Preclinical safety evaluation of rAd5-hTERTC27 by intravenous injection

Regulatory Toxicology and Pharmacology 67 (2013) 53–62

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Preclinical safety evaluation of rAd5-hTERTC27 by intravenous injection Pei-jian Yue a,1, Lei He a,1, Yi Li a, Qing-yu Shen a, Mei Li a, Da-quan Huang a, Jun-Jian Huang b, Ying Peng a,⇑ a b

Department of Neurology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107#, Yan Jiang Xi Road of Guangzhou, Guangzhou City 510120, China Laboratory of Tumor and Molecular Biology, Beijing Institute of Biotechnology, Beijing, China

a r t i c l e

i n f o

Article history: Received 9 January 2013 Available online 1 July 2013 Keywords: HCC hTERTC27 Gene therapy Single-dose toxicity Preclinical evaluation

a b s t r a c t The safety of rAd5-hTERTC27, a replication defective adenovirus vector carrying hTERTC27 for possible use against hepatocellular carcinoma (HCC) was assessed. In single-dose evaluations, intravenous dose levels of up to 2  1011 VP/kg in rats and 9  1010 VP/kg in monkeys were well tolerated with no abnormal changes in general signs, body weight and food consumption, and no significant differences in biochemical parameters, urinalysis, ECG, and systemic necropsy observations between the rAd5 groups and solvent control group except that slight hematological change was observed. No hemolytic effect using rabbit blood, local perivasculitis following intravenous injection in rabbits or systemic anaphylaxis in guinea pigs following intravenous dosing was seen. No effects on the central nervous system of mice occurred following intravenous dosing with the exception of an increase in sleep duration at the dose of 1.2  1011 VP/kg (p < 0.05) but not at lower doses of 2  1010 and 6  1010 VP/kg in the hypnotic synergism test. These results demonstrate that administration of rAd5-hTERTC27 was well tolerated in an initial set of safety studies as part of an evaluation to allow human trials for the treatment of HCC. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction HCC is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide (Forner et al., 2012; Jemal et al., 2011). Since over 80% of HCC cases are usually diagnosed at an advanced stage, the prognosis remains poor, despite advances in palliative treatments for the disease (Chen et al., 2011; Llovet, 2005). Thus, it is necessary to develop novel therapeutic strategies that inhibit disease progression and improve survival in patients with HCC. Gene therapy may provide a promising strategy for the treatment of HCC, and intensive clinical trials utilizing a variety of transgenes have been reported (Habib et al., 2002; Li et al., 2007; Sangro et al., 2010; Tian et al., 2009; Tolcher et al., 2006). In addition to selection of therapeutic genes, an effective gene delivery vector is crucial for the success of this strategy. Adenovirus (Ad) vectors are now the most commonly used vector in gene therapy clinical trials worldwide (23.3% of all 1843 trials) (Edelstein et al., 2007). This is a result of the multiple advantages offered by this vector system for gene therapy: wild-type adenoviruses are weakly or non-pathogenic for their natural hosts; they can mediate high transduction efficiency in both quiescent and proliferating cells; and Ad vectors do not integrate into host genome (Ahi et al., 2011; Campos and Barry, 2007; Vorburger and Hunt, ⇑ Corresponding author. Fax: +86 20 81332833. 1

E-mail address: [email protected] (Y. Peng). These authors are contributed equally to this work.

0273-2300/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2013.06.004

2002).On the basis of these merits, adenoviral vectors have great application value in the treatment of malignancies (Nicklin et al., 2005; Relph et al., 2005; Verma and Weitzman, 2005). Results of animal studies and clinical trials in humans for cancer therapy and other metabolic disorders using human Ad vectors are encouraging (Akbulut et al., 2003; Garber, 2006; Huang et al., 2007). A recombinant human adenovirus-p53 injection (Gendicine) and a conditionally replicative adenovirus (Oncorine) were approved by the State Food and Drug Administration of China (SFDA) for the treatment of head and neck carcinoma in 2003 and 2005, respectively, and thus set a new milestone in the history of gene therapy and biotechnology (Liang, 2012; Peng, 2005; Raty et al., 2008). Therefore, we constructed rAd5-hTERTC27, which was a recombinant, replication defective adenovirus vector carrying hTERTC27, and it was confirmed to significantly inhibit the growth of hepatocellular carcinoma in mice by intravenous injection with a single dose of 5  107 PFU compared with rAd5-EGFP only carrying Enhanced Green Fluorescent Protein (He et al., 2013). hTERTC27, a 27 kDa C-terminal polypeptide of human telomerase reverse transcriptase (hTERT), is capable of inducing telomere dysfunctions and anaphase chromosome end-to-end fusions in hTERT-positive HeLa cells. Overexpression of hTERTC27 also inhibits HeLa cell growth and tumorigenicity in nude mice xenografts (Huang et al., 2002). Importantly, the actions of hTERTC27 are mediated without perturbing the endogenous telomerase activity, thereby minimizing the potential side effects on telomerase positive reproductive cells and proliferative cells of renewal tissues in antitelomerase therapies (Huo et al., 2006; Shay et al., 2001).

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Further investigations have reported that intratumoral injection of recombinant adeno-associated virus (AAV) carrying hTERTC27 (rAAV-hTERTC27) is highly potent in inhibiting the growth of human U87-MG glioblastoma cells in athymic nude mice (Ng et al., 2007). The objective of this study was to evaluate the safety of rAd5hTERTC27 to understand the toxicity of rAdv-hTERTC27 on animals and to provide experimental data to support use in clinical studies. In vivo experiments were conducted with intravenous administration as this corresponds to the clinical route of administration.

2. Materials and methods 2.1. Adenovirus vector rAd5-hTERTC27 and rAd5-EGFP, which are first-generation E1/ E3-deleted adenovirus vectors under control of the cytomegalovirus (CMV) promoter, were constructed and prepared under Good Manufacturing Practice (GMP) by AGTC Gene Technology Company Ltd. (Beijing, China). Briefly, hTERTC27 cDNA was inserted into the shuttle plasmid pDC316-mCMV to yield pDC316-CMV-hTERTC27. HEK-293 cells were then cotransfected with the shuttle plasmid and the helper plasmid pBHGlox_E1,3Cre using Lipofectamine2000 to produce rAdv-hTERTC27. rAdv-EGFP was produced similarly as a control vector. The recombinant adenoviruses were purified by ion exchange column and gel chromatography. The particle number and titer of the recombinant viruses were measured by absorbance of ultraviolet (UV) light (wavelength, 260 nm) and 50% tissue culture infectious dose (TCID50) assay, respectively. Adenovirus concentration was 1  1012 VP/mL (activity unit was 2.56  1011 IU/ mL). Adenovirus vectors were sterilized by 0.22 lm sterile filters with PVDF membranes (Merck Millipore, Darmstadt, Germany), stored in sealed EP tubes at 80 °C, and diluted to the corresponding concentration by Tris buffer before use.

rAd5-EGFP, which only carried Enhanced Green Fluorescent Portein (EGFP) without hTERTC27, had been used as a control treatment previously, and was included as an additional control group to differentiate toxicity occurring from the adenovirus particle vs. toxicity associated with the expression vector in this study. 2.2. Experimental animals and housing condition All studies were performed at the National Chengdu Center for Safety Evaluation of Drugs (NCCSED) (Chengdu, Sichuan) with the approval of the Institutional Animal Care and Use Committee (IACUC), which has passed the international certification. Species of animals in our studies included Kunming mice, Sprague–Dawley rats, Rhesus monkeys, Common rabbits and Hartley guinea pigs obtained from various breeders (Table 1). The animals were housed in air-conditioned rooms (temperature: 21 ± 2 °C; humidity: 55 ± 4% and light: 12 h light/dark cycle), and were fed with commercial primate complete feed, fresh fruit (for monkey); complete pellet feed (for mice, rats, rabbits and guinea pigs) and reverse osmosis water ad libitum. All study protocols were conducted according to Chinese Guidance for Human Gene Therapy and Product Quality Control, the Guideline of General Pharmacological Study Technology, and the Regulations of Good Laboratory Practice (GLP) for nonclinical laboratory studies of drug issued by the State Food and Drug Administration (SFDA) of China. 2.3. Single-dose toxicity in rats Sprague–Dawley rats (25 males, 179.0–198.2 g and 25 females, 160.0–179.2 g; 6 weeks old) were randomly divided into five groups with 10 per group, equally by sex (Table 1). We defined the day of administration as day1.General signs were observed and recorded daily for 14 days after administration. Observations included appearance, general behavior, mental status, respiration, appearance and color of urine and stool, genitals, and death. Body

Table 1 Design and implementation of studies. Studies

Species of animals; breeders

Effective dose

Grouping and treatments

Multiple of the effective dose

Route of administration

Volume of administration (mL/kg)

Single-dose toxicity

SD rat; Slac Laboratory Animal Co., Ltd. (Shanghai, China)

8.0  109 VP/ kg

rAd5hTERTC27 low-dose, 6.25; mid-dose, 12.5; high-dose, 25

i.v.

2

Single-dose toxicity

Rhesus monkey; Ping’ an research base of animal breeding (Chengdu, China)

3.6  109 VP/ kg

rAd5-hTERTC27 low-dose, 5.6; high-dose, 25

i.v.

1

Local tolerance study

Common rabbit; laboratory animal farm of sichuan professional committee (Chengdu, China) Hartley guinea pigs; laboratory animal farm of sichuan professional committee (Chengdu, China) Kunming mouse; Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China)

–

rAd5-hTERTC27 low-dose 5  1010 VP/kg; mid-dose 1  1011 VP/kg; high-dose 2  1011 VP/kg; rAd5-EGFP group 2  1011 VP/kg, and solvent control group (Tris buffer) rAd5-hTERTC27 low-dose 2  1010 VP/kg, 2 male and 1 female; high-dose 9  1010 VP/ kg, 1 male and 2 female; rAd5-EGFP group 9  1010 VP/kg, 1 male and 2 female; solvent control group (Tris buffer), 2 male and 1 female rAd5-hTERTC27 low-dose 1  1010 VP/mL; high-dose 1  1011 VP/mL; rAd5-EGFP group 1  1011 VP/mL; solvent control group (Tris buffer); negative control group, (normal saline) Sensitizing dosages: rAd5-hTERTC27 lowdose7  109 VP/kg; high-dose 3.5  101 VP/ kg positive control group (Ovalbumin), and negative control group (Tris buffer)

–

i.v.

1 mL per ear marginal veins of both sides

rAd5-hTERTC27 low-dose, 1; high-dose, 5

1 (except 0.5 mL per pig in positive control group)

rAd5-hTERTC27 low-dose 2.0  101 VP/kg; mid-dose 6.0  101 VP/kg; highdose1.2  1011 VP/kg rAd5-EGFP group 1.2  101 VP/kg, and solvent control group (Tris buffer)

rAd5-hTERTC27 low-dose, 2; mid-dose, 5; high-dose, 10

i.v. (except intraperitoneal injection in positive control group) i.v.

ASA test

Effects on spontaneous activity; subthreshold hypnosis; Hypnotic synergism

7.0  109 VP/ kg

1.12  1010 VP/kg

20

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weight determinations were conducted on days 1(before injection), 4, 8, 11, and 15. Food consumptions were determined on days 1, 4, 8, and 11. On day 15, blood was obtained from the abdominal aorta. Parameters of hematology, blood coagulation and biochemistry were analyzed by Automatic Five Classification Blood Cell Analyzer (Siemenz: ADVIA2120, German), Automatic Thrombus/Hemostasis Analyzer (Sysmex: CA-7000, Japan) and Automatic Biochemistry Analyzer (Roche: Cobas C311, Switzerland), respectively. Systemic necropsy was performed after rats were killed. 2.4. Single-dose toxicity in Rhesus monkeys Rhesus monkeys (6 males, 4.20–6.75 kg and 6 females, 4.55– 6.10 kg; 2–4 years old) were allowed to acclimatize for 19 days before treatment and randomly divided into four groups with 3 per group (Table 1). General signs were conducted just as that in rats. Body weight determinations were conducted on days 1 (before injection), 7, and 14. Food consumptions were determined on days 3, 7, 13, and ear temperature was on days 1 (before and 2, 4 h after injection), 7, and 14. Hematological, blood coagulation, biochemical parameters were assayed on days 2, 6, and 15. Electrocardiographic (ECG) examinations (standard lead II) were performed with a Four Channels Physiological Recorder (RM6240, Chengdu Instrument Factory) and heart rate(HR), R–R, Q–T, corrected Q–T and P–R intervals, amplitudes of P, R and T waves, time of P and QRS waves, offset of ST were recorded after animals were anesthetized on day 14. Urinalyses were performed on urine samples obtained directly collected from bladders when all animals were sacrificed on day 15. Monkeys were sacrificed by blood depletion after being fasted for 12 h and intravenously anesthetized with 30 mg/kg Pentobarbital Sodium, then systemic necropsy was performed. Weights of heart, liver, spleen, lung, kidney, brain and thyroid were measured and relative organ weight (organ/body or organ/brain weight ratio) were calculated and recorded. 2.5. In vitro hemolytic assay A male rabbit was provided by the Laboratory Animal Farm of Sichuan Professional Committee (Chengdu, China). In vitro hemolytic assay was conducted according to the Guideline of Hemolytic Study Technology by SFDA and the reported method previously (Li et al., 2009). After the rabbit was anesthetized, 20 mL fresh rabbit blood was obtained from the heart, and then the red blood cells were isolated and purified. The purified RBC was resuspended in normal saline to obtain 2% (v/v) of RBC suspension. Then 2.5 mL of the RBC suspension was mixed and incubated with 2.5 mL of the sample solutions (containing 0.5, 0.4, 0.3, 0.2, 0.1 rAd5hTERTC27, 0.5 mL rAd5-EGFP, respectively), 2.5 mL of Tris buffer, 2.5 mL of normal saline or 2.5 mL injection water at 37 °C. Hemolytic effect was observed at 0, 15, 30, 45, 60, 120, and 180 min. Normal saline was used as the negative control with no hemolysis, and injection water was used as the positive control with holohemolysis. 2.6. Local tolerance study in rabbits Rabbits (10 males, 2.305–2.645 kg and 10 females, 2.405– 2.685 kg; 4–5 months old) were randomly divided into four groups with 4 per group, equally by sex (Table 1). The injection site was ear marginal veins of both sides, and the volume of administration was 1 mL per side. General signs were observed and recorded daily for 16 days after administration, especially the changes at the sites of injection. One male and one female rabbits from each group were killed after 48 h, and the rest of the rabbits were sacrificed on day 16 by CO2 inhalation. Vessels and peripheral tissues of

injection sites were histopathologically.

examined

macroscopically

and

2.7. Active systemic anaphylaxis (ASA) test in guinea pigs Guinea pigs were randomly divided into four groups with 6 per group, equally by sex. We defined the day of the initial administration as day 1. Guinea pigs were sensitized with ovalbumin by intraperitoneal injection in positive control group, rAd5-hTERTC27 or Tris buffer by intravenous injection in other groups, every other day, for 5 times (Table 1). On day 19, the sensitizers were injected via the caudal vein of the foot with twice sensitizing dose for ASA challenge. Allergic reaction symptoms were observed and recorded in 30 min after administration of challenge, which included restlessness, piloerection, shivering, scratching nose, sneeze, cough, tachypnea, involuntary urination or diachoresis, lacrimation, dyspnoea, rales, purpura, gait instability, jumping, gasping, spasm, circling, tidal respiration, and even death (Meng et al., 2012; Su et al., 2008). 2.8. Effects on spontaneous activity, hypnotic synergism combined with pentobarbital sodium and effects on hypnosis induced by subthreshold pentobarbital sodium in mice Kunming mice (75 males and 75 females, 17.1–22.4 g; 4 weeks old) were randomly divided into fifteen groups with 10 per group equally by sex. Each five groups were for one of the three tests. Mice were fasted for more than 12 h before test, and were intravenously administered with corresponding samples or Tris buffer (Table 1). 15 min later, spontaneous activity frequency was determined and recorded by ZZ-6 Spontaneous Activity Tester (Taimeng Technology Co., Ltd., Chengdu) in the following 10 min, including activity frequency and standing up frequency; For the hypnotic synergism combined with pentobarbital sodium and the effects on hypnosis induced by subthreshold pentobarbital sodium, each animal was given a predetermined dose of pentobarbital sodium (70 mg/kg or 35 mg/kg) intraperitoneally after 15 min. During the following 30 min, the sleeping duration for the former and the number of mice which had fallen asleep for more than 1 min for the latter were evaluated and recorded by righting reflex (Su et al., 2008; Gerhard Vogel et al., 2013). 2.9. Statistical analysis The quantitative data such as body weight, hematological and blood biochemical parameters were expressed as mean ± standard deviation (SD). Statistical evaluations were performed using the one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test when normality and homogeneity of variance are satisfied, otherwise non-parametric test (Kruskal–Wallis H rank test and Mann–Whitney U rank test) would be used. The qualitative indexes such as routine urinalysis were expressed as frequency. Fisher’s exact probability, Kruskal–Wallis H rank test and Mann–Whitney U rank test were used for statistical analysis. All data were processed and analyzed using SPSS for Windows 13.0. P < 0.05 was considered statistically significant. 3. Results 3.1. Single-dose toxicity in rats 3.1.1. General observation and body weight, food consumption measurement During the observation period of 14 days, rats in all groups showed sound general health, including normal behavior, clean

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Fig. 1. Effects on body weight and food consumption following single-dose administration in rats. A and B show the changes of body weights of rats after administration, which were measured on days 1, 4, 8, 11 and 15. There was no statistical significance between all of the rAd5 groups and solvent control group at the corresponding testing time points (p > 0.05). C and D show food consumption after administration, which was determined on days 1, 4, 8 and 11. There were no obvious abnormal changes comparing all of the rAd5 groups with solvent control group at the corresponding testing time points.

skin and fur, no nausea, vomiting, no abnormal changes in urination or defecation. There was no dead or dying rat at the end of the observation period. Body weights of all rats increased normally throughout the test except that they tended to decrease or remained very little increase at the last weight measurement interval. No significant differences in body weight and food consumption were observed between all of the rAd5 groups (rAd5-hTERTC27 and rAd5-EGFP) and solvent control group at the corresponding testing time points (Fig. 1). 3.1.2. Effects on hematology At the end of the observation period, white blood cell (WBC) count and its differential counts (except eosinophilic granulocyte count), red blood cell (RBC) count, hemoglobin (HGB), platelet count (PLT), prothrombin time (PT), and activated partial thromboplastin time (APTT) fluctuated within normal ranges. There were no statistically significant differences between rAd5 groups and solvent control group. Eosinophilic granulocytes (EOS) of male rats decreased in all of the Ad5 groups, including Ad5-EGFP group. Decreases of EOS percentage were also observed in rAd5-hTERTC27 middle, high-dose groups and rAd5-EGFP group. There were significant differences between the changes above and solvent control group (p < 0.05) (Tables 2 and 3). 3.1.3. Effects on blood biochemistry For blood biochemistry, serum levels of alanine aminotransferase (ALT), aspartate Aminotransferase (AST), creatine kinase (CK), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), glucose (GLU), total bilirubin (TBIL), total cholesterol (CHOL), triglyceride (TG), blood urea nitrogen (BUN, Urea), creatinine (Crea) and electrolytes such as sodium (Na+), potassium (K+) and chloride (Cl ) fluctuated within normal ranges. No significant differences were observed, compared with solvent control group (p > 0.05) (Table 4, Supplement 1).

3.1.4. Systemic autopsy No abnormalities were observed at necropsy in all groups, including the size, appearance, color, texture, etc. of the heart, brain, lung, liver, spleen, kidney, reproductive organs, stomach, intestines and other major organs. There were no abnormal bleeding points or secretions on the surface of these organs. There was no abnormal hydrops in brain. Brain gyri were clear. No bleeding or inflammatory exudation or thickening was observed in the subarachnoid cavity or cerebral pia mater blood vessels. The structure of parenchymatous tissues was clear. 3.2. Single-dose toxicity in Rhesus monkeys 3.2.1. General observation and body weight, food consumption measurement During the observation period of 14 days, there was no death in any of the groups. No obvious adverse effects were observed. Rhesus monkeys in all groups showed sound general health just as the rats above. No significant differences in body weight and temperature were observed between all of the rAd5 groups and solvent control group at the corresponding testing time points. Although decreases in food consumption were observed in both of the rAd5-hTERTC27 groups on day 13 (p < 0.05) compared with the solvent control group, there was no effect on food consumption of all test groups at other testing time points. And the reduced amount of 4 g/day per animal was within normal ranges (Tables 5–7). 3.2.2. Effects on hematology At the end of the observation period, WBC count and its differential counts, RBC count, HGB, hematocrit (HCT), reticulocyte count (RET), PLT, PT and APTT fluctuated within normal ranges. There were no statistically significant differences between the rAd5 groups and solvent control group (p > 0.05) (Supplement 2).

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P.-j. Yue et al. / Regulatory Toxicology and Pharmacology 67 (2013) 53–62 Table 2 Hematological parameters of rats on day 15 following single-dose administration (Female). Parameter

9

WBC (10 /L) NEU (109/L) LYM (109/L) MONO (109/L) EOS (109/L) BASO (109/L) LUC (109/L) NEU% (%) LYM% (%) MONO% (%) EOS% (%) BASO% (%) LUC% (%) RBC (1012/L) HGB (g/L) HCT (%) MCV (fL) MCH (pg) MCHC (g/L) RET RET% (%) PLT (109/L) PT (s) APTT (s)

Solvent control

rAd5-EGFP 2  1011 VP/kg

rAd5-hTERTC27 low-dose 5  1010 VP/kg

rAd5-hTERTC27 middle-dose 1  1011 VP/kg

rAd5-hTERTC27 high-dose 2  1011 VP/kg

n

  SD X

n

  SD X

n

  SD X

n

  SD X

n

  SD X

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

3.97 ± 1.78 0.36 ± 0.11 3.42 ± 1.64 0.048 ± 0.025 0.094 ± 0.025 0.006 ± 0.005 0.040 ± 0.026 9.9 ± 3.0 85.1 ± 3.4 1.34 ± 0.40 2.50 ± 0.61 0.200 ± 0.071 0.94 ± 0.22 6.71 ± 0.27 137 ± 6 39.7 ± 1.5 59.2 ± 2.1 20.5 ± 0.7 346 ± 6 0.198 ± 0.022 2.96 ± 0.45 1274 ± 55 9.0 ± 0.3 11.1 ± 0.7

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

3.14 ± 0.72 0.22 ± 0.12 2.80 ± 0.63 0.040 ± 0.012 0.050 ± 0.016 0.010 ± 0.007 0.026 ± 0.011 6.9 ± 2.9 89.2 ± 3.2 1.22 ± 0.37 1.62 ± 0.40 0.280 ± 0.164 0.80 ± 0.25 6.45 ± 0.27 134 ± 7 38.0 ± 1.3 59.0 ± 1.6 20.8 ± 1.0 353 ± 15 0.192 ± 0.031 2.98 ± 0.55 1321 ± 152 8.8 ± 0.2 12.5 ± 0.9

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

3.35 ± 0.81 0.33 ± 0.08 2.86 ± 0.72 0.056 ± 0.025 0.066 ± 0.015 0.010 ± 0.007 0.034 ± 0.009 10.0 ± 2.3 85.2 ± 1.9 1.62 ± 0.58 2.04 ± 0.44 0.240 ± 0.167 0.98 ± 0.16 6.85 ± 0.22 140 ± 5 40.4 ± 1.7 59.0 ± 2.2 20.5 ± 0.9 348 ± 7 0.201 ± 0.055 2.93 ± 0.77 1315 ± 136 8.8 ± 0.3 12.4 ± 0.7

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

3.84 ± 0.93 0.38 ± 0.16 3.28 ± 0.78 0.064 ± 0.015 0.074 ± 0.030 0.010 ± 0.000 0.036 ± 0.018 9.8 ± 3.1 85.5 ± 2.6 1.64 ± 0.42 1.94 ± 0.56 0.240 ± 0.055 0.96 ± 0.33 6.74 ± 0.49 142 ± 6 40.4 ± 2.0 60.1 ± 2.4 21.1 ± 1.1 351 ± 6 0.217 ± 0.062 3.25 ± 1.04 1369 ± 129 8.8 ± 0.4 12.3 ± 0.8

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

3.14 ± 0.93 0.33 ± 0.10 2.65 ± 0.93 0.048 ± 0.019 0.070 ± 0.028 0.010 ± 0.007 0.034 ± 0.021 11.9 ± 5.7 83.2 ± 5.2 1.46 ± 0.28 2.12 ± 0.62 0.280 ± 0.084 0.96 ± 0.31 6.58 ± 0.52 138 ± 6 39.2 ± 1.8 59.8 ± 2.6 21.1 ± 0.9 352 ± 10 0.207 ± 0.054 3.16 ± 0.82 1262 ± 88 8.8 ± 0.3 11.8 ± 1.1

Table 3 Hematological parameters of rats on day 15 following single-dose administration (Male). Parameter

9

WBC (10 /L) NEU (109/L) LYM (109/L) MONO (109/L) EOS (109/L) BASO (109/L) LUC (109/L) NEU% (%) LYM% (%) MONO% (%) EOS% (%) BASO% (%) LUC% (%) RBC (1012/L) HGB (g/L) HCT (%) MCV (fL) MCH (pg) MCHC (g/L) RET RET% (%) PLT (109/L) PT (s) APTT (s) *

Solvent control

rAd5-EGFP 2  1011 VP/kg

rAd-hTERTC27 low-dose 5  1010 VP/kg

rAd5-hTERTC27 middle-dose 1  1011 VP/kg

rAd5-hTERTC27 high-dose 2  1011 VP/kg

n

  SD X

n

  SD X

n

  SD X

n

  SD X

n

  SD X

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5.94 ± 0.72 0.78 ± 0.32 4.89 ± 0.44 0.114 ± 0.044 0.074 ± 0.005 0.014 ± 0.005 0.064 ± 0.021 12.9 ± 3.9 82.6 ± 3.9 1.88 ± 0.64 1.22 ± 0.08 0.240 ± 0.055 1.06 ± 0.36 6.60 ± 0.44 144 ± 3 40.3 ± 1.8 61.1 ± 2.2 21.9 ± 1.3 358 ± 12 0.357 ± 0.014 5.42 ± 0.37 1311 ± 68 9.1 ± 0.3 12.6 ± 1.2

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5.34 ± 1.05 0.47 ± 0.02 4.71 ± 1.05 0.070 ± 0.012 0.032 ± 0.011* 0.016 ± 0.011 0.038 ± 0.015 9.2 ± 2.0 87.9 ± 2.4 1.30 ± 0.32 0.64 ± 0.25* 0.280 ± 0.110 0.68 ± 0.23 6.38 ± 0.20 141 ± 8 40.4 ± 1.4 63.4 ± 0.9 22.1 ± 0.7 349 ± 9 0.373 ± 0.008 5.85 ± 0.10 1242 ± 106 9.2 ± 0.3 13.2 ± 1.0

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5.03 ± 0.73 0.54 ± 0.13 4.28 ± 0.65 0.098 ± 0.019 0.050 ± 0.007* 0.012 ± 0.004 0.048 ± 0.008 10.8 ± 1.9 85.0 ± 2.0 2.00 ± 0.50 1.00 ± 0.19 0.260 ± 0.089 1.00 ± 0.30 6.58 ± 0.19 141 ± 4 40.5 ± 1.3 61.4 ± 0.7 21.4 ± 0.4 348 ± 10 0.395 ± 0.029 6.00 ± 0.44 1348 ± 90 9.2 ± 0.3 12.6 ± 0.6

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5.48 ± 1.35 0.61 ± 0.28 4.71 ± 1.20 0.070 ± 0.020 0.050 ± 0.010* 0.010 ± 0.007 0.038 ± 0.013 11.3 ± 4.1 85.7 ± 4.4 1.26 ± 0.36 0.94 ± 0.24* 0.140 ± 0.055 0.70 ± 0.21 6.30 ± 0.52 139 ± 2 38.9 ± 2.2 61.9 ± 1.8 22.2 ± 1.7 359 ± 18 0.360 ± 0.068 5.71 ± 0.82 1274 ± 61 9.2 ± 0.4 12.5 ± 1.0

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5.16 ± 1.19 0.61 ± 0.23 4.36 ± 1.12 0.086 ± 0.042 0.044 ± 0.009* 0.014 ± 0.009 0.048 ± 0.019 12.0 ± 4.2 84.3 ± 4.9 1.70 ± 0.73 0.84 ± 0.23* 0.280 ± 0.084 0.88 ± 0.24 6.71 ± 0.22 144 ± 3 41.9 ± 0.7 62.5 ± 2.4 21.5 ± 1.0 344 ± 6 0.373 ± 0.034 5.57 ± 0.55 1392 ± 155 9.2 ± 0.3 13.4 ± 0.7

Compared with solvent control, there was statistical significance in means (p < 0.05).

3.2.3. Effects on blood biochemistry Serum levels of ALP, ALT, AST, TP, ALB, GLU, TBIL, CHOL, TG, CK, BUN, Crea and electrolytes fluctuated within normal ranges. No significant differences were observed, compared with solvent control group (p > 0.05) (Supplement 3). 3.2.4. Urinalysis The level distributions of glucose (GLU), bilirubin (BIL), ketobody (KET), specific gravity (SG), occult blood (BLO), PH value, protein (PRO), urobilinogen (UBO), nitrite (NIT), WBC were within

normal ranges during the observation period of 14 days. No significant abnormalities were noted, compared with that of solvent control group (p > 0.05) (Supplement 4). 3.2.5. ECG examinations (lead II) Obvious increased heart rate and reduced duration of R–R interval were recorded in the rAd5-hTERTC27 high-dose group and rAd5-EGFP group, and there were statistically significant differences compared with that of solvent control group (p < 0.05). No abnormalities were found in Q–T interval, corrected Q–T interval

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Table 4 Blood biochemistry parameters of rats on day 15 following single-dose administration (Male). Parameter

Solvent control

ALP (U/L) ALT (U/L) AST (U/L) CK (U/L) LDH (U/L) Urea (lmol/L) Crea (lmol/L) TP (g/L) ALB (g/L) A/G GLU (mmol/L) TBIL (lmol/L) CHOL (mmol/L) TG (mmol/L) K+ (mmol/L) Na+ (mmol/L) Cl (mmol/L)

rAd5-EGFP 2  1011 VP/kg

rAd-hTERTC27 low-dose 5  1010 VP/kg

rAd-hTERTC27 middle-dose 1  1011 VP/kg

rAd5-hTERTC27 high-dose 2  1011 VP/kg

n

  SD X

n

  SD X

n

  SD X

n

  SD X

n

  SD X

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

238.8 ± 66.3 28.9 ± 2.4 105.8 ± 7.9 612 ± 67 839 ± 181 6.94 ± 0.73 20.8 ± 2.5 54.0 ± 1.6 38.8 ± 1.1 2.55 ± 0.10 6.85 ± 1.11 0.9 ± 0.3 1.44 ± 0.12 0.54 ± 0.21 3.89 ± 0.19 137.8 ± 1.8 100.4 ± 1.3

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

245.8 ± 36.2 29.7 ± 2.8 113.8 ± 7.6 685 ± 135 887 ± 255 5.88 ± 0.72 18.4 ± 1.9 53.3 ± 0.3 38.2 ± 0.7 2.54 ± 0.16 6.63 ± 0.92 1.0 ± 0.2 1.44 ± 0.32 0.67 ± 0.12 3.73 ± 0.06 138.2 ± 1.8 99.9 ± 1.8

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

221.4 ± 35.4 28.1 ± 3.6 110.1 ± 18.5 691 ± 238 984 ± 397 7.10 ± 1.78 20.0 ± 4.1 53.0 ± 1.6 37.3 ± 1.4 2.38 ± 0.08 6.66 ± 0.77 1.0 ± 0.3 1.76 ± 0.26 0.52 ± 0.15 4.00 ± 0.30 136.8 ± 1.3 98.5 ± 1.6

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

242.2 ± 72.9 30.3 ± 2.6 104.1 ± 13.6 608 ± 111 890 ± 189 7.72 ± 1.33 22.2 ± 4.1 52.9 ± 0.8 37.7 ± 1.2 2.50 ± 0.22 6.50 ± 0.63 1.0 ± 0.4 1.35 ± 0.20 0.58 ± 0.12 3.85 ± 0.28 139.0 ± 1.2 101.6 ± 1.0

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

255.2 ± 65.7 26.9 ± 3.2 107.1 ± 13.5 715 ± 188 941 ± 301 6.30 ± 0.66 19.8 ± 2.6 54.2 ± 1.0 39.3 ± 1.0 2.65 ± 0.18 7.05 ± 0.90 1.0 ± 0.3 1.31 ± 0.11 0.61 ± 0.18 4.04 ± 0.18 138.2 ± 1.3 100.3 ± 1.4

Table 5 Body weight of Rhesus monkeys following single-dose administration (kg). Time

Solvent control

Acclimation period(first) Acclimation period(second) Day 1 Day 7 Day 14

rAd5-EGFP 9  1010 VP/kg

rAd5-hTERTC27 low-dose 2  1010 VP/kg

rAd5-hTERTC27 high-dose 9  1010 VP/kg

n

  SD X

n

  SD X

n

  SD X

n

  SD X

3 3 3 3 3

5.63 ± 0.99 5.57 ± 1.11 5.32 ± 1.11 5.35 ± 1.05 5.42 ± 1.09

3 3 3 3 3

5.43 ± 1.16 5.15 ± 0.95 5.07 ± 0.95 4.98 ± 0.83 4.97 ± 0.73

3 3 3 3 3

5.83 ± 0.35 5.72 ± 0.28 5.52 ± 0.23 5.52 ± 0.21 5.58 ± 0.20

3 3 3 3 3

5.50 ± 0.28 5.42 ± 0.23 5.07 ± 0.16 5.07 ± 0.12 5.13 ± 0.14

Table 6 Food consumption of Rhesus monkeys following single-dose administration (g/day). Time

Day 3 Day 7 Day 13 *

rAd5-EGFP 9  1010 VP/kg

Solvent control

rAd5-hTERTC27 low-dose 2  1010 VP/kg

rAd5-hTERTC27 high-dose 9  1010 VP/kg

n

  SD X

n

  SD X

n

  SD X

n

  SD X

3 3 3

201.0 ± 0.9 201.2 ± 0.7 204.6 ± 4.4

3 3 3

200.7 ± 0.4 200.7 ± 0.5 201.3 ± 0.6

3 3 3

200.9 ± 0.5 201.1 ± 0.8 200.9 ± 0.1*

3 3 3

200.6 ± 0.2 201.6 ± 0.2 200.7 ± 0.0*

significant difference in the mean compared with the solvent control group (p < 0.05).

Table 7 Temperature of Rhesus monkeys following single-dose administration (°C). Time

Acclimation period(first) Acclimation period(second) Before administration 2 h after administration 4 h after administration Day 7 Day 14

Solvent control

rAd5-EGFP 9  1010 VP/kg

rAd5-hTERTC27 low-dose 2  1010 VP/kg

rAd5-hTERTC27 high-dose 9  1010 VP/kg

n

  SD X

n

  SD X

n

  SD X

n

  SD X

2 3 3 3 3 3 3

37.7 38.4 38.9 ± 0.5 37.0 ± 0.3 37.3 ± 0.5 37.2 ± 0.2 37.1 ± 0.4 37.1 ± 0.1

2 3 3 3 3 3 3

38.3 39.0 38.5 ± 0.9 37.7 ± 0.7 37.1 ± 0.0 37.2 ± 0.3 37.2 ± 0.2 37.4 ± 0.3

3 3 3 3 3 3 3

38.2 ± 0.2 38.8 ± 0.5 37.7 ± 0.4 36.9 ± 0.1 37.4 ± 0.6 37.2 ± 0.4 37.3 ± 0.3

3 3 3 3 3 3 3

38.6 ± 0.7 38.3 ± 1.0 38.3 ± 1.0 37.1 ± 0.2 37.2 ± 0.3 37.0 ± 0.2 37.2 ± 0.3

(QT/RR1/2), amplitude of R wave, amplitude and time of P wave, P– R interval, time of QRS wave, amplitude of T wave and ST-T wave (p > 0.05) (Fig. 2, Supplement 5).

significant differences in major organ weight and coefficient between the treated and control groups (p > 0.05) (Supplement 6, Tables 8 and 9).

3.2.6. Systemic autopsy No abnormalities were observed at necropsy in all groups, including the size, appearance, color, texture of the heart, brain, lung, liver, spleen, kidney and other major organs. There were no

3.3. In vitro hemolytic assay After 3 h incubation at 37 °C, it was observed that RBCs in the negative control tube (NO. 8) subsided, the supernatant became

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Fig. 2. ECG examination of Rhesus monkeys following single-dose administration. 1st and 2nd stand for the first and second time ECG examination in the acclimation period of Rhesus monkeys, respectively. Data were presented as the mean ± SD (n = 3). ⁄p < 0.05, as compared with that of solvent control group. No abnormalities were found in P–R interval, time of QRS wave, Q–T interval (p > 0.05).

Table 8 Organ/body weight ratio of Rhesus monkeys on day 15 following single-dose administration (g/100 g body weight). Parameter

Brain Heart Liver Spleen Lung Thymus Kidneys Adrenal glands Ovaries Uterus Testes Epididymis

Solvent control n

  SD X

3 3 3 3 3 3 3 3 1 1 2 2

2.032 ± 0.211 0.436 ± 0.019 1.827 ± 0.202 0.092 ± 0.011 0.731 ± 0.093 0.040 ± 0.008 0.391 ± 0.037 0.015 ± 0.005 0.005 0.064 0.053 0.034 0.028 0.025

rAd5-EGFP 9  1010 VP/kg

rAd5-hTERTC27 low-dose 2  1010 VP/kg

n

  SD X

n

  SD X

rAd5-hTERTC27 high-dose 9  1010 VP/kg n

  SD X

3 3 3 3 3 3 3 3 2 2 1 1

2.097 ± 0.407 0.424 ± 0.026 1.942 ± 0.134 0.094 ± 0.030 0.759 ± 0.120 0.043 ± 0.028 0.364 ± 0.043 0.017 ± 0.004 0.005 0.010 0.087 0.224 0.032 0.017

3 3 3 3 3 3 3 3 1 1 2 2

1.865 ± 0.043 0.435 ± 0.027 1.832 ± 0.077 0.082 ± 0.020 0.683 ± 0.085 0.078 ± 0.041 0.373 ± 0.046 0.013 ± 0.002 0.003 0.094 0.022 0.049 0.023 0.026

3 3 3 3 3 3 3 3 2 2 1 1

1.997 ± 0.219 0.421 ± 0.022 1.812 ± 0.201 0.073 ± 0.007 0.639 ± 0.009 0.058 ± 0.032 0.386 ± 0.027 0.016 ± 0.004 0.006 ± 0.006 0.253 ± 0.092 0.035 0.017

Table 9 Organ/brain weight ratio of Rhesus monkeys on day 15 following single-dose administration (g/g brain weight). Parameter

Heart Liver Spleen Lung Thymus Kidneys Adrenal glands Ovaries Uterus Testes Epididymis

Solvent control n

  SD X

3 3 3 3 3 3 3 1 1 2 2

0.216 ± 0.023 0.900 ± 0.065 0.045 ± 0.003 0.366 ± 0.087 0.020 ± 0.004 0.194 ± 0.031 0.007 ± 0.002 0.002 0.029 0.030 0.016 0.015 0.011

rAd5-EGFP 9  1010 VP/kg

rAd5-hTERTC27 low-dose 2  1010 VP/kg

n

  SD X

n

  SD X

n

  SD X

3 3 3 3 3 3 3 2 2 1 1

0.209 ± 0.054 0.942 ± 0.119 0.047 ± 0.024 0.375 ± 0.108 0.019 ± 0.010 0.175 ± 0.014 0.009 ± 0.003 0.002 0.006 0.042 0.132 0.013 0.007

3 3 3 3 3 3 3 1 1 2 2

0.233 ± 0.013 0.982 ± 0.032 0.044 ± 0.011 0.366 ± 0.037 0.042 ± 0.021 0.200 ± 0.024 0.007 ± 0.001 0.002 0.052 0.012 0.026 0.013 0.014

3 3 3 3 3 3 3 2 2 1 1

0.212 ± 0.021 0.913 ± 0.123 0.037 ± 0.004 0.323 ± 0.041 0.029 ± 0.015 0.196 ± 0.036 0.008 ± 0.002 0.003 0.003 0.116 0.052 0.017 0.008

colorless and transparent. And there were no hemolytic and aggregation phenomenon when RBCs were dispersed again by shaking appropriately. There was no RBC left in the bottom of the positive

rAd5-hTERTC27 high-dose 9  1010 VP/kg

control tube (NO. 9), and solution became red completely, which indicated that holo-hemolysis phenomenon occurred and this test system was reliable. No hemolytic effect was observed in all of the

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P.-j. Yue et al. / Regulatory Toxicology and Pharmacology 67 (2013) 53–62 Table 10 Spontaneous activity of mice following single-dose administration (times/10 min). Group

Solvent control group Ad5-EGFP group Ad5-hTERTC27 (low-dose) Ad5-hTERTC27 (middle-dose) Ad5-hTERTC27 (high-dose)

Fig. 3. Representative hemolytic effect after 3 h incubation at 37 °C. Tubes 1–2 to 5–2 were rAd5-hTERTC27 groups, Tube 6–2 was rAd-EGFP group, Tube 7–2 was Tris buffer group, Tube 8–2 was negative control group, Tube 9–2 was positive control group (The ‘‘2’’ of Tube x-2 stands for the second hemolytic assay). There were no hemolysis and aggregation phenomenon in Tubes 1–2 to 7–2, which was just the same as that of the negative control group (Tube 8–2). In Tube 9–2, solution became red completely, which indicated holo-hemolysis phenomenon occurred.

rAd5-hTERTC27 tubes, rAd5-EGFP tubes and Tris buffer tubes. The results were the same as that of the negative control tube (Fig. 3). 3.4. Local tolerance study During the observation period of 16 days, there were no related incompatibility reactions such as reddening and swelling at the administration site. For the histopathological examination, slight perivasculitis was observed at one injection site in the negative control group on day 16. The abnormality only happened in the negative control group and was very slight, which was probably resulted from injection procedure itself. On the whole, a single administration of rAd5-hTERTC27 into ear marginal veins caused no macroscopical or microscopic effects which differed from the control. 3.5. ASA test in guinea pigs During the period of sensitization, guinea pigs in all groups were in good condition. No abnormalities were observed. In the initial 30mins after challenge, no animal showed any signs of anaphylaxis except for positive controls, in which systemic symptoms such as restlessness, jumping, convulsive spasms were observed and all 6 animals died unexpectedly within 10 min before proper euthanasia could be administered. The result of ASA challenge test by rAd5-hTERTC27 at dose of 3.5  1010 and 7  109 VP/kg showed no evidence of hypersensitivity. 3.6. Effects on spontaneous activity, hypnotic synergism combined with pentobarbital sodium and effects on hypnosis induced by subthreshold pentobarbital sodium in mice Activity frequency and standing up frequency in the period of 10 min in all groups showed no significant difference comparing the frequencies of all rAd5 groups with that of the control group (p > 0.05) (Table 10). After the administration of pentobarbital sodium intraperitoneally, the sleeping duration in high-dose group of rAd5-hTERTC27 (116 ± 29 min) and rAd5-EGFP group (115 ± 33 min) was longer than that in control group (70 ± 21 min) (p < 0.05). There was no significant difference in sleeping duration between middle-dose (90 ± 33 min), low-dose group (81 ± 34 min) of rAd5-hTERTC27 and control group

Frequency of spontaneous activity (times/10 min)

Frequency of standing up (times/ 10 min)

n

  SD X

n

  SD X

10 10 10 10 10

53 ± 28 41 ± 23 46 ± 27 53 ± 36 43 ± 26

10 10 10 10 10

108 ± 22 100 ± 36 105 ± 30 101 ± 44 110 ± 52

(p > 0.05). There was no significant difference in the ratios of mice which had fallen asleep between all rAd5 groups (2/10, 3/10, 3/10, 2/10 animals in rAd5-hTERTC27 low-dose, middle-dose, high-dose groups and rAd5-EGFP group respectively) and control group (2/10 animals) (p > 0.05). So rAd5-hTERTC27 at the dose of 1.2  1011 VP/ kg might enhance the effect of hypnosis of pentobarbital sodium, and prolonged the sleeping duration of mice; rAd5-hTERTC27 at the dose of 2  1010, 6  1010 and 1.2  1011 VP/kg had no effect on the hypnosis induced by subthreshold pentobarbital sodium in mice.

4. Discussion The safety assessment of rAd5-hTERTC27 was evaluated in a series of in vivo and in vitro experiments in accordance with Chinese Guidance for Human Gene Therapy and Product Quality Control, and the Guideline of General Pharmacological Research Technology issued by the SFDA. Determination of the dose levels in single-dose toxicity studies was based on results from our previous in vivo tumor treatment study in C57BL/6 mice model of HCC, in which 5  107 PFU rAd5-hTERTC27 per mouse inhibited the tumor growth significantly(Huang et al., 2007; He et al., 2013; Su et al., 2008). In the single-dose toxicity test, body weights of all rats increased normally during days 1 through 11 and remained stable between days 11 and 15. There were no abnormal signs of toxicity in the animals and the failure to gain weight between days 11 and 15 was considered possibly due to the low body weights of the young animals and effect of the 12 h fasting prior to collection of blood samples on day 15. For hematological response, decreases of EOS counts in all of the rAd5 groups, EOS percentage in rAd5-hTERTC27 middle, high-dose groups and rAd5-EGFP group of male rats had significant differences compared with that in solvent control group. In view of transient, dose-related suppression of lymphocytes, monocytes, eosinophils and platelets had been observed in the clinical trials of rAd.p53 (SCH58500) (Reid et al., 2002), the decrease of EOS count and percentage might be due to rAd5-hTERTC27 and rAd5EGFP. Furthermore, there was a similar change trend of EOS count and percentage in female rats, although no statistical differences were found compared with that of the solvent control group. However, the decrease of cell count and percentage only occurred in EOS among all the hematological parameters, and no abnormal clinical signs were observed in rats, so it is hard to draw a conclusion from the data in hand. For ECG examinations, the abnormalities of heart rate and R-R interval in the rAd5-hTERTC27 high-dose group and rAd5-EGFP group which fluctuated within broader ranges during the acclimation period, were within normal ranges. So the abnormalities may be resulted from physiological fluctuations. Single-dose toxicity studies demonstrated that the highest dose levels, 2  1011 VP/kg for rats, 9  1010 VP/kg for monkeys were

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not toxic evidently. No drug-related adverse effects were observed in all animals. Previous studies have reported that tail vein administration of Ad to normal SD rats caused obvious dose-dependent symptoms within 5–10 min, including prostration, lethargia, tachypnea, and cyanosis. At a dose of 1.2  1012 VP/kg, the symptoms were reversible and rats recovered within 1 h with no apparent lasting symptoms (Smith et al., 2004a; Smith et al., 2004b; Xu et al., 2010). The dose 1  1012 VP/kg for Rhesus monkeys has been proved to be safe in another study with Ad5 vector (Nunes et al., 1999). The results of our present studies were consistent with these findings. In order to explore the potential influences of rAd5-hTERTC27 intravenously on the central nervous system in mice, we conducted three studies including effects of rAd5-hTERTC27 on spontaneous activity, hypnotic synergism, and hypnosis induced by subthreshold pentobarbital sodium. Effects of rAd5-hTERTC27 on spontaneous activity and hypnosis induced by subthreshold pentobarbital sodium in mice were negative. In the hypnotic synergism test combined with pentobarbital sodium, rAd5-hTERTC27 and rAd5-EGFP at the dose of 1.2  1011 VP/ kg might enhance the effect of hypnosis of pentobarbital sodium, and prolong the sleeping duration of mice. The dose was equivalent to 10 times the effective dose of mice, and hypnotic synergism was not observed at the doses of 2  1010 and 6  1010 VP/kg, which were 2 and 5 times higher than the effective dose in mice. In the in vitro hemolytic assay and ASA test, the results did not reveal any evidence for hemolysis or anaphylaxis, which further supported the potential of clinical use. According to our previous studies in mice, estimated effective dose of rAd5-hTERTC27 in human intravenously was approximately 1.2  109 VP/kg. In a Phase I clinical trial of intravenously administered Ad5CMV-p53 in patients with advanced cancer, the dose of 1  1012 VP IV daily for 3 days every 28 days was safe and recommended (Tolcher et al., 2006). The results from clinical trials of rAd.p53 (SCH58500) administered by hepatic artery infusion indicated that 2.5  1013 particles was the maximum tolerated dose (MTD) both with single and repeated doses, and frequent related adverse events included fever, lymphopenia, chills and thrombocytopenia (Reid et al., 2002). These adverse events occurred in the clinical trial above were not observed in our singledose toxicity evaluations in rats and monkeys. In addition, compared with the doses used in the clinical trials above, the estimated effective dose 1.2  109 VP/kg of rAd5-hTERTC27 in human was at a lower level, which also implied that it might be feasible in subsequent clinical trials. Although the results of this study are encouraging for systemic delivery of Ad vectors, an immunocompetent host will generate immune responses within hours of infection and then adaptive responses occur over the course of two weeks (Brenner, 1999; Khare et al., 2011; Liu et al., 2003), which may lead to systemic toxicity, inhibit uptake of the virus vector, promote clearance from the blood, and drastically limit vector transduction efficiency (Ahi et al., 2011). Thus, although the presented initial set of safety studies showed that rAd5-hTERTC27 was well tolerated, further studies are planned in rats and monkeys to evaluate the potential toxicity and immunogenicity as well as distribution before commencement of clinical work.

5. Conclusion Administration of rAd5-hTERTC27 was well tolerated when tested in an initial set of safety studies, although slight hematological change was observed in rats. Further studies will be conducted to assess more information on the potential toxicity, immunogenicity and distribution of rAd5-hTERTC27.

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