To begin the search for antineoplastons, the developer used human blood, separating and removing the peptides found there. Later it was discovered that the same peptide fractions existed in human urine. Each peptide fraction was tested in vitro against various normal and neoplastic cell lines to gauge their effect on DNA synthesis and growth. The fractions that had little or no inhibitory effect on normal cells but a substantial inhibitory effect on neoplastic cell lines were separated into two classes: those that were effective against a specific cell line and those that were active against a broad array of neoplastic cell lines. Those with a broad spectrum of activity were grouped together and called "antineoplaston A." Peptide fractions with specific antineoplastic activity were not investigated further.
Antineoplaston A was further purified and yielded antineoplastons A1, A2, A3, A4, and A5. These mixtures of 7 to 13 peptides were patented in 1985.In vitro tissue culture studies and in vivo toxicity studies in animal models were performed for antineoplastons A1 through A5. According to the developer, each individual fraction had a higher level of antitumor activity and lower toxicity level than antineoplaston A.
Phase I trials of this antineoplaston group in patients with various advanced cancers showed A2 as contributing to the highest tumor response rate, so it was selected for further study.
The active compound in A2 was found to be 3-phenylacetylamino-2,6-piperinedione, which was named antineoplaston A10. From antineoplaston A10, three other compounds have been derived:
- AS2-5, which is phenylacetylglutamine (PAG).
- AS2-1, which is a 4:1 mixture of phenylacetic acid (PA) and PAG.
- A5, which is PA and an aromatic fatty acid.
Other antineoplastons (A3, A4, A10-1, AS5) were added to this group after further studies.[2,3,4]
There have been no independent analyses of which amino acids comprise the antineoplastons used in any of the reported studies.
Antineoplastons are administered by different methods. Antineoplaston A has been given intravenously, intramuscularly, rectally, topically, intrapleurally, and by bladder instillation. Presently, antineoplastons A10, AS2-5, AS2-1, A2, A3, and A5 are given orally or by injection.[8,9,10,11,12,13,14,15,16,17,18,19,20]