Glyco-polypeptides in Treating of Various Types of Late-Stage Refractory Solid Carcinoma in Humans-A Double-Blind Study-Case Report of 126 Patients: Complete Study Report of Phase II Clinical Trial

and Summary Glyco-polypeptides (Comosain) induced leucocyte binding ability to tumor surface antigens, such as interleukin 2, 6, 8, and TNFs, is known as an immuno-target therapy. Using the different concentration of Bromelain proteinases in 6 types of cancer cell, it resulted in hydrolysis, fibrinolysis, necrosis, and anti-metastatic effects in tumor cells. Anti-cancer effects were achieved in carcinoma of lung, breast, colon, ovary, cervix, and uterus. Investigation of anti-metastatic effects in Bromelain was carried out in a double-blind study: low dose cohort was on 10 mg/kg/day and a high dose cohort which was on 50 mg/kg/day for over six months. A total of 151 patients with 3rd and 4th stage of refractory solid tumors were enrolled, 25 patients were post-surgery within 6 weeks which were excluded from entering this research who at least previously failed on two regimens of chemotherapy and/or failed on radiation therapy. The actual study patients were only 126 patients. The rates of Complete Response (CR) and Partial Responses (PR) in the high-dose cohort are astonishing with 52% and 27% respectively. The Progress Disease (PD) was 10%, and the Stable Disease (SD) was 11%. The implications and results of the findings are discussed in view of the reported anti-metastatic activity of orally administrated Bromelain.


Introduction
The administration of glycol-polypeptides (bromelain) in cancer treatment in nonclinical trials has been reported as early as 1968 by Wolf M, & Ransberger K [1]. Both in vitro and animal studies have suggested anti-metastatic effects through the use of bromelain.  reported that orally administered bromelain reduced the incidence of pulmonary metastasis in Lewis lung cancer cells in mice [2,3]. In recent years, Batkin & Taussig suggested the antitumor mechanisms are due to fibrinolytic effects in Bromelain [4]. Taussig   discovered that bromelain has anti-platelet aggregation effects [5]. Taussig and Batkin in 1985 also discovered the inhibition growth of tumor cells such as Lewis lung carcinoma, V-8 lymphoma, MC1-1 ascites, KATO-gastric carcinoma cells [5].
From the experimental studies above, we hypothesize that activation of bromelain proteinases in lymphocytes and T-cells has anti-metastatic effects both in vitro and in vivo. In our conducted study, we compared the modulation of low dose cohort and high dose cohort of bromelain administration to the patients with stage 3 and stage 4 refractory solid tumors, which include various types of carcinoma of lung, breast, colon, ovarian, cervix, uterine, prostate, melanoma, lymphoma, and gastrointestinal origins, etc. [16][17][18][19]. All patients have previously failed on at least two regimens of chemotherapy and/or failed with radiation therapy. The treatments were carried out for at least 24 to 30 weeks. The complete blood count, liver, renal function, hematopoietic elements, tumor markers were evaluated at an interval of every 4 to 6 weeks. The computerized tomography scans were performed at an interval of every 3 to 4 months. The size of tumors was measured, and the tumor markers were recorded for the evaluation of complete response (CR), partial response, (PR), stable disease (SD), and progressive disease (PD) according to the Standard Response Criteria of National Cancer Institute (NCI). The common toxicity was recorded by using NCI's Standard Toxicity Criteria. The results of CR and PR were promising and astonishing when Bromelain was administered in high-dose cohort patients [20,21].

Clinical Application and Study Protocol
The Phase II Clinical Study investigates the efficacy of low dose and high dose cohort bromelain (comosain) in human subjects diagnosed with advanced late-stage refractory cancers [23,24]. The bromelain (comosain) extract derived from the stem and fruit of Ananas comosus will be administered orally each day.

Patient Eligibility and Selection
I. Eligible patients are those suffering from late-stage solid cancer of breast, lung, colon, cervical, ovarian, and uterine, prostatic, lymphoma, bladder, etc. They are in stage III or IV with tissue proof of well-documented malignancies, whether by tissue biopsies, laparatomy, or thoracotomy. These individuals have not been cured by conventional methods such as radiation therapy or chemotherapies for at least two separate regimens.
II. Other eligibility requirements also require patients to have no other available therapy known to provide clinical benefit.
For example, the breast cancer patients must have failed at least 2 chemotherapy regimens in the metastatic setting. Additionally, if their tumors are HER2 positive or hormone receptor (ER, PR) positive, respectively, they must also have failed several anti-HER2 targeted therapies and no longer be eligible for hormonal therapy [25][26][27].
III. Additionally, the following conditions must be met: a) Patient's age is between 18 and above. b) Patient is not taking anticoagulants or on antiplatelet therapy. c) Patient does not have a history of abdominal fistula, gastroenteral perforation, peptic ulcer diseases, or intra-abdominal abscess within 4 months prior to study enrollment [28][29][30]. g) Female patients should not be pregnant or breastfeeding [31,32].
h) Patient's platelet counts must be greater than 100,000/uL.
i) Patient's hemoglobin must be greater than 9.0 g/dL. j) Patient does not have significant abnormal hepatic and/or renal function. k) Patient's tumors are measurable; between 0.2 -10 cm in size and number between 1-15. All measurable tumors that have spread to the bones, liver, lung, kidney, and abdomen will be included in the data analysis [33][34][35].
f) Patient currently has uncontrolled hypertension, diabetes, or clinically significant cardiac arrhythmia.
g) Patient who had major surgery performed within 4 weeks prior to entering the study; and patients who have not recovered from adverse events due to surgery performed more than 4 weeks earlier [39,40].
h) Patient with a history of allergic reaction to Bromelain or pineapple-containing products.
i) Female patients who are pregnant or breastfeeding.
j) Patient with tumors that are widely spread in the chest and abdomen that cannot be measured by CT scan.
Patients who are eligible for this study will be randomly assigned to either the low dose group or the high dose group by a coin toss. Each study subject will be assigned a patient number for the purpose of this study [41].

Drug Dosage and Schedule
The dose of glycol-polypeptide (bromelain) at 50 mg (125 GDU)/kg/day is extrapolated from in vivo animal studies. It is determined to be safe by a safety study on healthy human subjects [42,43]. For this clinical investigation, the high dose group will be given bromelain at 50 mg/kg/day (at a bodyweight of 50-60 kg) to a maximum of 2400 mg (5000 GDU)/ day and divided into 2 doses/day of 1200 mg/dose [44].
In both the high dose group and the low dose group, the number of patients suffering from well-documented refractory solid malignancies will be at least 60 and 30 respectively to be assigned to each group. All patients are diagnosed with different types of carcinomas. For example breast, lung, colon, ovarian, cervical, bladder, prostatic and uterine origin, etc. In the high-dose group, patients will be given bromelain at 5000 GDU (2400 mg)/ day divided into two doses of 1200 mg/dose and taken with meals. In the low dose group, patients will be given Bromelain at 1250 GDU (500 mg)/day divided into two doses of 250 mg/dose and taken with meals [45][46][47][48].

Duration and Route of Administration
Study subjects will be provided with bromelain for oral administration. The containers will be clearly labeled (see Section V-E). Bromelain will be taken orally twice daily with meals. On their bi-weekly visits to the doctor's office, the study patients will be provided with enough doses for two weeks. The study patients are required to keep a journal of the daily doses they take and any side effects they experience [49][50][51][52].
The study patients will be evaluated using blood tests and/or CT scans at the end of each cycle (i.e., 6 weeks) and at six months for signs of disease progression. If the disease did not progress, then treatment will continue and the patient will be evaluated every six months thereafter until the investigator determines otherwise. If the disease did progress, then the patient will be taken off the study. On the humanitarian basis, the low dose group patients will be transferred to the high dose group due to lack of efficacy in the treatment [53,54].

Evaluations to be conducted
A. Blood and Laboratory tests schedule: blood tests will be conducted every 4-6 weeks, Blood tests include CBC, Chemistry-7, Chemistry-24, liver and renal function, CEA, CA125, CA153, CA199, PSA, TSH, alfa-Feto-Protein, and other tumor markers. The test results will be recorded for discussion and evaluation.
B. Radiological tests schedule will be also assessed every 3 months for the result of CT scan and/or PET scan, C. Each study subject will be also assessed every four weeks for any side effects that they may have experienced during the previous four weeks. These side effects will be recorded for evaluation [55][56][57].

A. Adverse events and serious adverse events and reporting information:
The NCI listed Adverse Events in CMC (Common Toxicity Criteria) is based on pathological (Allergy/Immunology) and anatomical (Dermatology /Skin) categories to facilitate the location of related adverse events.

B. Grades of Adverse Events
For each adverse event, grades are assigned and defined using a scale from 0 to 5. With 0 representing no adverse event within normal limits and 5 representing death related to an adverse event [58,59].

Study Endpoints
At the end of six months, an assessment of the therapy results for each study patient will be performed to determine whether to continue with this therapy. Individual data sets will be combined to assess the efficacy of the therapy for the cancers studied ( Figure 2). The study endpoints for both groups are: 3-a.Use of Standard response Criteria: (NCI Chapter Standard Response Criteria 11.1.1 through 11.1.7 and 11.2, 11.3,).

Evaluation of Target Lesions
A. Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes must have a reduction in short axis to <10mm.

B. Partial Response (PR):
At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.

C. Progressive Disease (PD):
At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on the study). In addition to the sum to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions)

D. Stable Disease (SD):
Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as a reference to the smallest sum diameters while on the study [60][61][62].

A. Complete Response (CR):
Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10mm short axis) (if tumor makers are initially above the upper normal limit, they must normalize for a patient to be considered incomplete clinical response.

B. Non-CR/ Non-PD:
Persistence of one or more non-target lesion(s) and /or maintenance of tumor marker level above the normal limits.

C. Progressive Disease (PD):
Appearance of one or more new lesions and/ or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase [63][64][65].

Data Analysis
Data collected from all patients will be analyzed to determine the overall efficacy of Bromelain to treat advanced cancers. Statistical analysis such as Student t-test will be used. The results of the ongoing analyses will be reported to the FDA in an annual report. (4-a) Adverse events are mild to moderate anemia, leukopenia, thrombocytopenia, and/or liver and renal impairment. (4-b) Severe and serious adverse events are Liver and/or Renal damage or failure, anaphylactic reaction. All serious adverse events will be reported to the FDA [66].

Results and Conclusions
The results of the study will be reported as required to the FDA in an annual report and now report as following: 1. Age distribution: Both in the high dose group and low dose group patients are mainly ages 65 and above. 68% and 81% respectively of participants were over the age of 65 (Table 3). And the participants that are male gender are 75% and 67% respectively.  2. The disease classification and distribution are as following: breast carcinoma account for 25% in the high dose group and 28% in the low dose group, in lung carcinoma the incidence are 9.9%, and 19% respectively, in colon & G-I carcinoma the incidence are 3.7% and 7.1% respectively, in ovarian carcinoma the incidence are 8.6% and 9.5% respectively. The uterine and cervical carcinoma in the high-dose group is about 13% etc.
Please see Table 4 for overall disease distribution. Table 4 showed breast cancer incidence in low dose group and in high dose group are 28.6% and 25% respectively. In lung cancer, the incidence is 19% and 10% respectively. In colon cancer, the incidence is 7.1% and 3.7% respectively. In Ovarian, uterine, and cervix cancer, the incidence is 9.5% and 30.2% respectively. In bladder and prostate cancer, the incidence is 14.4% and 16% respectively. The incidence of melanoma cancer is 2.5%. The incidence of liver cancer are 2.4% and 1.2% respectively. The incidence of lymphoma are 9.5% and 5% respectively. In the incidence of thyroid cancer and sarcoma cancer, both are 2.4% and 1.23% respectively [67,68].  The overall clinical response rate in high dose group patients and low dose group patients are as follows: the Complete Response (CR) rates are 52% (66/126) and 0% respectively, the Partial Response (PR) rates are 27% (34/126) and 0% respectively. In the patients of the low dose group there was no Stable Disease (SD) and in the patients of the high dose group is 11% (13/126). The Progressive Disease (PD) in the high dose group is 10% (13/126), and in the low dose group is 100%.

Name of Cancer Low Dose Group High Dose Group
The overall adverse effects and toxicities are shown in Table 5A to Table 5D which all concluded that there were no hematological, renal, and hepatic toxicities in patients of all groups. The primary target lesion size is less than or equal to 2 cm in the low dose group and high dose group are 36% and 38% respectively. The lesion size between 2-5 cm are 38% and 39.5% respectively, the lesion size between 5-10 cm are 28.6% and 25% respectively [69].     The tumor markers such as CEA, CA-125, CA-153, CA-199, PSA, TSH, and alpha-fetoprotein are being monitored, their value corresponds to the tumor masses, they return to normal value when the tumor has been Complete Responded (CR), and when the tumor progress the tumor marker value is elevated.
The serious adverse effect in toxicity in the both low dose group and the high dose group are not observed as seen in Table 5A to  Table 5D. There is no serious hematopoietic toxicity, no hepatorenal toxicity, no anaphylactic reaction and life threaten events. There were very rarely minor or non-serious side effects such as nausea, vomiting, diarrhea, palpitation, headache, insomnia, pruritus, urticaria, and skin rash. We conclude that bromelain administered in an amount of 2500 to 3000 mg/day to patients with average body weight is effective and non-toxic. The following figures are self-explanatory for the above results.

Tumor Measurement in Phase -2 Clinical Trial Study
A. Low dose cohort with measurable disease was either by direct measurement, X-ray, CT scans, and /or PET scans. Table 4 showed tumors progressions in all low dose group patients without exception. ## Special Note ## : Due to the non-efficacy in this low dose treatment, all patients in this cohort were transferred to the high dose cohort to continuing therapy for humanitarian reasons.
B. High dose cohort measurable disease was either by direct measurement, X-ray, CT scans, and/or PET scans (Table 6).

Discussion
In summary, throughout the 6 to 10 months course of the double-blind study of bromelain administration for high and low dose groups, only the high dose group patients of 50 mg/kg/ day regimen showed effectiveness. The low-dose group patients showed no efficacy at all. Both groups did not show serious adverse effects such as leukopenia, anemia, hepato-renal toxicity, anaphylactic reaction, and life-threaten events. Minor adverse effects such as nausea, vomiting, diarrhea, urticaria, insomnia, palpitation, pruritus, and headache occurred in rare instances [70,71] (Table 7 and Table 8).   The glycopeptides of stem Bromelai were obtained from proteolytic digestion with pronase as described by Murachi, et al. in 1967 and later found that there were four kinds of glycopeptides that only differed from each other in the peptide part [29]. The amount of the glycopeptide was calculated from its content of glutaminic acid as determined by amino acid analysis. The average molecular weight was assumed to be 1.5x10^3 DA. Bromelain contains nine different glycol-polypeptides. Each polypeptide contains amino acids in a double benzene ring structure and one of twelve different monosaccharides fractions [22]. Specifically, breakthrough fractions such as Comosain (F9) account for 80%, ananase account for 10%, the rest of 10% was derived from Bromelain F1, F2, F3, F5, F6, and so forth [4]. They mainly comprise glycosylated multiple enzyme species of the papain superfamily with different proteolytic activities, molecular masses between 20 to 31 kDa, and isoelectric points >10 and 4, 8 respectively. Two major basic proteinases, F4 and F5, were further characterized and shown to have molecular masses of 24397 Da and 24472 DA, respectively [22,25]. Napper and Bennett et al in 1994 further purified and characterized multiple forms of bromelainases derived from cysteine proteinases Ananain and Comosain. Lee and Albee in 1997 postulated the complete amino acids sequence of Ananian and comparison with Bromelain and other plant cysteine proteinases. They all have protein electronic density between 272 to 282 mu [32,72] (Table 9) (Figures 1A-1C).   Table 9: Outcome Measurement-Serious Adverse Outcomes in Toxicity (One patient withdraw from testing).