Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1991-12-13
1995-01-10
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
424 9465, C12N 950, A61K 3754
Patent
active
053806565
DESCRIPTION:
BRIEF SUMMARY
This invention relates to chymopapain, improved pharmaceutical compositions containing chymopapain and to methods of treating damaged, herniated or otherwise abnormal intervertebral mammalian spinal discs which comprise injecting into said discs a solution of the improved composition. The invention further relates to processes for preparing the chymopapain of the invention, to peptides and affinity chromatography matrices of use in such processes, and to monospecific antibody preparations raised against chymopapain.
Chymopapain is a cysteine proteinase present in the latex of the paw-paw (Carica papaya) plant. It has been found to be of clinical use, particularly for the treatment of prolapsed or herniated discs or sciatica in a process known as "chemonucleolysis", Smith, L. (1964), J. Amer. Med. Assoc. 187, 137-140.
Purification and characterisation of chymopapain was first attempted by Jansen and Balls (1941), J. Biol. Chem., 137, 405-417 who used acid precipitation and a salting-out procedure to prepare the More recently, purification methods based on ion-exchange chromatography have been employed. Thus, for example, GB2098997 (Smith Laboratories Inc.) and GB2156821 (Simmons) both describe the use of cation-exchange resins to separate chymopapain from other known cysteine proteinases and these processes have both been used to prepare chymopapain on an industrial scale. However, the resulting material is found to be of relatively low specific activity compared to the chymopapain prepared by Buttle and Barrett (1984), Biochem. J., 223, 81-88 using a multi-stage process on a small scale incorporating inter alia a cation-exchange chromatography step. Whilst this material was of high specific activity it was obtained in only very low yields and the process is not suitable for application on a commercial scale. Buttle et al., Biochem. J. (July 1989) 261, 469-476, have now found that this material is contaminated with a recently isolated and characterised proteinase, papaya proteinase IV, which co-elutes with chymopapain from cation-exchange resins. Cation-exchange chromatography is unable to separate chymopapain from papaya proteinase IV to an extent which could be of use in the preparation of chymopapain substantially free of contamination with papaya proteinase IV.
The literature also contains references to methods employing affinity chromatography steps. Polgar (1981), Biochim. Biophys. Acta, 658, 262-269, describes the purification of chymopapain using, amongst a number of other techniques, affinity chromatography on an agarose-mercurial column and Dubois et al., Biol. Chem. Hoppe-Sevler (1988), 369, 733-740, have reported separation of cysteine proteinases from Carica papaya latex using a similar technique. This type of affinity chromatography relies on the interaction between mercuric ligands bound to an inert matrix and the thiol groups of the proteins exposed thereto. Thus, proteins other than chymopapain, particularly other cysteine proteinases such as papaya proteinase IV, can bind to such columns and can subsequently be co-eluted with chymopapain.
Recently, scientific papers have described the use of affinity chromatography using immobilised peptide derivatives to purify specific proteinases such as human cathepsin B (Rich et al., 1986, Biochem. J. 235, 731-4) and histolysin from Entamoeba histolytica (Luaces & Barrett, 1988, Biochem. J. 250, 903-909). This approach had not hitherto been applied to procedures for purifying chymopapain and the need for an improved process for purifying chymopapain, suitable for use on an industrial scale, remains apparent.
Certain synthetic peptide derivatives which are inhibitors of the serine proteinase chymotrypsin have been described in WO84/00365.
The inventors have now discovered a novel process to purify and isolate highly active chymopapain free from contaminants and, in particular, free from the other cysteine proteinases present in papaya latex, including papaya proteinase IV.
The present invention provides chymopapain which is substantially pure and which
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Barrett Alan J.
Buttle David J.
Rich Daniel H.
Robinson Douglas W.
The Boots Company PLC
Weber Jon P.
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