ToxSci Advance Access originally published online on August 14, 2008
Toxicological Sciences 2008 106(1):242-250; doi:10.1093/toxsci/kfn168
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Toxicology Profiles of a Novel p53-Armed Replication-Competent Oncolytic Adenovirus in Rodents, Felids, and Nonhuman Primates
,1,1
,2
* Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Second Military Medical University, Shanghai 200438, China
Vector Gene Technology Company, Ltd., Beijing 100176, China
Xinyuan Institute of Medicine and Biotechnology, Zhejiang Sci-Tech University, Hangzhou 310009, China
2 To whom correspondence should be addressed at Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, No. 225 Changhai Road, Shanghai 200438, China. E-mail: qianqj{at}sino-gene.cn.
Received June 11, 2008; accepted August 6, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Conditionally replicating adenovirus (CRAd) has demonstrated to be safe in clinical studies. We generated a triple-regulated p53-armed CRAd, SG600-p53, in which the partially deleted E1a and E1b genes are regulated under the human telomerase reverse transcriptase promoter and the hypoxia response element. SG600-p53 was proven to be effective both in vitro and in vivo. In this study, the preclinical safety profiles of SG600-p53 in animal models were investigated. SG600-p53 had no adverse effects on mouse behavioral and nervous systems at 1.0 x 1011 viral particles (VP)/kg, 2.0 x 1011 VP/kg and 4.0 x 1011 VP/kg doses, and on cat cardiovascular and respiratory systems at 2.0 x 1010 VP/kg, 4.0 x 1010 VP/kg, and 8.0 x 1010 VP/kg doses. In acute toxicity test in mice, the maximum tolerated dose (2.5 x 1013 VP/kg) induced cachexia, decreased activity, and eye closure in 9/20 mice which could be self-resolved within 30 min. Sensitized by five repeated ip injections at 1.0 x 1010 VP/kg each ip and excitated by one iv injection at 1.0 x 1011 VP/kg, guinea pigs did not show any sign of systemic anaphylaxis. In repeat-dose toxicological studies, the no-observable-adverse-effect levels of SG600-p53 in rats (1.0 x 1011 VP/kg) and cynomolgus monkeys (5.0 x 1011 VP/kg) were 12-fold and 60-fold of the proposed clinical dose, respectively. Intramuscular injections of SG600-p53 in cynomolgus monkeys caused inflammation at injection sites, which was alleviative at the end of observation period. The anti-virus antibody was produced in animal sera and decreased gradually 4 weeks later. No histopathological changes were found by bone marrow examination. Our data in different animal models suggest that SG600-p53 is a safe antitumor therapeutic agent.
Key Words: conditionally replicating adenovirus; human telomerase reverse transcriptase; hypoxia response element; p53 gene; safety evaluation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Viruses are frequently engineered as gene transfer vectors for cancer gene therapy. Recently, modification of the viral genome is occurring to make the virus vectors target the tumor cells, and propagate specifically in tumor cells but not in normal cells, thus the tumor cells are lysed (Mathis et al., 2005
). This is the concept of oncolytic therapy by the conditionally replicative adenovirus (CRAd), such as H101 item (Lu et al., 2004), which is an E1B-55 kDa gene-deleted adenovirus and similar to Onyx-015, by Shanghai Sunway Biotech Company. H101 oncolytic adenovirus had already completed phase III clinical trials and was granted to markets in 2005. Although antitumor efficacy has been inconsistent, the encouraging safety of CRAd vectors has been demonstrated in clinical trials to date. Data from Onyx-015 in phase I–III trials reported that the virus was generally well-tolerated by intratumoral, intraperitoneal, hepatic arterial, and intravenous administration, and no maximum tolerated dose (MTD) was identified (Kirn, 2001). The most common associated toxicities were flu-like symptoms. No dose-limiting toxicity (DLT) was identified at doses up to 2.0 x 1012 viral particles (VP) by intratumoral injections (Ganly et al., 2000) and by hepatic artery administration (Reid et al., 2001), or at doses up to 2.0 x 1013 VP by intravenous administration (Nemunaitis et al., 2000). Further studies at dose escalation were limited by virus supplies. Data from other CRAds in clinical trials were also demonstrated to be well-tolerated at high doses (Page et al., 2007; Nettelbeck, 2003). The clinical development of CRAds has great potential in cancer therapy. However, this novel therapeutic approach still demands an optimization in the improvement of CRAd efficacy and safety.
Because the occurrence of cancers involves some gene changes (Cha and Dematteo, 2005; Giehl, 2005; Ha and Califano, 2006; Payne and Kemp, 2005), treatment with gene therapy by transferring the therapeutic gene into the cancer cells or other somatic cells to rectify the gene blemish may be a viable option. In the candidates of therapeutic genes, p53 was studied extensively (Lutz and Nowakowska-Swirta, 2002; Soussi and Lozano, 2005). A great deal of data confirmed that p53 can really repress the tumor cell growth, induce cancer cell apoptosis (Haupt and Haupt, 2004) and cellular senescence that was associated with differentiation and the upregulation of inflammatory cytokines (Xue et al., 2007). p53 also can increase the sensitivity of cancer cells to chemotherapy and radiotherapy (Matsubara et al., 1999; Pirollo et al., 2000). In addition, normal cells are well-tolerated of overexpression of wild type p53 in tumor-bearing mice (Ventura et al., 2007). Based on the received preclinical validations, Gendicine, a recombinant adenovirus p53 injection, was approved to come into the market in China in 2004 (Guan et al., 2005), and has not yet noted DLT or adverse events for the treatment of cancers, except transient fever after Gendicine administration.
Based on studies of CRAd vectors and cancer gene therapy for several years, we constructed a triple-regulated gene-oncolytic adenovirus SG600-p53. It was modified from human adenovirus type 5, in which the E1a gene is controlled by the promoter of human telomerase reverse transcriptase (hTERT), and the E1b promoter is replaced by a cis-element of five copies of hypoxia regulatory element (HRE). The E1a gene was synchronously deleted of 24 nucleotides (nt 923–946). The p53 expression cassette (CMV promoter, p53 cDNA, SV40 poly A) was inserted into the adenovirus genome between the E1a and E1b genes. Our preclinical studies demonstrated that SG600-p53 exerts much more antitumor efficacy by combining viral oncolytic therapy with p53 gene therapy, and provides a new strategy for human cancer treatment (Wang et al., 2008).
In the proposed open-label phase I clinical trial, we will determine the safety and feasibility of intratumoral SG600-p53 injections in patients who are between ages of 18–78 and histopathologically confirmed unresectable stage IIIB–VI nonsmall cell lung cancer (NSCLC) with chest invasion. The intended clinical dose of SG600-p53 is 5.0 x 1011 VP per time per tumor focus (equal to 8.3 x 109 VP/kg). The phase I clinical trial is a dose escalation study, and escalating dosage levels in five dose groups will be investigated from 1.0 x 108 to 1.0 x 1010 VP/kg. The sample size of six patients in every group will be evaluated. A total of five intratumoral injections of SG600-p53 once every other day will be administered to the participating patients under the guidance of computed tomography. The MTD is defined as the dose at which no more than one third of patients experience DLT after treatment. Immunogenicity will be evaluated by adenoviral-specific neutralizing antibody. Patients will be discharged one week after the last study injection and be followed weekly for 3 weeks and monthly thereafter for 3 months. In this study, in preparation for clinical trials of this recombinant gene-oncolytic adenovirus injection, the potential adverse effects, or the safety, of SG600-p53 were assessed in mice, rats, cats, guinea pigs and cynomolgus monkeys in accordance with Chinese Good Laboratory Practice standards and Chinese guidance for human gene therapy and its product quality control.
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MATERIALS AND METHODS |
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Safety pharmacology test in mice and cats.
When observing the influence of drugs on behavioral and nervous systems, the drug response of mice are close to that of human beings. One hundred and twenty Kunming mice (Experiment Animal Center of Chinese Military Medical Academy, Beijing, China), aged 4–6 weeks, weighed 18–22 g, male and female each occupying one half, were assigned into four groups semirandomly by sex, and injected with SG600-p53 at 1.0 x 1011 VP/kg, 2.0 x 1011 VP/kg, 4.0 x 1011 VP/kg doses in three experimental groups, and the matched control group was given 0.9% sodium chloride (Raowang Pharmaceutic Company of Qinzhou, Shandong, China) by single injection through tail vein. The effects on the behavioral and nervous systems were evaluated by assessing the moving activity in 10 mice in every group. In addition, 10 mice in every group were managed following ip injection of 30 mg/kg sodium pentabarbital 1 h after SG600-p53 treatment, and the number of mice with loss of retroflexion reflex and sleep delitescence were recorded. The other 10 mice in every group were observed their abilities to climb a 45°-gradient wire netting 50 min after SG600-p53 treatment.
To observing the influence of drugs on cardiovascular and respiratory systems, the response of cats is steady and close to that of human beings. Sixteen cats (Experiment Animal Breeding Center of Beijing Fuhao Company, Beijing, China), aged 2–3 years, weighed 2–3 kg, male and female each occupying one half, were also separated into four groups and injected SG600-p53 at 2.0 x 1010 VP/kg, 4.0 x 1010 VP/kg, 8.0 x 1010 VP/kg doses in three experimental groups, and the control group was given 0.9% sodium chloride by single injection through tail vein. Cats in every group were anesthetized with 5 ml/kg of 20% urethane and observed for changes of cardiovascular and respiratory systems, including the heart rate, P wave voltage and interval, P–R interval, QRS voltage and interval, Q–T interval, systolic pressure, diastolic pressure, mean arterial pressure, breath frequency and depth, at 0, 5, 10, 15, 30, 45, 60, 90, 120 min after injection.
Acute toxicity test in mice.
Mouse is one of approved experimental animals, and has advantages of steady response to drugs and convenience in breeding. Forty Kunming mice, aged 3–4 weeks, weighed 18–22 g, male and female each occupying one half, were divided into two groups. Mice in the experimental group were given a maximal concentration of SG600-p53 (1.0 x 1012 VP/ml, 25 ml/kg), which was calculated to be 2.5 x 1013 VP/kg and 3000-fold of the proposed clinical dose, by im single injection in both hind legs. Mice in the control group were given 0.9% sodium chloride in the same volume. After injection, the average food consumption, body weight and lethality of mice were observed for 2 weeks.
Systemic anaphylaxis test in guinea pigs.
Guinea pigs are sensitive to various allergens and easy to establish the models of anaphylactic shock. Fifteen male Hartley guinea pigs (Animal Breeding Center of Beijing Keyu Company, Beijing, China), aged 2–3 months, weighed 250–400 g, were divided into three groups. Guinea pigs in the SG600-p53 group were given 5 ip injections of SG600-p53 at 5.0 x 1010 VP/kg dose once every other day to prime for anaphylaxis. Ten days after the final priming injections, 1.0 x 1011 VP/kg of SG600-p53 was injected by iv to excitated. Guinea pigs in the positive control group were given 20 mg/1.0 ml of human albumin (Beisheng Pharmaceutic Company, Guangxi, China) and those in the negative control group were given 0.9% sodium chloride in the same volume. The animals were weighed on the day of anaphylaxis-priming and -excitation injections, and the positive anaphylaxis symptoms were observed and recorded continuously for 3 h after the iv excitation injection. The positive anaphylaxis symptoms included 20 exhibitions of the uneasy behavior, hair standing, trembling, scratching nose, sneeze, cough, short of breath, micturition, defecating, lachrymation, dyspnea, rale, purpura, unsteady pace, jumping, breathless, convulsion, thwartwise turn, tide-type breath, and death.
Repeat-dose toxicity test in rats.
Wistar rats are internationally approved experimental animals for drug repeat-dose toxicity test, with advantages of steady response to drugs, clear genetic background, and small individual difference. One hundred and twenty Wistar rats (Experiment Animal Center of Chinese Military Medical Academy), aged 6–7 weeks, male (weighed 230.42 ± 0.18 g) and female (weighed 249.97 ± 2.06 g) each occupying one half, were divided into four groups, 30 mice in every group, and injected with SG600-p53 by im injections at 1.0 x 1011 VP/kg, 3.0 x 1011 VP/kg, 1.0 x 1012 VP/kg doses, respectively, daily for 2 weeks. Rats in the control group were given the same volume of Dulbecco's phosphate buffered saline (PBS) buffer. After finishing injections, the general symptoms, body weight, body temperature and food consumption of rats were observed continuously for 4 weeks. The hematological indices, including white blood cell count (WBC), reticulocyte count (Ret), platelet count (PLT), aspartate aminotransferase (AST), red blood count (RBC), hemoglobin (HGB), hematocrit (HCT), albumin (ALB), total bilirubin (TBil), mean corpusular hemoglobin (MCH), mean corpusular volume (MCV), mean corpusular hemoglobin concentration (MCHC), alkaline phosphatase (ALP), creatine kinase (CK), glucose (GLU), blood urea nitrogen (BUN), total protein (TP), creatinine (Cr), triglyceride (TG), and total cholesterol (TCHO), were observed at the end of 2-week injection period and the end of 4-week observation period. The parameters were assessed increase or decrease on the basis of absolute changes that overran mean ± 2 SD of the control group. Ten rats in every group were anesthetized with ether 24 h after the last injection, killed and examined for macroscopic pathological changes, organ weight and organ index (organ index = organ weight/body weight x 100%). Quantitative PCR by TaqMan fluorescent probe was performed to examine the distribution of viruses in rat organs. The probe was labeled FAM reporter gene in the 5' end and TAMRA quenching gene in the 3' end, the forward primer: 5'-aaa tcc ggt gac tga aaa tga-3', the reverse primer: 5'-ggc cat ttc ttc ggt aa-3', the probe: 5'-cat att atc tgc cac gga ggt gtt-3'. The PCR products were 65 bp in length. The specific anti-virus antibody IgG titers in 40 µl of rat sera, and p53 expression in every organ tissue were measured by the enzyme-linked immunosorbent assay (ELISA). Fifty milligrams of organ tissues were homogenized and assessed for p53 expression with human p53 ELISA kit (Bender Medsystems, Inc., Burlingame, CA). Ten rats in every group were killed 24 h after the last injection for examination of hematology, blood biochemistry and anatomic pathology. The remaining 10 animals were killed at the end of observation period to observe the recovery of toxic effects and possible delayed toxicity.
Repeat-dose toxicity test in cynomolgus monkeys.
Cynomolgus monkeys are internationally approved experimental animals for drug repeat-dose toxicity test, and their physiological characteristics are similar to those of human beings. Twenty four cynomolgus monkeys (Luchen Science and Technology Company, Guangxi, China), aged 3–6 years, weighed 3.5–5.3 kg, male and female each occupying one half, were divided into four groups. SG600-p53 was given by im injections at 5.0 x 1010 VP/kg, 1.5 x 1011 VP/kg, 5.0 x 1011 VP/kg doses in three groups, respectively, daily for 2 weeks. Animals in the control group were given the same volume of Dulbecco's PBS buffer. After finishing injections, the general symptoms, body weight, body temperature, electrocardiogram, hematological indices, blood biochemistry, prothrombin time, urine examination, ophthalmic examination and any evidence of systemic or partial anaphylaxis symptoms of animals were observed and noted continuously for 4 weeks. The anti-virus antibody titers in sera were measured. At the end of 2-week injection period and the end of 4-week observation period, the animal organ weight and index were calculated, and the bone marrow and organs were examined histopathologically. Quantitative PCR by TaqMan fluorescent probe was performed to examine the distribution of viruses in every organ tissue, and ELISA assay was performed to examine the p53 expression in every organ tissue and mouse sera as described above.
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RESULTS |
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Safety Pharmacology Test
The safety pharmacology test was assessed in mice by single injection of SG600-p53 through tail vein at 1.0 x 1011 VP/kg, 2.0 x 1011 VP/kg, 4.0 x 1011 VP/kg doses in three experimental groups, respectively. The results demonstrated that SG600-p53 had no adverse effects on behavioral and nervous systems. The quantity of independent activity within 10 min in the high-dose group (4.0 x 1011 VP/kg) was similar to that in the control group (283.4 ± 180.6 times versus 300.6 ± 131.7 times, p > 0.05). The activity quantity in the mid-dose group (2.0 x 1011 VP/kg) and in the low-dose group (1.0 x 1010 VP/kg) was slightly decreased compared with the control group (231.7 ± 81.87 times in the mid-dose group, 210.8 ± 56.68 times in the low-dose group, versus 300.6 ± 131.7 times in the control group), but these were not statistically significant (p > 0.05). Within 30 min in the test of hypnotic effects of pentobabital sodium, lost retroflexion reflex appeared in 2/10 animals in the mid-dose group and 1/10 animal both in the low- and high-dose groups. Within 10 min after treatment in the test of wire netting climb, no animal in any group fell from the wire netting.
The safety pharmacology test was studied to observe the effect of SG600-p53 on cardiovascular and respiratory systems in cats. 2.0 x 1010 VP/kg in the low-dose group, 4.0 x 1010 VP/kg in the mid-dose group, 8.0 x 1010 VP/kg in the high-dose group of SG600-p53 were injected by tail vein. At the time-points of 0, 5, 10, 15, 30, 45, 60, 90, 120 min after injection, the heart rates were ranging from 283.00 ± 141.65 to 318.63 ± 170.45, 178.79 ± 20.74 to 240.97 ± 85.37, 240.98 ± 118.35 to 288.65 ± 130.10 times/min in the low-dose, mid-dose and high-dose groups, respectively, compared with 280.96 ± 145.23, 175.82 ± 11.71, 237.32 ± 124.31 times/min in three groups before injection. The average artery pressures were ranging from 17.46 ± 1.82 to 19.47 ± 2.51, 17.90 ± 0.85 to 18.66 ± 0.18, 16.24 ± 3.40 to 18.43 ± 2.82 KPa in the low-dose, mid-dose and high-dose groups, respectively, compared with 19.44 ± 2.38, 18.54 ± 0.66, 17.81 ± 2.22 KPa in three groups before injection. The breath frequencies were ranging from 62.04 ± 10.08 to 76.65 ± 17.52, 59.91 ± 4.16 to 80.33 ± 4.98, 64.18 ± 15.42 to 75.15 ± 15.88 times/min in the low-dose, mid-dose and high-dose groups, respectively, compared with 69.50 ± 15.39, 68.45 ± 9.20, 72.94 ± 19.77 times/min in three groups before injection. After treatment with different doses of SG600-p53, the index signs of the cardiovascular and respiratory systems of cats at any monitoring time-point were not different from those before treatment (p > 0.05).
Acute Toxicity Test
The acute toxicity test was assessed by giving the maximal concentration of SG600-p53 (2.5 x 1013 VP/kg) by im single injection. Seventeen minutes after injection, 4/10 female mice appeared lethargic, with activity decrease and eye closure, but were sensitive to sound stimulation. The symptoms recovered gradually 30 min after injection, and basically recovered to normality 35 min after injection. The same symptoms appeared in 5/10 male mice 5–27 min after injection, and recovered 34 min after injection. All animals began to eat 40 min after injection. No mouse showed any signs of cyanosis, hyperspasmia, as well as any other abnormalities, for example, activities, fur, response, breath, and defecation, within 2 h after injection. There were no inflammation, ulceration and induration in the injection sites within 2 weeks. Because no mice died during the 2-week observation period, it is considered that the MTD of SG600-p53 is 2.5 x 1013 VP/kg.
Body weight gain was decreased 1 week and 2 weeks after injection of SG600-p53, and there were significant differences compared with the control group (p < 0.01 for female and male 1 week later, and for male 2 weeks after injection; p < 0.05 for female 2 weeks after injection). Mouse food consumption was decreased obviously 1 week later (p < 0.05 for female; p < 0.01 for male), but not different 2 weeks later compared with the control group (p > 0.05, Table 1).
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Systemic Anaphylaxis Test
In the systemic anaphylaxis test, guinea pigs were sensitized by 5 ip injections of SG600-p53 at 5.0 x 1010 VP/kg dose, and excitated by iv injection of SG600-p53 at 1.0 x 1011 VP/kg dose 10 days later after the last ip injection. Synchronously, 20 mg/1.0 ml of human albumin by ip and iv injections was installed as a positive control group and 0.9% sodium chloride by ip and iv injections as a negative control group. No guinea pigs appeared to exhibit any abnormal behavior in the SG600-p53 group or the negative control group within 3 h after excitation injection. In the positive control group, 2/5 animals showed strong positive anaphylaxis with appearance of the former 19 symptoms in 20 designed symptoms mentioned above, and 3/5 animals presented positive anaphylaxis with appearance of the former 10 symptoms. No animal died in any group during the observation period.
Repeat-Dose Toxicity Test in Rats
In the repeat-dose toxicity test in rats, 1.0 x 1011 VP/kg, 3.0 x 1011 VP/kg, and 1.0 x 1012 VP/kg doses of SG600-p53 were administrated by im injection daily for 2 weeks. The associated slight toxicities, which included the decreased food consumption, increased WBC, Ret, PLT, AST, decreased RBC, HGB, HCT, ALB, and TCHO were proved at the end of injection period and the end of observation period (Table 2). The liver was the main target organ of toxicity and the liver cells showed vacuolar degeneration at the end of injection period (Fig. 1). This change was mitigated at the end of observation period compared with that at the end of injection period. Animals in the low-dose group rarely showed abnormality in the indices of hematology and blood biochemistry. Hence, the no-observable-adverse-effect level (NOAEL) of SG600-p53 in rats is 1.0 x 1011 VP/kg.
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The anti-virus antibody was produced in each animal in the SG600-p53–treated groups at the end of injection period and the end of observation period, and the antibody had a function of neutralizing adenovirus. SG600-p53 could be measured in the injection sites of muscle and in the spleen tissues, but not in the brain, heart, lung, liver, kidney, stomach, testicle, prostate, ovary, and womb at the end of injection period. At the end of observation period, the vectors also could be found in the spleen tissues, but under the detection limit in the injection sites of muscle. Under the condition of this experiment, no p53 expression was showed in the injection sites or any organ tissue both at the end of injection period and the end of observation period.
Repeat-Dose Toxicity Test in Cynomolgus Monkeys
In the repeat-dose toxicity study in cynomolgus monkeys, SG600-p53 was injected at 5.0 x 1010 VP/kg, 1.5 x 1011 VP/kg, 5.0 x 1011 VP/kg doses in low-, mid-, and high-dose groups, respectively, daily for 2 weeks. During the 2-week injection period and 4-week observation period, there were no changes of toxicity in general symptoms, anaphylaxis, body weight, body temperature, electrocardiogram, hematological indices, blood biochemistry, prothrombin time, urine, and ophthalmic examination. The specific anti-virus antibody was produced in sera in 5/6 animals of the low-dose group, 6/6 animals of the mid-dose group, and 5/6 animals of the high-dose group one week after finishing injections, with the titers ranging from 1:50 to 1:200. Two weeks after finishing injections, the anti-virus antibody titers were up to 1:800, and one animal in the low-dose group had a titer of 1:3200, which descended gradually four weeks after finishing injections (Fig. 2).
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At the end of injection period, the organ weight and organ index of the thymus in the SG600-p53-treated groups were decreased compared with those in the control group, and there were significant differences between the control group and the low-dose group (p < 0.05) or high-dose group (p < 0.01). But in the organ weight and index of the spleen, there were no differences between the control group and the low-dose group (p > 0.05, Table 3). At the end of injection period and the end of observation period, no obvious histological changes were found by pathological examination of bone marrow and other organ tissues, except the slight vacuolar degeneration of liver cells and the focal necrosis. At the end of injection period, inflammation in the injection sites of SG600-p53–treated animals in the high-dose group was evident, but alleviative at the end of observation period (Fig. 3). Therefore, under the condition of this experiment, the NOAEL of SG600-p53 in cynomolgus monkeys was 5.0 x 1011 VP/kg.
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The distribution of SG600-p53 in organ tissues was detected by real-time quantitative PCR. The adenoviruses existed in the spleens of animals in the high-dose group and in the injection sites in every SG600-p53–treated group at the end of injection period, but its level was under the detection limit in heart, lung, liver, kidney, stomach, and ovary or testicle tissues. At the end of observation period, the level of SG600-p53 was under the detection limit in spleens and in the injection sites. No p53 expression was found by ELISA assay in organ tissues and animal sera both at the end of injection period and the end of observation period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Recombinant adenovirus is the most extensively used vector in gene therapy (Lai et al., 2002
), which shares some excellent features and can be modified to lack pathogenicity and increase safety due to its replication defectiveness (Nakatani et al., 2000; Palmer et al., 2002; Varnavski et al., 2005). But, compared with the cancer-selective replicating adenoviral vector, the replication-defective adenoviral vector has limitations such as low levels of gene expression, no target and poor anticancer potency. The oncolytic adenovirus is a biotherapeutic agent based on genetic engineering to be capable of selectively replicating within cancer cells, which can increase effectively the copies and expression of the transgene targeting cancer cells, and overcome the limitations of replication-defective virus. Virotherapy of using a variety of different adenovirus strains as oncolytic agents has led to several promising approaches for the treatment of human cancers. Such viruses have been demonstrated to be further improved in their safety and to produce antitumor effects in a number of phase I and II clinical trials and preclinical experiments (Bauerschmitz et al., 2002, 2004; DeWeese et al., 2001; Li et al., 2005; Makower et al., 2003).
Additionally, the tumor suppressor genes can be introduced into cancer cells by gene transfer to inhibit cancer cell growth (Horowitz, 1999; Roth, 2006). Among them, p53 disruption through mutation, or deletion, is a very frequent event and associated with a poor prognosis, with resistance to both chemotherapy and radiotherapy in various cancers (Munro et al., 2005; Somasundaram, 2000). The gene transfer by adenovirus vector that expresses p53 (rAd-p53) has been extensively studied clinically (Robson et al., 2005; Tango et al., 2004), and the data demonstrated that adenoviral-mediated expression of p53 showed a good safety profile (Wilson, 2002).
We previously constructed an hTERT-regulated oncolytic adenovirus, CNHK300, which demonstrated the characteristics of selectively replicating in cancer cells without cytotoxicity to normal cells and normal tissues (Su et al., 2004, 2006). We further constructed a novel triple-regulated oncolytic replicative adenovirus, SG600-p53, which carries the human p53 gene. This CRAd holds advantages of higher specificity to cancer cells and lower cytotoxicity to normal cells, and can mediate the effective copy and expression of the transgene in target cells (Wang et al., 2008). In the current study, the preclinical safety evaluation demonstrated that SG600-p53 has no adverse effects on cardiovascular and respiratory systems, but possibly has slight inhibition effect on behavioral or nervous systems in treated animals. At 2.5 x 1013 VP/kg dose of SG600-p53 by im single injection, no mice died within 2 weeks. So we considered that the MTD of SG600-p53 is 2.5 x 1013 VP/kg in mice. No systemic anaphylaxis was found in SG600-p53–treated guinea pigs. The NOAEL of SG600-p53 in rats (1.0 x 1011 VP/kg) or in cynomolgus monkeys (5.0 x 1011 VP/kg) was 12-fold and 60-fold of the proposed clinical dose, respectively. Although SG600-p53 might results in slight acute toxicity in mice, and slight vacuolar degeneration of liver cells in rats and cynomolgus monkeys, the changes were self-restored after stopping injections. Under the condition of this experiment, no p53 expression was showed in any organ tissue both at the end of injection period and the end of observation period in the repeat-dose toxicity tests in rats and cynomolgus monkeys. The main reasons are that the vast excess of animal tissue proteins in the specimens decreases the ratio of p53 protein, and the amount of transgene expressed in animal cells is under the detection level. In conclusion, the oncolytic adenovirus has an excellent safety profile demonstrated by our safety evaluation according to the Chinese guidance for human gene therapy and its product quality control. But, there is a difficulty in assessing the toxicity of CRAd, because the species specificity of the human adenovirus limits the choice of animal models. Considering that the wild type adenovirus has no serious side effects when injected intratumorally in clinic and the oncolytic adenoviruses are safe in clinical trials and clinical applications, the triple-regulated CRAd, SG600-p53, provides us a brand-new promising strategy for cancer gene therapy with good safety and antitumor effect. The preclinical safety evaluation demonstrated the safety, feasibility and optimal dose of intratumoral SG600-p53 injection, and the results of safety evaluation will guide the proposed phase I clinical trial in patients with advanced NSCLC.
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FUNDING |
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Important Project of National Natural Scientific Foundation of China (30730104 and 30572149); and the State 863 High Technology Project of China Grant (2007AA021108).
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NOTES |
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1 These authors contributed equally to this study.
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ACKNOWLEDGMENTS |
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We sincerely thank Beijing Joinn Pharmaceutical Center (Bejing, China) for their assistance with animal studies.
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