Cellular accumulation of antineoplaston AS21 in human hepatoma cells.

Antineoplaston AS2-1 is a mixture of sodium salts of phenylacetic acid (PAA) and phenylacetylglutamine (PAG) in the ratio 4:1. The uptake of both compounds has been examined in human hepatoma cell line, Hep G-2. The accumulation of PAA was characterized by temperature sensitivity, saturability and energy dependency. Organic anions (probenecid, p-aminonohippuric acid and stilbene) inhibited PAA uptake suggesting the involvement of organic anion system in PAA transport. PAG cellular uptake exhibited dependency on metabolic energy, since the accumulation was sensitive to lowered temperature as well as to replacement of sodium ions by choline in the incubation medium. In contrast, the process showed tolerance to lithium ions as a substitute to sodium ions. This finding, together with the strong inhibition of PAG accumulation by histidine and glutamine, indicates that system N, known to be specific for hepatic tissue and the glutamine-preferring amino acid transport system, mediates PAG uptake. We conclude that PAG, through competition with glutamine for the same membrane carrier, may reduce glutamine transport leading to intracellular glutamine depletion. The physiological consequence of this biochemical event could be critical to cancer cells and therefore might contribute to the mechanism of antineoplaston AS2-1 action.

The influence of antineoplaston A5 on particular subtypes of central dopaminergic receptors.

A new therapeutic strategy for the treatment of Parkinson's disease (PD) is based on providing trophic support for degenerating dopaminergic (DA) neurons. It can be accomplished by administration of neurotrophic factors, or inducing astrocytes to differentiate and produce such factors. Antineoplaston A5 (A5), which is a naturally-occurring cytodifferentiating agent, may induce astrocytes to undergo normal differetiation, produce neurotrophic factors and alleviate the symptoms of PD. This paper describes studies on the influence of A5 on subtypes of central DA receptors by measuring the potency of haloperidol catalepsy in rats. A5, D1 agonist, and D1 D2 antagonists were given i.p. and D2 agonist s.c. for three consecutive days. Haloperidol catalepsy was measured by the method of Costall and Nylor. The degree of catalepsy was assessed every 30 min for 24 h and statistically evaluated using the Student's t-test. The results confirmed that A5 significantly attenuated catalepsy and stimulates dopamine D2 receptors. It reverses catalepsy induced by haloperidol and D2 antagonists, but increases cataleptogenic activity if given in combination with the D2 agonist. This leads to the conclusion that A5 as a naturally-occurring agent neutralizes both hyper- and hypoactivity of central dopaminergic structures. Besides possible use as an antiparkinsonism agent, A5 may find application in the treatment of other disturbances of dopaminergic transmission.

The influence of antineoplaston A5 on the central dopaminergic structures.

Antineoplastons are naturally occurring cytodifferentiating agents. Chemically, antineoplastons are medium and small sized peptides, amino acid derivatives and organic acids which exist in blood, tissues and urine. In clinical trials in advanced cancer, in addition to the anticancer activity it was observed that patients suffering from both cancer and Parkinson's disease exhibited marked improvement in parkinsonian symtomatology when treated with antineoplaston A5. The present study was designed to analyse the influence of A5 on central dopaminergic structures. Mice and rats were given A5 intraperitoneally at three different dosage levels. Experiments conducted included spontaneous locomotor activity, amphetamine-induced yawning and erections, catalepsy, the effect on the level and utilization of noradrenaline and dopamine in the brain and the influence of prolonged and chronic treatment on the haloperidol-induced catalepsy. It has been demonstrated that A5 stimulates the central dopaminergic receptors. It diminishes the cataleptic response to haloperidol and enhances the incidence of apomorphine-induced yawning. Biochemical studies demonstrated increased concentration of dopamine and noradrenaline in the brain and diminished utilization of both catecholamines.

Treatment of hormonally refractory cancer of the prostate with antineoplaston AS2-1.

Successful treatment of advanced cancer of the prostate which no longer responds to hormonal manipulation continues to be a difficult task. The present study describes the results of treatment of the first fourteen patients in Phase II clinical trials with Antineoplaston AS2-1 (AS) and low-dose diethylstilbestrol (DES). The study involved thirteen patients diagnosed with stage IV and one patient with stage II adenocarcinoma of the prostate. The ages of the patients were between 54 and 88. The previous therapy included prostatectomy, orchiectomy, radiation therapy and treatment with DES, LHRH agonist (LH), flutamide (FL), aminoglutethimide and immunotherapy. After initial response to such treatments, the disease continued to progress. The majority of patients showed progression of the disease after treatment with LH and FL. The current treatment program consisted of administration of AS and DES. The treatment was given orally daily. The majority of patients received from 97 to 130 mg/kg/24 h of AS and from 0.01 to 0.02 mg/kg/24 h of DES. The treatment was administered from 64 to 425 days and was free from significant side effects due to AS. The dose of DES was lower than usual, and only some patients experienced mild side effects typical for DES. Only two patients showed progression of the disease. Complete remission was obtained in two patients and partial remission in three patients. Stabilization of the disease with objective improvement was determined in seven patients. The first patient enrolled in the program has been in complete remission for 17 months and off the treatment for 16 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial clinical study with antineoplaston A2 injections in cancer patients with five years' follow-up.

This paper describes Phase I clinical studies of Antineoplaston A2 injections. The studies involved 15 patients diagnosed with advanced neoplastic diseases including cancers of the breast, bladder, lung, kidney, oesophagus, colon and liver, mesothelioma and glioma. Antineoplaston A2 was administered in divided doses daily intravenously through a subclavian vein catheter. The treatment was given from 53 to 358 days. The highest dosage administered was 147 mg/kg/24 h. Only minimal adverse effects were noticed sometime during the treatment, including fever, chills and myalgia. Desirable side-effects included increase of platelet and white blood cell counts, hypertrophy of epidermis and decrease of cholesterol and triglyceride levels. Nine patients showed objective response to the treatment. Cases of complete remission included adenocarcinoma of the lung, mesothelioma, metastatic liver and bladder cancers. In an additional case of breast cancer, the patient obtained complete remission of liver metastasis and stabilization of bone metastases. Partial remission was accomplished in cancers of the breast and oesophagus. Three patients, including cases of adenocarcinoma of the lung, mesothelioma and bladder cancer, were in complete remission for over five years.

In vitro cancer growth inhibition and animal toxicity studies of antineoplaston A3.

Antineoplaston A3 is an oxidized mixture of small and medium size peptides and amino acid derivatives isolated from human urine. The purpose of this study was to evaluate the growth inhibitory effect of Antineoplaston A3 on cancer cells in vitro and its toxicity in mice. The growth inhibitory studies were carried out on breast carcinoma cells HBL-100. The cells were plated at 1000 cells per flask in L-15 medium and supplemented with 10% foetal bovine serum and antibiotics. Two different experiments were performed: to study dose response; and to investigate duration exposure. To establish dose response, Antineoplaston A3 was added to each flask to a final concentration of 0.05, 0.1, 0.2 and 0.4 mg/ml. Two flasks in each test were added with the same amount of medium to serve as control. The flasks were incubated at 37 degrees C for 6 days, then fixed and stained, after which colonies were counted. Results were expressed as percentage of control. In duration exposure experiments Antineoplaston A3 was added to the final concentration of 0.4 mg/ml. After 24, 48 and 72 h of exposure to Antineoplaston A3, the flasks were returned to normal growth medium and colonies were again counted. Dose response to Antineoplaston A3 was as follows (average of 3 replications +/- standard error): 0.05 mg/ml, 100% +/- 0; 0.1 mg/ml, 79.0% +/- 14.4; 0.2 mg/ml, 4.3% +/- 6.6; 0.4 mg/ml, 0% +/- 0.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I clinical studies of antineoplaston A3 injections.

Antineoplaston A3 is an oxidated mixture of small peptides and amino acid derivatives isolated from human urine which have shown antineoplastic activity in tissue culture and low toxicity in mice. Twenty-four patients diagnosed with 25 cases of neoplastic diseases were involved in the studies. The patients' diagnoses included: adenocarcinoma of the prostate, stage IV (7 cases); adenocarcinoma of the breast, stage IV (3); adenocarcinoma of the colon and rectum, stage IV (3); adenocarcinoma of the colon, status post resection (1); adenocarcinoma of the lung, stage III (2); squamous cell carcinoma of the lung, stage III (2); adenocarcinoma of the pancreas, stages II and IV (2); and single cases of adenocarcinoma of the kidney, stage IV; malignant fibrohistiocytoma, stage IV; glioblastoma multiforme, stage IV; basal cell epithelioma; and transitional cell carcinoma of the bladder, grade II. Only patients who had over six weeks' anticipated survival and who continued the treatment for over six weeks were eligible. In 23 patients, Antineoplaston A3 was administered in divided doses daily i.v. through a subclavian vein catheter. In one patient, the injections were given i.m. The length of treatment was from 44 to 478 days and the highest dosage was 76 mg/kg/24 h. Side-effects associated with treatment included febrile reaction (4 patients), vertigo (2), headache (2), flushing of the face, nausea and tachycardia (1 each). Adverse reactions were mild and occurred only once during the entire course of treatment. Desirable side-effects included increase of platelet count, increase of white blood cell count and increase of reticulocyte count. At the end of the study, there were 5 cases of complete remission, 5 of partial remission, nine of stable disease and six of increasing disease. The patients who obtained complete remission were diagnosed with cancers of the bladder, prostate, colon, and basal cell epithelioma. In view of its very limited toxicity and the interesting responses obtained, Antineoplaston A3 was submitted for Phase II clinical trials to establish its usefulness in cancer treatment.

Tissue culture and animal toxicity studies of antineoplaston A5.

Antineoplaston A5 is a mixture of small to medium size peptides and amino acid derivatives isolated from human urine. This report describes growth inhibition by Antineoplaston A5 of human cell carcinoma line HBL-100 and human leukaemic cell line Jurkat, and acute and chronic toxicity studies in mice. The inhibitory effect of Antineoplaston A5 on HBL-100 cells was tested at concentrations varying from 0.125 mg/ml to 0.5 mg/ml and at exposure durations varying from 24 to 96 h. The tests on Jurkat cells were carried out at concentrations varying from 0.1 mg/ml to 0.8 mg/ml and exposure durations varying from 24 to 120 h. The results from HBL-100 cells, shown as percentage of survival, were as follows: at 0.125 mg/ml 58%; at 0.25 mg/ml 14%; at 0.5 mg/ml 0. Exposure of HBL-100 cells to 0.5 mg/ml of Antineoplaston A5 for 24, 48, 72 and 96 h reduced survival to 55%, 10%, 4% and 0 of the control respectively. The results for suspension culture of Jurkat were as follows: The control cells showed a 7.7-fold increase in cell number during the incubation period, while the cells treated with 0.8 mg/ml of Antineoplaston A5 showed a steady decrease in cell number. At the end of 120 hours' incubation, only 1% of the cells was still viable. The growth inhibition patterns for dosages ranging from 0.4 mg/ml to 0.1 mg/ml were unique; instead of decreasing, the cell numbers in these flasks kept increasing, although at a slower pace during the initial 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I clinical studies of antineoplaston A5 injections.

Antineoplaston A5 is a mixture of small peptides and amino acid derivatives isolated from human urine which show a unique pattern of growth inhibition in the tissue culture of human neoplastic cells and no significant animal toxicity. Clinical trials described in this paper involved 15 patients diagnosed with advanced neoplastic diseases. The patients' diagnoses included: lung cancer, stage III (4 cases); breast, stages III and IV (3); colorectal, stage IV (2); bladder (2); and single cases of adenocarcinoma of the jejunum, stage IV; adenocarcinoma of the prostate, stage IV; adenocarcinoma of the ovary, stage IV and astrocytoma, grade IV. Antineoplaston A5 was administered daily in divided doses intravenously through a subclavian vein catheter. The formulation was given from 47 to 130 days. The highest dosage attained was 153 mg/kg/24 h. Adverse effects included febrile reaction in five patients, arthralgia in one patient and premature beats and pressure in the chest in one patient. The side-effects were very mild and usually experienced only once during the entire course of treatment. Desirable side-effects included increase of platelet count, increase of white blood cell count and hypertrophy of epidermis. Nine patients (60%) had objective response to the treatment. They were diagnosed with cancer of the lung (3 patients), breast (2), colorectal (2) and bladder (2). Four patients had increasing disease and two patients were classified as having stable disease without objective improvement.

Pharmacokinetic study of radioactive antineoplaston A10 following oral administration in rats.

Radiolabelled (3H-labelled) Antineoplaston A10 was administered in a single dose of 220 mg to 230 mg/kg to female Sprague Dawley rats. Blood and urine samples for determination of radioactivity were collected one hour prior to, and then at different time intervals after, the administration of the drug. Rats were sacrificed 6 h or 36 h later for the study of radioactivity in the various organs. The concentration of radioactivity in blood reached a maximum after 2 to 3 h after the administration of Antineoplaston A10, whereas the highest concentration of radioactivity in urine was observed in the 3.5-h to 4-h samples. It was observed by quantitative HPLC analysis that in rats sacrificed 6 h after Antineoplaston A10 administration, between 61% to 69% of the drug was absorbed, whereas between 37% to 28% was found in the stomach and between 2% to 3% was present in the intestinal contents and faeces. After 36 h, none could be detected in the stomach, intestinal contents or faeces. Organ distribution studies indicated greater accumulation of radioactivity in ileum, bladder, duodenum, kidneys and jejunum, and relatively low accumulation in the heart, lung, liver and brain. The concentration of radioactivity after 36 h was very low. By quantitative measurement, between 40% to 42% of the drug was excreted in the urine in 6 h and 75% of the radioactive material was in the form of Antineoplaston A10. The identification of the major radioactive material as Antineoplaston A10 was confirmed by TLC and analysis of the products of acid hydrolysis and by determination of melting range.

Chemoprevention by antineoplaston A10 of benzo(a)pyrene-induced pulmonary neoplasia.

The effect of Antineoplaston A10 (AA10), an amino acid derivative isolated from human urine, has been studied on pulmonary adenoma formation resulting from intragastric administration of benzo(a)pyrene(BP) to A/HeJ mice. Two doses of BP, 3 mg each, administered two weeks apart, induced an average of 6.86 tumours within 157 days in the control animals (Tumorigenic Index 437). One per cent of AA10 (w/w) given in mouse food for one week prior to, and then continued after the administration of BP, produced a 70% reduction in the total number of tumours in the test groups.

N,N'-disubstituted L-isoglutamines as novel cancer chemotherapeutic agents.

Previous studies carried out in the authors' Institute revealed that one of the active metabolites of Antineoplaston A10 is phenylacetylisoglutamine. The objective of this study is screening of N,N'-disubstituted L-isoglutamine derivatives for selection of novel anticancer agents. A series of seven derivatives of L-isoglutamine was synthesized as potential chemotherapeutic agents. Antitumor activity studies were performed in tissue culture of breast carcinoma cells HBL-100. The acute toxicity study was carried out on a group of 60 HA/ICR Swiss mice. Among the compounds tested N alpha-(phenylacetylamino)-gamma-(4-amino-N-benzyl-piperidinyl)-L-g lutamyl amide was found to have the best anticancer activity and no significant acute toxicity. The LD50 was calculated by the moving average method and determined as 4.5 g/kg i.p. in mice. It is concluded that the important structural features for good antineoplastic activity are lipophilic groups on both amine and amide nitrogen of isoglutamine. The activity is also increased when the phenyl group present at the side chain is at least two carbon atoms away from the amide nitrogen.

Quantitative assay of plasma and urinary peptides as an aid for the evaluation of cancer patients undergoing antineoplaston therapy.

Plasma and urinary peptides can be reproducibly assayed by a procedure involving reverse phase chromatography on a column of C18 and HPLC on a column of sulfonated polystyrene. The average plasma and urinary peptide levels of normal persons are 79.4 nmoles/ml and 73.6 nmoles/mg creatinine, respectively. Ninety-one percent of 108 confirmed cancer patients referred to the authors' Clinic for antineoplaston therapy showed various degrees of deficiency of peptides in the plasma, and 98% of the patients had plasma/urine peptide ratios below the normal ranges of 0.82 to 1.15, indicating that the overwhelming majority of cancer patients excreted greater than normal amounts of peptides in the urine. When a patient responded favourably to the antineoplaston therapy, the excessive urinary excretion of peptides was reversed. Plasma levels of peptides and plasma/urine peptide ratios showed a steady increase after one to two months and eventually approached those of normal persons. On the other hand, if a patient was not responding to the antineoplaston therapy, the excessive urinary excretion of peptides remained unaltered or even became greater. Patients in remission following antineoplaston therapy tended to show plasma and urinary peptide levels comparable to those of normal persons. Thus the quantitative assay of plasma and urinary peptides provides a useful parameter for the evaluation and prognosis of cancer patients undergoing antineoplaston therapy. A growing colon tumour was found to have the lowest level of peptides among mice tissues analysed, suggesting that deficiency of inhibitory peptide components may be associated with malignant growth. The results of this study are consistent with the interpretation that a deficiency of antineoplastons may be required for malignant cells to proliferate, and the consequent malignant growth further aggravates this deficiency. Antineoplaston therapy can halt such a "vicious cycle" and induce long-term remission in some cancer patients.

Chemo-surveillance: a novel concept of the natural defence mechanism against cancer.

Synthetic Antineoplaston A10, 3-phenylacetylamino-2,6-piperidinedione, has been shown to produce promising clinical results, similar to those obtained with antineoplastons derived from urine. Antineoplaston A10 is capable of reducing the excessive excretion of peptides often associated with cancer patients, thereby boosting endogenous levels of antineoplaston. Its therapeutic effect is partially attributable to endogenous antineoplastons, the level of which Antineoplaston A10 can help to raise. A patient must have a certain reserve of endogenous antineoplastons in order to benefit from therapy with Antineoplaston A10. The critical plasma peptide level seems to be above 30 nmoles/ml. Patients who responded favourably to the treatment with Antineoplaston A10 invariably showed an increase of plasma peptide levels and a decrease of urinary excretion of peptides. These results suggest that the body, under normal circumstances, is protected by antineoplastons against cancer. This chemical mechanism of protection is hereby termed chemo-surveillance.

Stereochemical modelling studies of the interaction of antineoplaston A10 with DNA.

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedione), a peptide analogue originally isolated from human urine, has been demonstrated to fit between base pairs in DNA. Examination of the fit of A10 into the 10 possible sites in unwound DNA using published criteria revealed a preference, but not absolute specificity, for the sequence 5'-dTdT-3' X 5'-dAdA-3'. Good fits were also observed for the sequences 5'-dTdC-3' X 5'-dGdA-3' and 5'-dCdT-3' X 5'-dAdG-3'. In each case at least one stereospecific hydrogen bond was possible between the imino proton of the piperidinedione ring stacked between the two pyrimidines and a neighbouring phosphate oxygen of the DNA backbone. These findings support the prediction that A10 may interact reversibly with DNA and thereby compete with carcinogens that form covalent linkages with DNA (e.g., arene oxides). It follows that such interactions should prevent the growth of tumours induced by various carcinogens.

Preclinical studies on antineoplaston AS2-1 and antineoplaston AS2-5.

Antineoplaston AS2-1 and Antineoplaston AS2-5 are degradation products of Antineoplaston A10. The initial hydrolysis of Antineoplaston A10 (3-phenylacetylamine-2, 6-piperidinedione) yields phenylacetylglutamine and phenylacetylisoglutamine. When hydrolysis is carried further the products of the reaction include phenylacetic acid, glutamic acid and ammonia. Sodium salts of phenylacetylglutamine, named Antineoplaston AS2-5, phenylacetic acid and a mixture of phenylacetylglutamine and phenylacetic acid in the ratio of 1:4, named Antineoplaston AS2-1, were submitted for tissue culture and animal toxicity studies. Tissue culture tests were carried out on breast carcinoma cell lines HBL-100 and Ki No. 1 and the results were expressed as the percentage of colony reduction based on control. Acute toxicity studies were done on a group of 88 HA/ICR Swiss mice with each of these formulations. An additional group of 160 mice was used for chronic toxicity studies of Antineoplaston AS2-1. In chronic toxicity studies the mice were injected with 92.3 mg/kg, 553.8 mg/kg and 1107.6 mg/kg of Antineoplaston AS2-1 intraperitoneally for 365 days and subjected to complete physical, gross pathology and microscopic examination. LD50 for tested formulations were as follows: Antineoplaston AS2-1: 2.83 g/kg, Antineoplaston AS2-5: 2.90 g/kg and phenylacetic acid: 2.71 g/kg. The results clearly demonstrated that Antineoplaston AS2-1 has interesting antineoplastic activity in tissue culture of breast carcinoma, and low acute and chronic toxicity in mice. Antineoplaston AS2-5 does not show significant activity in tissue culture of breast carcinoma HBL-100 and only slight antineoplastic activity in Ki No. 1. Both formulations were submitted for phase I clinical testing in cancer patients.

Toxicology studies on antineoplaston AS2-5 injections in cancer patients.

Antineoplaston AS2-5 is one of the degradation products of Antineoplaston A10. The chemical structure of Antineoplaston AS2-5 corresponds to phenylacetylglutamine. Toxicology studies of Antineoplaston AS2-5 injections involved 13 patients diagnosed with 15 types of neoplastic disease, including: lung cancer, 3 cases; breast, 3 cases; colon, 2 cases; and single cases of cancer of the larynx, prostate, stomach, pancreas, malignant fibrohistiocytoma, embryonal teratoma and lymphocytic lymphoma. Antineoplaston AS2-5 was injected i.v. daily through subclavian vein catheter in divided doses. The treatment was administered from 41 to 436 days. The highest dosage given was 167.6 mg/kg/24 h. The treatment was associated with only very mild side-effects, including febrile reaction in two patients and swelling of small joints in one patient. Two patients had beneficial side-effects, including increase of platelet count and increase of concentration of plasma globulin. The treatment resulted in two complete remissions, one mixed response, four cases of stabilization and six cases of increasing disease. Complete remission was achieved in squamous cell carcinoma of the larynx, stage II, and large cell undifferentiated carcinoma of the lung with lymph nodes and liver metastases. One patient had mixed response during the treatment of carcinoma of the breast with metastases to the lymph nodes, liver and skin and obtained complete remission of liver metastases but increasing disease of skin metastases. Eight patients discontinued the treatment and three patients died during the trials. The patient diagnosed with lung cancer who obtained complete remission continues to be free from the disease over 5 years after the beginning of the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicology studies on antineoplaston AS2-1 injections in cancer patients.

Antineoplaston AS2-1 is a mixture of two products of hydrolysis of Antineoplaston A10 and consists of sodium salts of phenylacetylglutamine and phenylacetic acid in the ratio of 1:4. Antineoplaston AS2-1 injections were administered to 20 patients diagnosed with 21 types of neoplastic diseases. The patients' diagnoses included: lung cancer, stage III, 4 cases; colorectal, stage IV, 3; breast, stage IV, 2; breast in remission, 1; glioblastoma, 3; head and neck, stage IV, 3; uterine cervix, stage IA, 1; chronic myelocytic leukaemia, 2; lymphocytic lymphoma, stage IV, 1; and leiomyosarcoma of the uterus, stage IVB, 1. Antineoplaston AS2-1 was administered every 6 h i.v. through subclavian vein catheter. The treatment was administered from 38 to 872 days. The highest dosage taken was 160 mg/kg/24 h. The treatment was associated with minimal side-effects, including slight nausea and vomiting in one patient, mild allergic reaction in the form of maculopapular rash in another patient and moderate elevation of blood pressure in an additional patient. One patient developed febrile reaction and three patients had mild electrolyte imbalance. Only one patient showed slight decrease of WBC. Desirable side-effects included improved healing of chronic atrophic ulceration. The response to the treatment included 6 complete remissions, 2 partial remissions, 7 cases of stabilization and 6 cases of increasing disease. Three patients are alive, well and free from cancer 5 years after the beginning of the study. The hypothetical mechanism of action of Antineoplaston AS2-1 as an anticancer agent is described.