Research ArticleResearch Article

Phase III Clinical Trials of the Cell Differentiation Agent-2 (CDA-2): Therapeutic Efficacy on Breast Cancer, Non-Small Cell Lung Cancer and Primary Hepatoma

Fengyi Feng, Qing Li, Changquan Ling, Yang Zhang, Fengzhan Qin, Huaqing Wang, Wenxia Huang, Shunchang Jiao, Qiang Chen, Mingzhong Li, Yunzhong Zhu, Meizhen Zhou, Jun Ren, Yetao Gao, Jingpo JZhao, Rongsheng Zheng, Wenhua Zhao, Zhiqiang Meng, Fang Li, Qizhong Zhang, Dongli Zhao, Liyan Xu, Yongqiang Zhang, Yanjun Zhang, Zhenjiu Wang, Shuanqi Liu and Ming C. Liau
Chinese Journal of Clinical Oncology August 2005, 2 (4) 706-716;

Abstract

OBJECTIVE The objective of this study was to explore the effect of CDA-2, a selective inhibitor of abnormal methylation enzymes in cancer cells, on the therapeutic efficacy of cytotoxic chemotherapy.

METHODS Advanced cancer patients, all of whom had previously undergone chemotherapy, were randomly divided into 2 groups, one receiving chemotherapy only as the control group, and the other receiving CDA-2 in addition to chemotherapy as the combination group. The therapeutic efficacies and the toxic manifestations of the 2 groups were compared based on the WHO criteria.

RESULTS Of 454 cancer patients enrolled in phase III clinical trials of CDA-2, 80, 188, and 186 were breast cancer, NSCLC, and primary hepatoma patients, respectively. Among them 378 patients completed treatments according to the protocols. The results showed that the overall effective rate of the combination group was 2.6 fold that of the control group, 4.8 fold in the case of breast cancer, 2.3 fold in the case of primary hepatoma, and 2.2 fold in the case of NSCLC. Surprisingly, the combination therapy appeared to work better for stage IV than stage III patients. CDA-2 did not contribute additional toxicity. On the contrary. it reduced toxic manifestations of chemotherapy, particularly regarding white blood cells, nausea and vomiting

CONCLUSION Modulation of abnormal methylation enzymes by CDA-2 is definitely helpful to supplement chemotherapy. It significantly increased the therapeutic efficacy and reduced the toxic manifestation of cytotoxic chemotherapy on breast cancer and NSCLC.

KEYWORDS:

keywords

Aberrant promoter hypermethylation of CpG islands has been widely accepted as a hallmark of cancer,[1-6] and DNA demethylation has gained momentum as a logical approach for cancer therapy.[7-13] Whereas 5-azadeoxycytidine is undoubtedly the most effective agent to achieve DNA demethylation, its clinical application has raised grave concerns because of non-selectivity. DNA demethylation is a dangerous therapeutic approach, because DNA hypomethylation is known to promote mutation and chromosomal instability, [14,15] and is a condition associated with the evolution of cancer.[16-19] Thus, successful execution of DNA demethylation to accomplish cancer therapy must rely on finding agents that can cause DNA demethylation only in cancer cells. CDA-2 can fulfill such a requirement.

Biological methylation is carried out by a ternary enzyme complex consisting of MAT-MT-SAHH. [20] Methylation enzymes are altered in cancer cells. Cancer cells generate a cancer-specific factor that becomes associated with MAT and SAHH. The association of the cancer-specific factor changes the kinetic properties of MAT and SAHH and also the regulation of the ternary enzyme complex. [20-23] Normal methylation enzymes are subject to the regulation by exogenous growth factors, whereas cancer cells generate a cancer-specific factor to lock the methylation enzymes in an active and stable configuration that drives cancer cells to keep on dividing and denies their ability to undergo terminal differentiation. [24] Abnormal methylation enzymes are, therefore, right at the heart of the cancer problem. Aberrant hypermethylation of CpG islands,[1-6] evolution of cancer,[21,22] blockade of differentiation,[24] tumor progression, [25-27] and even the failure of cytotoxic chemotherapy [28-31] can all attribute to abnormal methylation enzymes.

CDA-2 is a perfect solution of the abnormal methylation enzymes of cancer cells. CDA-2 is purified from fresh human urine. CDA-2 contains differentiation inducers capable of selectively antagonizing the cancer-specific factor of abnormal methylation enzymes.[32] It also contains differentiation helper inducers which work synergistically to potentiate the activity of differentiation inducers.[33,34] In addition it is comprised of anti-cachexia chemicals which can prevent cancer patients from excreting too much endogenous anticancer metabolites such as those present in CDA-2.[35] These multiple components work cooperatively to achieve a good cancer therapy. Preclinical studies of CDA-2 have yielded results showing that abnormal methylation enzymes could be effectively eliminated by CDA-2,[32] and as a consequence cancer cells were induced to undergo terminal differentiation through induction of DNA hypomethylation. [7] But this was not the whole story. CDA-2 also brought delightful bonuses which included down-regulation of oncogenes and up-regulation of suppressor genes, [36] prevention of recurrence and metastasis,[37] reversal of drug resistance,1381 and abrogation of telomerase.[39] These unexpected results are actually more precious than the intended objectives.

CDA-2 was approved by the SFDA of China on October, 1999 to undergo clinical trials (Approvable Certificate 1999×L0101). It was classified as a chemical drug category 1, meaning a brand new drug originating from China. Phase III clinical trials started in September, 2001 and concluded in November, 2002. It was approved in July, 2004 (New Drug Certificate H20040768) to advance to phase IV clinical trials. During phase II clinical trials, CDA-2 was employed as a single agent. When CDA-2 was used as a single agent, its therapeutic endpoint was terminal differentiation. Therefore, tumor shrinkage was rather limited, but the improvement on clinical benefit response and quality of life was much better.[40-42] However, when CDA-2 was used in combination with vitamin C the therapeutic endpoint shifted from terminal differentiation to apoptosis or cell killing, then the therapeutic efficacy based on tumor shrinkage improved dramatically.[43] Phase Iff clinical trials were then attempted as a combination therapy with cytotoxic drugs to achieve a better result of tumor shrinkage. The phase III study focused on breast cancer, NSCLC and primary hepatoma which were found responding more favorably to CDA-2 during phase II clinical trials.

PATIENTS AND METHODS

CDA-2 injection

CDA-2 injection, also named uroacitides injection, was purified from fresh human urine as previously described. [44] Briefly, the procedure included filtration of the urine to remove precipitates, reverse phase chromatography on XAD-16, extraction with ethanol, incubation of the ethanol eluant at 25℃ to inactivate viruses if present, vacuum evaporation to remove ethanol, and finally ultrafiltration of the final redissolved solute to remove pyrogens and microbials. CDA-2 injection, 4 g/100 ml, Was provided by the Everlife Pharmaceutical Co. free of charge. Three batches (000901, 000902, 000903) used in the phase III clinical trials were inspected and approved by the Bureau of Inspection of Drugs and Biological Products.

Selection of patients

Phase III clinical trials of CDA-2 were multicenter, open label, and randomized clinical trials on breast cancer, NSCLC, and primary hepatoma. Patients were either advanced patients refractory to the previous chemotherapy or recurrent patients after a certain interval from the previous chemotherapy. Patients were randomly divided into the control group receiving chemotherapy only or the combination group receiving CDA-2 in addition to chemotherapy.

Patients eligible for phase III clinical trials of CDA-2 had to fulfill the following criteria

1) Diagnosis had to indicate pathological and cytologi cal confirmation. In cases of primary hepatoma, an AFP assay was included. 2) Patients were post-surgery pa tients due to recurrence or metastasis, or inoperable pa tients of the confirmed case. 3) Tumor sizes ≥ 1 cm were clearly revealed by CT, MRI, X-ray or ultrasound. 4) KPS scores were ≥60, and expected survival was more than 2 months. The age was limited between 16 and 78.5) The last chemotherapy treatment had elapsed more than 4 weeks. 6) The functioning of vital organs including heart, liver, kidney, and bone marrow was within normal range. In the case of hepatoma patients, parameters of liver function did not exceed 2.5 fold that of the normal range. 7) Patients were cooperative, and willing to participate in the follow-up studies. 8) Pa tients had to sign the informed consent form.

A week before treatment, each eligible patient under went a physical examination, laboratory analyses of blood and urine samples, analyses of biochemical mark ers in the blood to evaluate liver and kidney function, determination of blood sugar, electrocardiogram exam ination, and analyses of tumor markers. Photographic examination of the tumors such as X-ray, CT, MRI or ultrasound was also conducted. Photographic examina tion of the tumors was again conducted at the conclu sion of each cycle of treatment for the evaluation of therapeutic efficacy. Eligible patient's symptoms and previous diagnosis and treatments were kept on file.

Patients with the following conditions were not eligible for phase III clinical trials of CDA-2

1) Women who were pregnant or lactating. 2) Tumors were not clearly measurable due to ascites fluid or pleu ral effusion, or less than ≤ 1 cm. 3) Patients who were not conscious due to severe brain metastasis. 4)Patients who had a severe infection. 5) Function of vital organs was severely damaged. 6) Patients who were participating in a clinical trial of another chemotherapeutic drug. 7) Patients who had another primary tumor. 8) Primary hepatoma patients who were incompatible for embolization treatment.

Exclusion of patients

Enrolled patients if found to use other anticancer medications, or not closely following the treatment protocols were excluded from evaluation for therapeutic efficacy. But those patients were included in the evaluation under ITT. Their adverse effects were also recorded.

Protocols

The protocols for breast cancer included the CAF regimen: CTX 500 mg/m2 iv d1,5; ADM 49 mg/m2 iv d1 or THP-ADM 40 mg/m2 or E-ADM 60 mg/m2; 5-FU 500 mg/m2 iv d1,5; and the NP regimen: NVB 25 mg/m2 iv d1,5; DDP 30 mg/m2 iv d1–3. The protocols for NSCLC included the MVP regimen: MMC 6 mg/m2 iv d1; VDS 25 mg/m2 iv d1,5; DDP 30 mg/m2 d1–3; and the NP regimen: NVB 25 mg/m2 iv d1,5; DDP 30 mg/m2 d1–3. The protocols for primary hepatoma included the HF regimen: HCPT 10 mg/m2 iv d1–5; 5FU 450 mg/m2 iv d1–5; CF 60 mg/m2 iv d1–5; and an artery embolization CFF regimen: Iodine oil 5~30 ml, E-ADM 40~80 mg, 5-FU 500~1000 mg, CBP 100~200 mg.

Method of drug administration

Patients in the control group were given chemotherapeutic drugs according to the protocols mentioned above. Those in the combination group were given 300 ml of CDA-2 per day by iv infusion through a subclavian catheter. CDA-2 was administered for 7 days prior to the start of chemotherapy. The treatment with CDA-2 was discontinued during the day in which chemotherapy was given, and started again the next day after chemotherapy. Twenty-one days constituted a cycle of chemotherapy treatment, whereas 28 days constituted a cycle of embolization treatment. Responding patients who developed at least MR during the first cycle of treatment were encouraged to receive the second cycle of treatment.

Evaluation of therapeutic efficacy and toxicity

Therapeutic efficacy was evaluated according to the criteria set up by the WHO for solid tumors. Objective tumor responses were recorded as follows:

Complete response (CR) indicated disappearance of all measurable tumor, within a time factor of at least a 2 month duration.

Partial response (PR) indicated greater or equal to 50% regression of one or more viable lesions, with no progression of any lesion.

Moderate response (MR) indicated greater or equal to 25% but less than 50% regression of one or more viable lesions, with no progression of any lesion.

Stable disease (SD) indicated less than 25% reduction in tumor volume or lack of progression.

Progressive disease(PD) indicated an increase in size by 25% of one or more lesions, and/or the occurrence of any new lesion.

The effective rate was defined as CR+PR/eligible patients or all enrolled patients to give the respective ITT rate. Improvement rate was defined as CR+PR+MR/eligible patients or all enrolled patients to give the respective ITT rate.

Toxicity was evaluated according to the criteria of stage 0, I, II, III, and IV set up by the WHO. Zero meant no toxicity at all. I meant very minor toxicity. II meant moderate toxicity. III meant serious toxicity. IV meant very severe, sometime fatal toxicity.

Statistical analysis

SPSS software was employed to sort out various subgroups of patients under the 2 major treatment groups. Mean value and statistical significance were performed by the 2-tailed Student t test for paired data and considered significant if P values were <0.05. χ2

2 or Fisher's exact test was used to compare the distribution of categorical variables in the observed endpoints.

RESULTS

General information and break down into groups of the participating patients

General information on the participating patients is presented in Table 1. A total of 454 patients participated in the phase III study, and 378 patients completed the treatment according to the protocols resulting in a completion of 83%. Patients in the combination group and the control group were more or less similar with respect to sex, age, KPS scores, and body surface area as shown in Table 1. Break down of the patients in group assignments is presented in Table 2. About half of the participating patients were stage III patients and the other half stage IV. Most patients had been previously treated with cytotoxic drugs. There were 193 on-going patients who had become refractory to the previous treatment and then enrolled in the present study. A total of 261 were recurrent patients who had been in remission for certain periods. The number of patients with tumors in each organ site were 80, 188 and 186 for breast cancer, NSCLC, and hepatoma, respectively. The assignment of patients in the combination group and the control group was roughly 2:1. Of the 454 enrolled patients, 76 did not complete the study as shown in Table 3. Half of them simply refused to undergo further treatment, 14 declined due to progression, and 11 left the study due to adverse effects. There were 7 deaths which will be reported in detail later.

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Table 1. General information on the patients participating in phase III clinical trials of CDA-2.
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Table 2. Groups assignment of the patients participating in phase III clinical trials of CDA-2.
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Table 3. Exclusion of patients during phase III clinical trials of CDA-2.

Therapeutic efficacy

Overall therapeutic efficacy is presented in Table 4. The evaluation was based on the WHO chemotherapy criteria. The effective rate in the combination group obviously fared better than the control group by 2.6 fold. The break down of therapeutic efficacy on organ sites as presented in Table 5 shows that breast cancer patients benefited best by the combination therapy, followed by NSCLC. The increase of effective rate was 4.8 fold and 2.2 fold, for the breast cancer and NSCLC groups, respectively.

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Table 4. Overall therapeutic efficacy: Combination group vs control group
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Table 5.

Break down of therapeutic efficacy on organ sites.

It is noteworthy that of both groups, NSCLC had the highest percentage of patients responding to the treatments. The differences in therapeutic efficacy between the combination group and the control group among breast cancer and NSCLC patients were statistically significant. The therapeutic efficacy on hepatomas based on tumor shrinkage was dismal, but again the combination group fared better than the control group. Therapeutic efficacies among recurrent patients and on-going refractory patients were almost the same as shown in Table 6. Results presented in Table 7 show that the combination therapy unexpectedly worked much better on stage IV patients than on stage III patients. However, the control groups did not show any difference. Seven patients listed in Table 8 among the 454 enrolled cases, died during the phase III clinical trials. Four patients were in the combination group, but no deaths were related to CDA-2.

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Table 6. Break down of therapeutic efficacy on recurrent vs on going refractory patients.
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Table 7. Break down of therapeutic efficacy on clinical stages
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Table 8. List of deceased cases

Since cytotoxic drugs were employed in these clinical trials, toxic manifestations were unavoidable. CDA-2 as a single agent during phase-II clinical trials did not show severe toxic manifestations.[40-42] The most frequent complaint was blood vessel irritation at the site of infusion when CDA-2 was administered through a peripheral vein. All patients who participated in the phase III clinical trials were equipped with a subclavian catheter to avoid vessel irritation. Results presented in Table 9 clearly show that CDA-2 did not contribute additional toxic effects caused by the chemotherapy. On the contrary, it reduced toxic manifestations of chemotherapy, particularly on white blood cells, nausea and vomiting, about which the differences were statistically significant.

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Table 9. Comparison of toxic manifestations between the combination group and the control group

DISCUSSION

For cytotoxic chemotherapy to be successful all cancer cells have to be destroyed, because if some cancer cells survive they will likely progress to become insensitive to any assault. Abnormal methylation enzymes of cancer cells play a key role in the progression resulting in resistance.[28-31] Silencing of hMLHl marks the point of no return.[28,29]Elimination of abnormal methylation enzymes is, therefore, very important for the success of cancer therapy. CDA-2 plays such an important role.

The design of protocols for phase III clinical trials was based on the rationale of eliminating abnormal methylation enzymes in order to avoid tumor progression triggered by cytotoxic drugs. [31] CDA-2 treatment was, therefore, initiated 7 days prior to the start of chemotherapy. Since most patients enrolled in phase III clinical trials had been previously treated with chemotherapy, tumor progression attributable to aberrant DNA hypermethylation had already taken place to some extent. Pretreatment with CDA-2 could only prevent further progression. The rescue of aberrant DNA hypermethylation may not take place under the present setting, because demethylation requires 2 cell cycles to complete.[45,46] The application of cytotoxic drugs simply prevents the demethylation of already methylated DNA, because DNA replication is not taking place to a great extent. The combination of CDA-2 with chemotherapy works best on new patients whose aberrant DNA hypermethylation is still limited to genes which are not causing fatal damage such as hMLHl. The prevention of tumor progression due to DNA hypermethylation is the major objective of CDA-2 in the combination therapy. This objective, however, requires long-term observation to evaluate, which must await phase IV study.

The phase III study was designed to observe short term effects. On short term effects, the enhancement of therapeutic efficacy is encouraging, which is probably attributable to the ability of CDA-2 to reverse drug resistance.[38] The synergistic effect of the combination therapy may also contribute to the enhancement of therapeutic efficacy, since CDA-2 alone in phase II clinical trials did result in 13.3%, 3.4% and 8.1% of PR responses in breast cancer, NSCLC, and primary hepatoma, respectively.[40-42] The uncharacteristically low effective rates of control groups may be due to the fact that most patients in the phase III study are late-stage refractory or recurrent patients. Nevertheless, the differences between combination and control groups among breast cancer and NSCLC patients are statistically significant. The combination therapy appears to work better for stage IV than stage III patients. It is possible that abnormal methylation enzymes play a more dominant damaging role at later stages, and that the modulation of abnormal methylation enzymes becomes more beneficial for therapy. The finding that CDA-2 works better for stage IV patients is particularly encouraging, since patients in this category do not respond well to any therapy.

ACKNOWLEDGMENTS

We gratefully acknowledge financial support provided by Mr. Ming Lieh Chang, President of Everlife Pharmaceutical Company. He was particularly generous in providing support for conferences which were instrumental to the successful execution of clinical trials. Dr. Po Shou Xu of Anhui Medical University, Dr. Shan Quan Shi of Eastern Hepatobilliary Hospital and Dr. Zhe Chen of Changhai Hospital of the second Army Medical University of Shanghai, Dr. Xue Ren Lin of the Cancer Hospital of Fujian Province, Dr. Gi Ming Lai of the National Institute of Health of Taiwan, Mr. Yi Shin Chang and Dr. Homg-Jen Fan Chiang of Everlife Pharmaceutical Company all played essential roles in clinical trials equally as important as the coauthors. We appreciate the help of Miss Tong Hua Wang of Everlife Pharmaceutical Company in the preparation of manuscript.

Footnotes

  • ↵Sponsored by Everlife Pharmaceutical Company of Hefei，Anhui 231202． China.

ABBREVIATIONS

ADM
adriamycin
E-ADM
epirubidn
THP-ADM
perarubicin
CBP
carboplafin
CTX
cytoxan
DDP
cisplatin
5-FU
5-fluorouracil
HCPT
hydroxy camptothecine
MMC
mitomycin C
NVB
navelbine
VDS
vindesine
AFP
alpha-fetal protein
CDA-2
cell differentiation agent-2
CF
citrovorum factor
ITT
intended to treatment
KPS
Karnofsky performance status
MAT
methionine adenosyltransferase
MT
methyltransferase
NSCLC
non-small cell lung cancer
SAHH
S-adenosylhomocysteine hydrolase
  • Received April 27, 2005.
  • Accepted June 8, 2005.

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