Research Article
Evaluation of Adverse Events in Dogs with Adenoviral Therapy by Intralymphonodal Administration in Canine Spontaneous Multicentric Lymphosarcoma
L. Núñez-Ochoa1*, V. Madrid-Marina2 and A. Gutiérrez-López3
1Department of Pathology, Faculty of Veterinary Medicine and Zootechnics, CDMX, Mexico
2Direction of Molecular Virology, Infectious Diseases Research Center, National Institute of Public Health, CDMX Mexico
3Cellular Therapy Unit, Rehabilitation National Institute, CDMX, Mexico. Current address: Oncolytics Biotech Inc.
*Corresponding author: Luis Núñez-Ochoa, Department of Pathology, Faculty of Veterinary Medicine and Zootechnics, Circuito Exterior s/n. Ciudad Universitaria, Cto. Exterior s / n, Cd. Universitaria, 04510 Mexico City, CDMX, Mexico
Published: 03 Apr, 2017
Cite this article as: Núñez-Ochoa L, Madrid-Marina V,
Gutiérrez-López A. Evaluation of
Adverse Events in Dogs with Adenoviral
Therapy by Intralymphonodal
Administration in Canine Spontaneous
Multicentric Lymphosarcoma. Clin
Oncol. 2017; 2: 1258.
Abstract
Background: Therapy administration in cancer is mainly performed by intravenous, oral, and in
situ routes. Adverse Events (AE) are a significant limitation of adenoviral vectors during somatic
gene therapy. There are some partial evaluations of AE in canine cancer research. The objective
of this study was to evaluate AE in adenoviral vector-mediated gene therapy administered by
Intralymphonodal Route (ILNR) in canine lymphosarcoma.
Methods: AE were determined in five dogs with spontaneous multicentric lymphosarcoma. A non
replicative recombinant adenovirus vector with a LacZ reporter gene was administered once by
ILNR at a starting dose of 1.35 X 1010 pfu/kg, with a dose-escalation model to 1.25 X 1012 pfu/kg
considered under these conditions, as the Maximum Tolerated Dose (MTD). AE was evaluated by a
canine scale for attribution of AE based in selected clinical findings, hemogram, biochemistry, and
urinalysis.
Results: No significant AE were observed during the study, therefore, no Dose-Limiting Toxicity
(DLT) and MTD were found in any dog.
Conclusion: Administration of adenovirus vector exhibited no clinical, nor laboratory significant
AE in this canine ILNR clinical trial. This suggests that adenoviral gene therapy by ILNR is safe for
use in dogs with lymphosarcoma and a potential model of administration in animals and human
beings with metastasis to lymph nodes.
Keywords: Adverse events; Dogs; Lymph node; Intralymphonodal administration; Adenoviral vector; Gene therapy; Lymphosarcoma; Lymphoma; Hemogram; Clinical biochemistry
Introduction
The treatment of Lymphosarcoma (LSA) in human and canine patients has predominantly been
based on conventional chemotherapy and radiotherapy [1-3]. Although an improvement in response
rates and survival has been obtained with these therapies over the years, a significant number of
patients do not respond or relapse. In addition, conventional chemotherapy is often associated with
morbidity, toxicity [4] and chemoresistance [5,6]. During the last decade, advancements brought
about by the introduction of new biotechnological therapies have entered into the human oncology.
These targeted therapies are dominated by the monoclonal antibodies, which have emerged as
important therapeutic agents in the treatment of several malignancies including non-Hodgkin
lymphosarcoma (NHL) [7,8]. Viral vectors have frequently been applied in somatic gene therapy
with the final goal of treating various genetic diseases in the areas of neurology, metabolic disease,
hemostasis and cancer. Vectors have been engineered based on AAV, adenoviruses, alphaviruses,
herpes simplex viruses, lentiviruses, and retroviruses [9,10]. One of the challenges of current gene
therapy vector development, concerns targeting a therapeutic gene to diseased cells with the aim of
achieving sufficient gene expression in the affected tissue, while minimizing toxicity and expression
in other tissues. Human adenovirus serotype 5 of subgroup C (Ad5) vectors are very popular in
somatic gene therapy because the most is known about the structure and biology, it can be easily
modified and there are convenient biological reagents available to produce recombinant Ad5
vectors in large quantities without modifying their ability to infect cells, which are not oncogenic in humans [11,12] and remain an attractive tool for gene therapy approaches because of their nonintegrating nature and the ability to
infect dividing and non-dividing cells [13]. Dogs have been used as
animal models of many human diseases, and several gene therapy
approaches, such as strategies for hemophilia A [14], hemophilia B
[15], cancer [16-18], hematopoietic growth factors [19], lesions in the
avascular portion of the meniscus [20], biological pacemaker activity
of heart [21], diabetic canine pancreas [22], cerebral vasospasm [23],
canine muscular dystrophy [24], and hereditary retinal degeneration
[25] have been assayed.
It is now well accepted that there is a dose-dependent toxicity
associated with systemic delivery of adenoviral vectors, the risk of
hepatotoxicity is a major concern. Vectors that can target specific
tissues following systemic or minimally invasive administration
would enhance their therapeutic potential and expand their
application [26]. LSA is the most frequently occurring hematological
malignant neoplasm in the dog. The multicentric form of LSA is
most common, with varying degrees of involvement of lymph nodes,
liver, spleen, blood, and bone marrow [27]. During clinical trials
one important feature is the drug safety assessment monitoring of
possible drug-induced organ injury. The evaluation of Adverse Events
(AE) considered as any unfavorable and unintended sign including
abnormal laboratory values, symptoms, or disease findings temporally
associated with the use of a medical treatment or procedure that may or
may not be considered related to the medical treatment or procedure.
An AE is a term that is a unique representation of a specific event
used for medical documentation and scientific analyses [28]. There
are some partial or organ specific recommendations for assessment of
AE as the one proposed by the Regulatory Affairs Committee of the
American Society for Veterinary Clinical Pathology for the selection
and interpretation of clinical pathology of liver-specific analytes data
for a consistent and rigorous approach to the use in the identification
and assessment of the potential for drug-induced hepatic injury in
animals and the potential for hepatic injury in humans [29].
Table 1
Table 1
Selected mesurands for adverse events evaluation in liver, kidneys, pancreas, muscle, hearth, general cellular integrity and acid-base.
Objective
The present study was conducted to assess the clinical, hematological, biochemical and urinary AE associated with a single intralymphonodal administration of adenovirus vector in dogs with spontaneous multicentric lymphosarcoma.
Materials and Methods
The study was carried out at the Small Animal Teaching Hospital
of UNAM, Rehabilitation National Institute and Experto Sur
Veterinary Clinical Pathology Laboratory in Mexico City. Dogs were
evaluated before and monitored for selected clinical and laboratory
AE throughout the trial in the clinic on the 12, 24, 48, and 72 h after
receiving intralymphonodal (ILN) adenoviral vector in D-MEM/F-12 vehicle (11039-021 GIBCO™ Invitrogen Co. USA).
Animals
Five adult dogs (1 male and 4 females) with spontaneous
multicentric lymphosarcoma were used and housed individually in
raised metal cages during this study. Prior to each sampling dogs
were fasted for 12 hours and water retired 4 hours before sampling.
Design of the study was approved by the local ethic committee at
College of Veterinary Medicine of UNAM.
Lymphosarcoma diagnosis
All cases had a clinical, radiological, hematological and
cytological diagnosis of WHO’s clinical stage III a to V b multicentric
lymphosarcoma [30].
Adenovirus
The non-replicative recombinant Ad5 vector (E1 and E3 regions
deleted) with a E. coli lacZ reporter gene (provided by Dr. Curiel, Gene
Therapy Center Alabama University), which expresses the enzyme
beta-galactosidase (Ad5B-gal) was amplified and tittered (AdEasy™
Vector System Quantum Technologies. Application Manual. Version
1.2) and purified (Adeno-X™ Virus Mini Purificator kit. Clontech
Laboratories, Inc).
Adenovirus infectivity test
Prior the ILN administration Ad5B-gal infectivity was
demonstrated in Hela cell line (VCA-1001, Amaxa, Inc. USA) using
the detection of β-galactosidase by overnight X-Gal staining at
37ºC (AdEasy™ Vector System Quantum Technologies. Application
Manual. Version 1.2. Montreal, Qc. Canada).
Clinical signs
The clinical examination included anorexia, body temperature,
cardiac frequency, respiratory frequency, vomit, dehydration,
diarrhea and edema. A complete laboratory panel was used for AE
research. The animals were observed every two hours during the 72
hours of study.
Clinical Pathology
A complete clinical pathology profile was established to evaluate
hematological (hematocrit, hemoglobin, erythrocytes, MCV, MCHC,
reticulocytes, nucleated and abnormal erythrocytic morphology,
leukocytes, neutrophils, band neutrophils, lymphocytes, monocytes,
eosinophils, platelets, total solids, and fibrinogen), hepatic, renal,
pancreatic, muscular, cardiac, acid-base balance changes, and general
cellular integrity (Table 1) [31].
Experimental design
A total of five Ad5B-gal ILN dose escalation levels were explored
(Table 2), one dose by dog (following the AE guide of Table 3). The attribution grade of adverse events was applied according to Table 4 from which the adjustment value was subtracted from the
Table 5 indications, to ensure that treatment-related conditions are
distinguished from disease-related conditions to obtain the real grade
(modified from National Cancer Institute NCI CTEP version 4.03.2010).
Dose-limiting toxicity
The dose-limiting toxicity was defined as a grade 3 or grater for
clinical or laboratory AE.
Laboratory analysis
Blood was collected for serum biochemistry and hemogram
evaluation. The biochemistry analytes were determined by Cobas Mira® Chemistry Analyzer (Roche Diagnostic Systems, Inc. New
Jersey, USA), and EasyLyte Plus Na+/K+/Cl- (Medica Corporation
MA USA), the hemogram, was evaluated by the Coulter Counter®
T-540 (Coulter Electronics, Inc. Florida, USA). Urine voided samples
was collected and chemistry was evaluated with Combur10 Test® M
(Roche, Germany).
Data analysis
Data were classified in accordance with our adverse events guide
for dogs (Table 3) and the grading adjustment values related with
the attribution of adverse events (Table 5). Statistical significance
of experimental results was analyzed by two-tailed Student’s t-test
(SPSS) to compare paired data in dogs with LSA. Differences were considered significant if P was <0.05.
Table 2
Table 3
Table 4
Table 5
Table 6
Table 6
Descriptive statistics and grades of clinical adverse events in 5 dogs with spontaneous multicentric lymphosarcoma. Grade adjustment after Ad5B-gal intralymphonodal administration.
Table 7
Table 7
Descriptive statistics and grades of hematological adverse events in 5 dogs with spontaneous multicentric lymphosarcoma. Grade adjustment after Ad5B-gal
intralymphonodal administration.
Table 8
Table 8
Descriptive statistics and grades of hepatic adverse events in 5 dogs with spontaneous multicentric lymphosarcoma. Grade adjustment after Ad5B-gal intralymphonodal administration.
Results
None of dogs that received Ad5B-gal died during the course of the study.
No DLT was observed in all patients, and therefore, the MTD was not established.
Clinical evaluation
All doses of Ad5B-gal were well tolerated; no significant AE was
related to the administration of vector (Table 6). Anorexia was from
grade 2 to 0 in 24 h after intralymphonodal treatment indicating a
favorable event. In dogs receiving higher doses (18.38 X 1010/VP/kg
and 153.85 X 1010/VP/kg) a small single vomit was present in the first
12 h post adenoviral administration. Cardiac frequency was elevated
during most evaluations. Respiratory frequency was statistically
different in 12-24 h but not dose-dependent. Four dogs had predose
submandibular or limb edema, therefore these results were
not considered secondary to adenoviral vector. Diarrhea and fever
changes were clearly related to disease (Table 6).
Hematological evaluation
The gradual decrease from pre-dose to 72 h (9.2%) of hematocrit
was found. Two dogs manifested anemia at 0 h and another at 72
h. Leukocytosis and neutrophilia was present during the 5 sampling
times in two dogs, one dog only at 12 hours (high dose dog). The
progressive decrease of total solids was statistically significant at 12-
24 h and 12-48 h comparison (Table 7).
Liver evaluation
The levels of glucose and triglycerides were statistically different
but within the reference values. The high values of ALT, AST and
AP enzymes were similar pre and post-dose. Even if the decrease
of proteins was significant between 0-72 h, the results are within
reference values. This was associated with gradual decrease of albumin
statistically different at 0-48 h and 0-72 h (Table 8).
Acid-base evaluation
No observable changes were seen with K+ and SIDclin. Electrolytes
(Na+, Cl- and HCO3
-) decreased at 12 h post-dose and were different at
0 and 24 h. There was a slight increase at grade 1 in the concentration
of NVA after 72 h of dosing.
Cell integrity
Total LDH was found increased at 12 and 24 h post-dose at grade
1 but not statistically significant.
No significant variations were seen in kidneys, urinalysis,
pancreas, muscle and heart evaluations.
Discussion
To our knowledge, there are no reports in the literature about
ILNR oncolytic adenovirus administration. The different routes
utilized for adenovector-mediated gene transfer administration in
vivo have had different tolerance and limitations. Major disadvantages
of human adenovirus vectors in gene therapy include preexisting
or induced immune responses, and possible coreplication of
recombinant Ad in the presence of wild-type Ads [32]. One of these
limitations is the low gene transfer rate into organs other than the
liver after systemic intravenous vector injection [33]. In a single-dose
intravenous injection of 6X1012 viral particles in dogs with hemophilia
A for human factor VIII transfer [14], 2X1012 into hepatic artery [17],
in situ administration into primary gastric cancer [18], 1X1012 of
intraprostatic injection [34], doses of 8.57X1011/VP/kg in dogs with
hemophilia B injected intravenously have had no significant AE [13].
Transient hepatic integrity (ALT, AST, ALP), muscular (CK), and
primary hemostasis (platelet counts) abnormalities were found after
administration of high (3X1012/VP/kg) [35] and low dose (6X1011/VP/
kg) [36]. A high i.v. dose of vector (>1013 VP) has been leading to a
systemic cytokine shock and may be resulted in the death [37]. AE was
also observed in some studies after Ad administration in situ, not for
Ad but for their proteins expressed [18]. The route of administration
and the vector affect the level and duration of expression [38]. In situ route is best for transferring the therapeutic gene into cancer cells [18]. The rationale for this approach is to increase the dose effects in a
specific tissue, improving antineoplasic efficacy, and reducing or even
eliminating the immunosuppression period and other critical AE.
Therefore, the neoplasic tissue receives higher quantity of therapeutic
product, and its systemic distribution to other sensible healthy organs
is reduced. The mild decrease of hematocrit is associated with an
evolution of anemia in natural lymphosarcoma cases [39-43] and
not related to adenoviral administration, because anemia is one of
the most common paraneoplastic syndrome seen in veterinary and
human oncology [44]. Cardiac frequency was elevated secondary to
anemia which also happens in other cancer patients [45]. Neutrophilic
leucocytosis corresponded in our study, with findings reported in
dogs with lymphosarcoma [43]. The gradual decrease of total solids,
proteins and albumin was none related to treatment. Frequently,
hypoproteinemia and hypoalbuminemia are considered as secondary
toxic responses to experimental drugs [46,47]. As in this work, these
changes are normally present in lymphosarcoma [48]. The decrease
of proteins is associated to constant decrease of albumin. In this case
the hypoalbuminemia is caused by its decreased synthesis, since it is
a negative acute-phase protein [31]. The significant difference in total
solids samples was related to mild hemoconcentration found at 12
h. The evaluation of hepatic integrity was similar all times and was
dose-independent as opposed to published paper [36]. The difference
of electrolyte concentrations among 12 h and 0 h or 24 h was related
to the degree of animal hydration, because their appetite and water
consumption improved from 12 h post-dose to the end of study. The
marginal AE of NVA evaluation at 72 h is consistent with a mild
pseudometabolic acidosis or spurious decrease of HCO3
- due to in vitro loss because the serum sample was analyzed 12 hours after the
sampling [49].
Most of clinical and laboratory changes were considered not
treatment-related, but disease-related conditions.
Conclusions
To the author’s knowledge, this is the first report about intralymphonodal administration of adenovirus for gene therapy. The administration of Ad5 vector in canine spontaneous multicentric lymphosarcoma at high dose exhibited no clinical, nor laboratory significant adverse events. This suggests that Ad5B-gal is a safe vector for use in lymphosarcoma gene therapy. This data provides the basis to consider the lymphonodal route as an appropriate way for therapy administration without significant adverse events in canine lymphosarcoma and a potential model for applied therapy research in lymphonodal metastasis in animals and human beings.
Acknowledgments
This project was supported by National Council for Science and Technology of Mexico and National Autonomous University of Mexico and funded by grant from Experto Sur Veterinary Laboratory, Mexico city (LE01-0206).
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