Pharmaceutical for treating tumors and method for making it
AssigneeAlfacell Corporation
InventorsKuslima Shogen, Stanislaw M. Mikulski, Wojciech J. Ardelt
Life of this patent
Estimating the term…
Abstract
Rana pipiens eggs are subjected to fertilization and the embryos are grown to a predetermined stage of development. The embryos and unfertilized eggs are then subjected to mechanical processing in the presence of a weakly acidic buffer to produce an extract. The extract is subjected to ion-exchange chromatography and size-exclusion chromatography. The resulting pharmaceutical has activity against certain cancer cells. A partial amino acid sequence of the pharmaceutical is disclosed.
Claims21
A substantially pure protein having a molecular weight of approximately 14,000 to 16,000 Daltons and an amino acid composition approximately as follows:
- ______________________________________ APPROX. # OF RESIDUES AMINO ACID PER MOLECULE OF MATERIAL ______________________________________ Aspartic acid/Asparagine 20 Threonine 13 Serine 11 Glutamic acid/Glutamine 9 Proline 6 Glycine 4 Alanine 4 Cystine/2 10 Valine 11 Methionine 1 Isoleucine 8 Leucine 7 Tyrosine 3 Phenylalanine 8 Histidine 4 Lysine 17 Arginine 4 Tryprophan 2 Approximate Total 142 ______________________________________
A substantially pure protein derived from frog eggs which have been subjected to fertilization, the protein having a molecular weight of approximately 15,000 Daltons, an isoelectric point pI of 9.5 to 10.5 and a blocked amino terminal group, the protein being substantially free of carbohydrates and other proteins.
A substantially pure protein which has a single methionine residue and which, when cleaved with cyanogen bromide at said residue, produces a shorter fragment and a longer fragment,
- the shorter fragment having a blocked amino terminal, a homoserine plus homoserine lactone carboxy terminal and a length of approximately 23 residues, and
- the longer fragment having a free amino terminal group and a length of approximately 121 residues,
- and the longer fragment containing an amino acid sequence expressed as ##STR3## in which sequence amino acid residues which have been identified with a level of confidence which is less than 100% and is at least 80% have been shown within parentheses, in which Xxx indicates an unidentified residue, in which underlined residues might be in inverted order, and in which there is an apparent polymorphism of Asn and Asp where they are listed in a common position.
A substantially pure protein which has a single methionine residue and which, when cleaved with cyanogen bromide at said residue, produces a shorter fragment and a longer fragment, the amino acid composition of the fragments being approximately as follows:
- ______________________________________ SHORTER FRAGMENT APPROX. # OF RESIDUES AMINO ACID PER MOLECULE OF MATERIAL ______________________________________ Aspartic acid/Asparagine 20 Threonine 6 Serine 3 Glutamic acid/Glutamine 0 Proline 2 Glycine 0 Alanine 0 Cystine/2 0 Valine 1* Methionine 1 Isoleucine 1** Leucine 2 Tyrosine 1 Phenylalanine 0 Histidine 1 Lysine 1 Arginine 1 Tryprophan 1 Approximate Total 23 ______________________________________ *(as carboxymethylcysteine) **(as homoserine and homoserine lactone)
- ______________________________________ LONGER FRAGMENT APPROX. # OF RESIDUES AMINO ACID PER MOLECULE OF MATERIAL ______________________________________ Aspartic acid/Asparagine 15 Threonine 10 Serine 10 Glutamic acid/Glutamine 7 Proline 6 Glycine 5 Alanine 4 Cystine/2 9 Valine 10 Methionine 0 Isoleucine 6 Leucine 6 Tyrosine 4 Phenylalanine 7 Histidine 3 Lysine 15 Arginine 3 Tryprophan 1 Approximate Total 121 ______________________________________
A process for producing a purified pharmaceutical, comprising the following steps:
- (a) subjecting eggs of a frog to fertilization and thereby producing embryos;
- (b) terminating development of the embryos prior to gastrualation;
- (c) mechanically homogenizing the embryos and any unfertilized eggs in the presence of a weakly acidic buffer;
- (d) centrifuging said homogenized embryos and eggs to derive a supernatant liquid therefrom;
- and
- (e) subjecting the supernatant liquid to ion-exchange chromatography
- to thereby obtain a purified pharmaceutical.
A process for producing a pharmaceutical, comprising the following steps:
- (a) subjecting eggs of a frog to fertilization and thereby producing embryos;
- (b) terminating development of the embryos prior to gastrulation;
- (c) mechanically homogenizing the embryos and any unfertilized eggs in the presence of a weakly acidic buffer;
- (d) centrifuging said homogenized embryos and eggs to derive a supernatant liquid therefrom;
- and
- (e) extracting, from the supernatant liquid, materials having an isoelectric point between 9.5 and 10.5.
A process for producing a pharmaceutical, comprising the following steps performed in the order listed:
- (a) subjecting eggs of a frog to fertilization and thereby producing embryos;
- (b) terminating development of the embryos prior to gastrulation;
- (c) mechanically homogenizing the embryos and any unfertilized eggs in the presence of a weakly acidic buffer;
- (d) centrifuging said homogenized embryos and eggs to derive a supernatant liquid therefrom;
- (e) subjecting the supernatant liquid to ion-exchange chromatography and thereby recovering a material;
- (f) subjecting said material to a second step of ionexchange chromatography under different conditions and thereby recovering a purer material;
- and
- (g) subjecting said purer material to size-exclusion chromatography.
A process for manufacturing a pharmaceutical, comprising the following steps performed in the order listed:
- (a) creating embryos of the Rana pipiens frog by fertilization of Rana pipiens eggs with Rana pipiens sperm outside the body of a female Rana pipiens;
- (b) terminating development of the embryos at approximately the 4 cell stage;
- (c) mechanically homogenizing the embryos and any unfertilized eggs in a weakly acidic buffer;
- (d) centrifuging said homogenized embryos and eggs to derive a supernatant liquid therefrom;
- (e) filtering the supernatant liquid;
- (f)subjecting the filtered supernatant liquid to ion-exchange chromatography and thereby recovering a material;
- (g) re-subjecting said recovered material to a second step of ion-exchange chromatography under slightly different conditions and thereby recovering a purer material;
- and
- (h) subjecting said purer material to size-exclusion chromatography and thereby recovering a purified material.
Dependencies are inferred from the claim text.