plasmid (VGX
Pharmaceuticals, Immune erapeutics Division, e Woodlands, Texas)
preparation of pSPc5-12-HV-GHRH was diluted in sterile water for injec-
tion + 1% poly--glutamate sodium salt to 5 mg/ml. e test doses and
placebo (sterile water + 1% poly--glutamate sodium salt, the vehicle used
for the plasmid preparation) were lled individually in Uniject (Becton-
Dickenson, Franklin Lakes, NJ)
47
and randomized. IM injection of 0.35 mg
of test article (in a total volume of 500 µl) or placebo was followed aer
2 minutes by EP using a ve-electrode array and a constant-current elec
-
troporator (CELLECTRA device, VGX Pharmaceuticals, e Woodlands,
TX) under the following conditions: ve pulses, 1 Amp, 50 ms/pulse.
Injection of plasmid was directly into the semimembranosus muscle with
a 1-cc syringe and a 26-gauge hypodermic needle (Becton-Dickenson,
Franklin Lakes, NJ). Dogs were injected with lidocaine with epinephrine
solution around the treatment site or underwent general anesthesia before
injection of the test article and EP in order to minimize discomfort. Any
concomitant treatments, such as radiotherapy and/or chemotherapy were
documented and test article injection was not given in an area that had
been irradiated or was adjacent to a chemotherapy injection site. Dogs did
not receive their chemotherapy immediately prior to or aer the test arti-
cle injection (5 days). Animals were allowed to recover for several hours
before rejoining their owners.
Quality of life. Evaluation of each dog’s quality of life was based on the
owner’s assessment as determined by a questionnaire completed at each
visit, with ratings of their dog’s condition as signicantly increased (5),
increased (4), no change (3), decreased (2), or signicantly decreased (1).
Conditions that were assessed included body weight, activity level, exercise
tolerance, mentation, appetite, thirst, urination frequency, number of bowel
movements per day, frequency of diarrhea and vomiting. Each dog was
also assessed by the investigator based on the Karnofsky performance scale
modied for veterinary use. e development of an adverse event as deter-
mined by the investigator or if the dog’s owner did not wish for his/her dog
to continue in the study at any time, resulted in removal of the subject.
Hematological and biochemical parameters. Complete blood count,
serum biochemistry, and urinalysis were obtained at baseline and at
each time interval as indicated and analyzed by an independent labora-
tory (Antech Diagnostics, Irvine, CA). Analysis of complete blood count
included the number of erythrocytes, Ht, Hb, total leukocyte count, and
dierential leukocyte counts (neutrophils, lymphocytes, monocytes,
eosinophils, and basophils), platelet count, mean corpuscular volume,
mean corpuscular Hb, mean corpuscular Hb concentration, and partial
prothrombin time. e following biochemical parameters were also exam-
ined: creatinine, total protein, chloride, potassium, sodium, blood urea
nitrogen, glucose, calcium, serum glutamate pyruvate transaminase, alka-
line phosphatase, bilirubin, inorganic phosphorus, globulin, cholesterol,
creatinine phosphokinase, and albumin. Urine analysis evaluated color,
consistency, volume, glucose, bilirubin, ketone (acetoacetic acid), specic
gravity, blood, pH, protein, urobilinogen, nitrite, leukocytes, and micro-
scopic examination of formed elements.
IGF-I analysis. IGF-I was measured using a heterologous human radio-
immunoassay following the manufacturer’s protocol (Diagnostic Systems
Laboratories, Webster, TX). e sensitivity limit of the assay was 0.8 ng/ml;
the intra- and inter-assay variations were 3.4 and 4.5%, respectively.
Statistical analysis. Data were analyzed by an independent third party
(Synergos, e Woodlands, TX), with all investigators blinded to treatment
and results until the study analysis was completed and results tabulated, and
reports released. Data were analyzed based on the “intent-to-treat” popula-
tion dened as all those animals that received an injection, treatment or
control. Treatment success was evaluated using a modied Karnofsky per-
formance scale for veterinary use and dened as a ≥5% increase above the
nadir in the RBC, Ht, and Hb levels on day 60 and remaining in the study
until at least day 90. Due to the fact that it may take several days for tran-
scription of the plasmid to begin and an additional 7–10 days for hema-
topoiesis to increase, the nadir for RBC, Ht, and Hb levels was selected
from the lower value on day 0 or day 20. Data analysis was carried out on
the baseline assessments with treatment comparisons using the Wilcoxon
signed-rank test, or Fisher’s exact test as indicated, where statistically signif-
icant results are dened as having a P value of <0.05. Survival was analyzed
by the Kaplan–Meier method. A post-hoc analysis compared GHRH treat-
ment responders to nonresponders (Student’s t-test). For biochemical anal-
ysis, the data was analyzed to baseline (day 0) using a Student’s t-test, where
statistically signicant results are dened as having a P value of < 0.05.
ACKNOWLEDGMENT
We thank the dogs and dog owners for their participation and cooperation
in this study. We thank the clinical staff at all the participating veterinary

870 www.moleculartherapy.org vol. 16 no. 5 may 2008
© The American Society of Gene Therapy
Plasmid GHRH Therapy for Cancer-associated Anemia
clinics in Houston, TX, San Antonio, TX and Tampa, FL especially the site
directors Glen King, Kevin Hahn, and Tracy LaDue, respectively, for their
excellent patient care. We thank the staff at InVentiv Clinical Solutions,
LLC, Synergos, The Woodlands, TX for their assistance with the statisti-
cal analysis. We would like to especially thank Robert Carpenter, Study
Director, and Catherine Tone, Clinical Monitor, for their participation
and assistance with the project, and Amir Khan, Jonathan Prigge and
Niranjan Sardesai for critically reviewing this manuscript.
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