Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Patent
1995-12-12
1998-05-19
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
424 945, 4351721, 4351723, 4352523, 43525411, 4352542, 43525421, 43525423, 4352543, 4352546, 4353201, 530402, 536 232, 935 22, C12N 988, A61K 3851, C07K 100, C07H 2104
Patent
active
057534870
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a PCT/DK94/00224 filed Jun. 9, 1994, which is incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a stabilised phenylalanine ammonia lyase, a phenylalanine ammonia lyase variant, a method of preparing the variant and a pharmaceutical composition containing phenylalanine ammonia lyase.
BACKGROUND OF THE INVENTION
Hyperphenylalaninemia, which may be defined as a plasma level of phenylalanine of more than 120 .mu.mol/l, is a hereditary disease caused by a deficiency in the hepatic enzyme phenylalanine hydroxylase or (in rare cases) its cofactor tetrahydropterin or the cofactor-regenerating enzyme dihydropterin reductase. The disease exists in different forms, phenylketonuria (PKU) which, if the patient is on a normal diet, has plasma phenylalanine levels of more than 1200 .mu.mol/l, and non-PKU hyperphenylalaninemia which has lower levels of plasma phenylalanine.
In normal subjects, phenylalanine hydroxylase converts phenylalanine to tyrosine. Highly increased plasma levels of phenylalanine (>600 .mu.mol/L) result in mental retardation. The effect appears to be ascribable to phenylalanine itself (not any metabolites thereof), but the mechanism is not yet fully understood. In most industrialised countries, newborn children are routinely screened for hyperphenylalaninemia. The negative effects of increased plasma levels of phenylalanine may, to a large extent, be prevented if a low-phenylalanine diet is introduced shortly after birth and continued well into adolescence. The aim is to obtain plasma phenylalanine levels of 180-425 .mu.mol/l. After adolescence, the low-phenylalanine regimen may be somewhat relaxed, although phenylalanine-free products are still a significant component of the diet. Pregnant hyperphenylalaninemic patients are required to go back on a strict low-phenylalanine diet in order to avoid the effects of excessive intrauterine phenylalanine, i.e. congenital malformation, microcephaly and mental retardation of the fetus.
The strict low-phenylalanine regimen is tiresome for the patients and their families, and is very difficult to enforce beyond childhood. Enzyme therapy to make up for the phenylalanine hydroxylase deficiency would therefore provide a great improvement in the treatment of hyperphenylalaninemia. Unlike phenylalanine hydroxylase, another phenylalaninedegrading enzyme, phenylalanine ammonia lyase, requires no cofactors to be active. Phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid which, via coenzyme A, is converted to benzoic acid which reacts with glycine and is then excreted via urine primarily as hippurate. The enzyme may, for instance, be obtained from the yeast Rhodotorula glutinis (also known as Rhodosioridium toruloides). It has previously been suggested to use phenylalanine ammonia lyase for treatment of hyperphenylalaninemia, vide for instance, J. A. Hoskins et al., Lancet, Feb. 23, 1980, pp. 392-394. Proteolytic degradation of the enzyme in the gastrointestinal tract has been recognized, e.g. by H. J. Gilbert and G. W. Jack, Biochem. J. 199, 1981, pp. 715-723. Various attempts to overcome this problem have been published. Thus, L. Bourget and T. M. S. Chang, Biochim. Biophys. Acta 883, 1986, pp. 432-438, propose microencapsulation of the enzyme in "artificial cells" composed of phenylalanine ammonia lyase mixed with hemoglobin and enclosed in microspheres covered by a cellulose nitrate membrane. H. J. Gilbert and M. Tully, Biochem. Biophys. Res. Comm. 131(2), 1985, pp. 557-563 propose using permeabilised cells of Rhodosporidium toruloides containing the enzyme. However, both of these approaches may have drawbacks such as low specific phenylalanine ammonia lyase activity of the final preparation or high cost due to processing or formulation.
DESCRIPTION OF THE INVENTION
The object of the present invention is to overcome the drawbacks of the previously suggested methods of stabilising phenylalanine ammonia lyase.
Accordingly, the present inv
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Clausen Ib Groth
Eigtved Peter
Agris, Esq. Cheryl T.
Novo Nordisk A S
Stole Einar
Wax Robert A.
Zelson Esq. Steve T.
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