Liposomes and Erythrocyte Ghosts for Enzyme And

LIPOSOMES AND ERYTHROCYTE GHOSTS FOR

ENZYME AND CHELATION THERAPY

Penelope Patricia Powell

A Thesis Submitted for the Degree of Doctor of Philosophy

Charing Cross Hospital Medical School University of London

November 1983 1

ABSTRACT

Three animal models of inborn errors of metabolism were used to investigate strategies for enzyme or chelation therapy using liposomes and erythrocyte ghosts.

Histidase, the first enzyme in the major pathway of histidine catabolism, was purified from a species of Pseudomonas for replacement in a murine model of histidinaemia. The enzyme was encapsulated in reverse phase liposomes for delivery to liver, but was found to be inactivated in hepatic lysosomes. Smaller and more stable liposomes for prolonged circulation and protection of entrapped histidase were both inefficient at histidase encapsulation and impermeable to histidine, which is 20-fold elevated in the plasma of histidinaemic mice. Liposomes were incapable of decreasing histidine levels in vivo. Histidase was entrapped in erythrocyte ghosts, prepared by a hypotonic dialysis method, for prolonged survival and the degradation of histidine in the circulation. Pseudomonas histidase was shown to be subject to substrate and product inhibition. Erythrocyte ghost entrapped histidase activity was found to be limited in vitro by the rate of diffusion of product from and substrate into the cells. In vivo, 125i-pvp-containing erythrocyte ghosts had a half- life in the circulation of 28 hours. l^Sj-higtidase entrapped in ghosts had a similar circulation time, but underwent an initial rapid lysis. On administration to histidinaemic mice, erythrocyte ghost entrapped histidase decreased plasma histidine levels 2-fold, whereas the same amount of free histidase decreased plasma histidine levels 30-fold. PRO/Re mice were used as a model for hyperprolinaemia, where proline levels are elevated due to a deficiency of mito chondrial proline oxidase. Proline oxidase was purified from E. coli and entrapped in liposomes and erythrocyte ghosts. The activity of entrapped proline oxidase was characterised. Lipospmally-entrapped, erythrocyte-entrapped and free proline oxidase all decreased plasma proline levels. Entrapped enzyme was more effective than the free enzyme. Rats given an iron overload were used as a model of haemochromatosis. Liposomally-entrapped desferrioxamine increased urinary excretion of iron and decreased hepatic iron levels more effectively than free desferrioxamine. - 2 - CONTENTS Pa ^ c Abstract 1 Contents 2 Acknowledgements 8 Abbreviations 9 Chapter 1 . General Introduction 10 Section A 11 1. General Introduction - Inborn Errors of Metabolism 11 2. Models for Therapy 14 (i) In vitro Cell Culture 14 (ii) In vivo Animal Models 16 3. Free Enzyme Therapy in vivo 19 4. Complications in Enzyme Replacement Therapy 22 (i) Source 22 (ii) Delivery 22 (iii) Stability 23 (iv) Immunological Effects 23 5. Enzyme Therapy for Amino Acid Depletion in vivo 25 6. Therapy for Metal Depletion 27 Section B Enzyme Carriers for Replacement Therapy 28 1. Antibodies 29 2. Receptor Mediated Targetting 30 3. Artificial Cells 31 4. Albumin 33 5. Nanoparticles and Microspheres 34 Section C Liposomes 33 1. Formation 35 2. Characteristics of Liposomes 37 (i) In vitro 37 (ii) In vivo 40 3. Enzyme Delivery in Liposomes in vitro 43 4. Enzyme Delivery in Liposomes in vivo 46 5. Chelation Therapy with Liposomes 49 6. Problems with Liposomes in vivo 51 (i) Serum 51 (ii) ImmunologicalInteractions 54 (iii) Targetting 55 7. Other Therapeutic Uses 57 Section D Erythrocyte Ghosts 58 1. Hypotonic Dilution Haemolysis 59 2. Electrical Haemolysis ^ - 3 - Pago

3. Hypotonic Dialysis Haemolysis 64 4. Erythrocyte Entrapped Enzymes in vitro 65 5. Erythrocyte Entrapped Enzymes in vivo 69 6 . Immunological Effects of Carrier Erythrocytes 71 in vivo 7. Human Administration of Erythrocyte Entrapped 72 Enzymes and Chelating Agents 8. Conclusions 73 Section E Objectives of the Project 74

Chapter 2 . The Possible Use of Liposomes and Erythrocyte Ghosts for Enzyme Therapy in 75 Murine Histidinaemia Section A Introduction 76 1. Histidinaemia in Man 76 2. The Histidinaemic Mutant Mouse 78 (i) Biochemical Characteristics 78 (ii) Genetic Characteristics 81 (iii) Endogenous Teratogenesis in Maternal Histidin- 83 aemia in Mice 3. The Physical and Chemical Characteristics of 84 Histidase (i) Mammalian Histidase 84 (ii) Bacterial Histidase 85 4. Synopsis of this Study 87 Section B Materials and Methods 89 Materials 89 1. Animals 89 2. Bacteria 89 3. Reagents 89 4. Apparatus 90 Methods 90 1. Assays 90 (i) Histidase Assay 90 (ii) Phenylalanine Ammonia Lyase Assay 92 (iii) Pauly Assay for Histidine and its Imidazole 92 Derivatives (iv) Protein Assay (Lowry Method) 93 (v) Protein Assay (Coorriassie Method) 93 (vi) Phosphorus Assay 93 (vii) Cholesterol Assay 93 (viii) Subcellular Fractionation Assays c-‘> Pago

2. Purification of Phosphatidylcholine from Egg Yolk 96 3. Thin Layer Chromatography 97 4. Preparation of Liposomes 98 (i) Multilamellar Liposomes MLV 98 (ii) Sonicated Liposomes 98 (iii) Reverse Phase Vesicles REV 100 (iv) Double Emulsion Technique (Battelle) 102 5. Efflux of Histidine from Liposomes 105 6. Preparation of Erythrocyte Ghosts 105 7. Estimation of Histidase Entrapped in Erythrocyte 106 Ghosts 8. Influx of Histidine into Erythrocyte Ghosts 109 9. Iodination of Histidase 110 10. Polyacrylamide Disc Gel Electrophoresis 110 11. Sub-Cellular Fractionation of Mouse Liver 111 12. Growth of Pseudomonas 112 13. Animal Experiments 114- 14. Amino Acid Analysis 114- 15. Scintillation Counting 115 16. Preparation of Column Resins 115 17. Expression of Experimental Results 115 Section C Histidase Purification 117 1. Purification of Mammalian Histidase 117 2. Polyacrylamide Gel Electrophoresis of Rat Liver 122 Histidase 3. Characterisation of Liver Histidase Activity 125 4. Growth of Pseudomonas ATCC 11,299b 130 5. Purification of Bacterial Histidase 130 6. Polyacrylamide Gel Electrophoresis of Bacterial 138 Histidase 7. Kinetic Studies on Pseudomonas Histidase 138 8. Inhibition of Pseudomonas Histidase by ATP 143 9. Radioiodination of Pseudomonas Histidase 143 Section D Entrapment and Activity of Histidase in 145 Liposomes 1. Entrapment and Latency of Histidase in Liposomes 145 (i) Multilamellar Liposomes MLV 145 (ii) Sonicated Liposomes 146 (iii) Reverse Phase Vesicles REV 146 2. Passage of ~^C Histidine Through the Liposome 150 Me rn bran e - 5 - Page

Section E Distribution of Liposomes in Mice 153 125 1. Liposome-Entrapped I PVP in vivo 154. (i) Distribution of ^ ^ 1 pyp j_n ^ice 154 125 (ii) Distribution of J1 PVP Entrapped in Reverse 151 Phase Vesicles in Mice (iii) Distribution of ^ ^ 1 pyp Entrapped in Sphingo- 154 myelin-Cholesterol Reverse Phase Vesicles (iv) Subcellular Fractionation of Mouse Liver.after 154 125 Administration of I PVP Liposomes 2. Liposome-Entrapped 125 I Histidase in vivo 158 12 5 (i) Distribution of Histidase Entrapped in 158 Reverse Phase Liposomes in Mice (ii) Subcellular Fractionation of Mouse Liver after 160 125 Administration of I Histidase in Liposomes 3. Administration of Histidase in Reverse Phase Lipo- 160 somes to Histidinaemic Mice (i) Subcellular Fractionation after One Hour 160 (ii) Effect on Blood Histidine Levels over 24 Hours 160 Section F Entrapment and Activity of Histidase and Phenylalanine Ammonia Lyase in Erythrocyte 1^4 Ghosts in vitro 1. Entrapment of Histidase in Erythrocyte Ghosts 164 2. Entry of Histidine into Erythrocyte Ghosts 164 3. Activity of Histidase Entrapped in Erythrocyte Ghosts 166 4. Entrapment of Phenylalanine Ammonia Lyase in Erythro- 168 cyte Ghosts o 5. Passage of ^H Phenylalanine into and out of Erythro- 169 cyte Ghosts 6. Activity of Phenylalanine Ammonia Lyase Entrapped 169 in Erythrocyte Ghosts Section G Administration of Erythrocyte Ghosts to Mice 172 1. Distribution of ^ ^ 1 PVP Entrapped in Erythrocyte 172 Ghosts in vivo 2. Distribution of Erythrocyte Ghosts containing 125 I 172 Histidase in vivo 3. Administration of Histidase Entrapped in Erythrocyte 175 Ghosts to Histidinaemic Mice Section H Discussion 182 - 6 - Page

Chapter 3 • The Possible Use of Liposomes and Erythro cyte Ghosts for Enzyme Therapy in Murine 197 Hyperprolinaemia Section A Introduction 298 1. Hyperprolinaemic Model Mouse I93 2. Proline Oxidase 203 (i) Mammalian Proline Oxidase 203 (ii) Bacterial Proline Oxidase 2O4. 3. Objectives of this Study 206 Section B Materials and Methods 207 Materials 207 1. Animals 207 2. Bacteria 207 3. Reagents 207 4-. Apparatus 207 Methods 208 1. Proline Oxidase Assays 208 (i) o-Aminobenzaldehyde 208 (ii) Iodonitrophenyl Tetrazolium Violet (INT) 208 (iii) A Radioisotopic Assay for Proline Oxidase 209 2. Entrapment of Proline Oxidase in Liposomes 211 3. Entrapment of Proline Oxidase in Erythrocyte Ghosts 211 4.. Detection of Hyperprolinaemic Mice 212 5. Passage of Proline Through the Erythrocyte Ghost 212 Membrane Section C Proline Oxidase 213 1. Growth of Bacteria 213 2. Purification of Proline Oxidase (E,coli) 213 3. Characteristics of E. coli Proline Oxidase 215 4-. Purification of Mouse Liver Proline Oxidase 215 Section D Entrapment of Proline Oxidase in Liposomes 220 and Erythrocyte Ghosts 1. Entrapment of Proline Oxidase in Liposomes 220 2. Entrapment of Proline Oxidase in Erythrocyte Ghosts 222 3. Passage of ^^C Proline Through the Erythrocyte 224- Membrane Section E Administration of Proline Oxidase to PRO/Re 226 Mice 1. Liposomally-Entrapped Proline Oxidase Administered 226 to PRO/Re Mice 7 Page 2. Erythrocyte Entrapped Proline Oxidase Administered 230 to PRO/Re Mice 3. Isatin Test on Urine of PRO/Re Mice 230 Section F Discussion 233 Chapter 4. The Possible Use of Liposomes for Chelation 238 Therapy in Iron Overload Section A Introduction 239 Section B Materials and Methods 244 Materials 244 1. Animals 24/ 2. Reagents 244 Methods 244 1. Animal Model for Haemochromatosis 244 2. Assay for Iron 244 3. Assay for Desferrioxamine 245 4- Entrapment of Desferrioxamine in Reverse Phase 245 Vesicles 5. Subcellular Fractionation of Rat Liver 246 6. Radioactive Iron Excretion 246 Section C Re suits 247 1. Entrapment of Desferrioxarnine in Reverse Phase 247 Vesicles 2. Animal Model for Haemochromatosis 247 3. Administration of Liposomally-Entrapped Desferri- 249 oxamine to Iron Overloaded Rats 4* Iron Content in Liver of Iron Overloaded Rats after 250 Administration of Liposomally-Entrapped Desferrioxamine Section D Discussion 255 Chapter 5. Concluding Remarks ^ 9

References 263 8

ACKNOWLEDGEMENTS

This study has been carried out jointly between the Department of Biochemistry, Charing Cross Hospital Medical School, London University and the Department of Genetics, University of Edinburgh. I would like to express my sincere thanks to Professor B. E. Ryman and Dr. H. Kacser for their invaluable advice and guidance during the course of this research. I am grateful to Professor Ryman for her encouragement and interest, particularly during the preparation of this manuscript. I would like to thank Dr. H. Kacser for his help and discussion concerning enzyme kinetics and for all his efforts in making it possible to work in two laboratories 4-00 miles apart.

In London, I am indebted to Dr. T. N. Palmer for his help with bacteriological techniques, advice about enzyme purification and for his general encouragement throughout this work. I would like to thank Miss M. A. Caldecourt for providing electron micrographs of liposomes. I thank Mr. C. J. Moore for technical help.

In Edinburgh, I am very grateful to Mrs. J. E. Burns for her help in running the amino acid analyser and with the histidinaemic and PRO/Re mice. I would like to thank Mrs. K. Henderson for technical help and Mrs. L. McIntyre for looking after the mice.

I express my thanks to Dr. R. A. Chalmers and Dr. A. R. Hubbard for demonstration of the preparation of dialysis erythrocyte ghosts. I thank Professor P. Cohen and Dr. C. Klee for advice about histidase purification.

I am grateful to the Medical Research Council for a Partnership Award.

Finally, I am indebted to Liz Varge for her excellent and very prompt typing of this thesis. 9

ABBREVIATIONS

cAMP cyclic adenosine monophosphate ATP adenosine triphosphate Ch cholesterol CNS central nervous system CRM cross-reacting material DMPC dimyristoyl phosphatidylcholine DOPC dioeoyl phosphatidylcholine DPPC dipalmitoyl phosphatidylcholine DSPC distearoyl phosphatidylcholine DTPA diethylene triamine penta-acetic acid EDTA ethylene diamine tetra-acetic acid FAD flavin adenine dinucleotide HDL high density lipoprotein INT iodonitrophenyl tetrazolium iv intravenous MLV multilamellar vesicles NPC non-parenchymal cells PA phosphatidic acid PBS phosphate buffered saline PC phosphatidyl choline PMSF phenyl methyl sulphonyl fluoride PVP polyvinyl pyrrolidone RES reticuloendothelial system SE standard error SUV small unilamellar vesicles URO urocanate 10

CHAPTER 1

GENERAL INTRODUCTION 11

SECTION A

1 . GENERAL INTRODUCTION - INBORN ERRORS OF METABOLISM

When Sir Archibald Garrod in 1908 created the concept of inborn errors in metabolism, the relationship between gene action and enzymes was not understood (A.E.Garrod, Croonian lectures, 1908). Since then over 4-00 inborn errors of man have been found, of which 14-0 have been attributed to the genetic deletion or alteration of one or several enzymes. Many of these show no phenotypic effect, whereas others are lethal, either in early infancy or in the adult. Many of the blocked pathways are catabolic for mucopolysaccharides, amino acids, lipids and glycogen, but few, if any metabolic lesions occur in the central anabolic functions of the cell because these are not compatible with life. Although the incidence of lesions in individual pathways is quite rare, together they make up a formidable array of diseases for which therapy is virtually non-existent. Diagnosis of enzyme deficiencies has progressed rapidly over the past thirty years, as have attempts at therapy, which have met with little clinical use but which have involved great endeavours of technology and ingenuity.

Diagnosis of enzyme lesions was possible as soon as biochemical pathways had been elucidated and enzyme assays were available. When Beadle and Tatum studied Drosophila and Neurospora crassa in 194-5 and developed the one gene-one polypeptide concept, the genetic basis for inborn errors of metabolism was developed. In 194-8 the first enzyme defect in a human genetic disease was demonstrated by Gibson (194-8) which was a deficiency in NADH-dependent inethaemaglobin reductase in idiopathic methaemaglobinaemia.

The enzyme defects that cause the most clinical problems affect the lysosomes. The accumulation of substrate resulting from a slower rate of endogeneous turnover will affect the cellular organelle most involved in the endocytosis and - 12 - metabolism of those excess substrates. The diagnosis of these storage diseases is carried out in cells from which the enzyme is absent or in tissue where the substrate has accumulated, such as blood cells, fibroblasts, liver, muscle, skin, urine and plasma. Cytoplasmic enzyme defects can also cause severe clinical problems when the accumulating substrate is toxic.

Several types of mutation can cause enzyme deficiencies (Desnick et al, 1976). The genetic material can be modified in regions that encode for the structure of the enzyme or in regions that encode the regulation of enzyme expression or in regions that encode the post-translational modification of the enzyme. In structural modifications, there may be altered kinetic properties, elevated rates of enzyme degradation or altered secondary binding sites for cofactors or allosteric effectors. There may be altered subunit interactions or incomplete polypeptide synthesis. Abnormal post-translational modification may include altered glycosylation, phosphorylation or peptide cleavage or there may be altered tissue distribution or subcellular localisation. Very few human defects, unlike bacterial or fungal, result from a regulatory mutation. These abnormal enzymes are detected by altered electrophoretic mobility or by different assay requirements. Kinetic mutations are difficult to detect because a low substrate affinity in vivo can be masked by in vitro assay conditions.

In a few cases direct gene product therapy is unnecessary as substrate depletion or product replacement may be carried out easily. This is called environmental manipulation. Substrate depletion techniques have been carried out, for example, in familial hypercholesterolaemia (Levy et al, 1973). An anion exchange resin, cholestyramine, bound bile acid in the intestine and thus reduced circulating cholesterol levels by an increased conversion of cholesterol to bile acid. Dietary restriction and plasma pheresis are other examples of substrate depletion. Product replacement, such as glucose in glycogen storage disease I and replacement of hormones in disorders of hormone synthesis, is commonly performed. Sometimes elimination of the metabolite ahead of the block and substitution of the metabolite behind the block is needed. In homocystinuria, which is caused by the defective enzyme cystathione synthetase, methionine in the diet must be depleted to decrease homocysteine formation, but since there is a lack of cysteine, this amino acid must be supplemented. Many metabolic treatments involve manipulation of the effects of the genetic lesion, such as treatment of polydactyly. Some metabolic defects are becoming increasingly more common because of the progressive uniformity of the world's diet. The intestinal lactase deficiency found in most Oriental and Negroid populations has only developed due to the change in their eating habits.

This work looks at the effect of direct gene product replacement on two model mouse lines, each deficient in one enzyme in the catabolic pathway of an amino acid.

The effect of enzymes administered in biodegradable carriers or alone to these model mice, in order to reduce accumulated substrate in the tissues and plasma, was studied. In a model of an artificially induced iron storage conditions, the environmental manipulation of enhanced excretion was used to reduce accumulated substrate by administering chelating agents, either alone or entrapped in biodegradable carriers. 2 . MODELS FOR THERAPY

(i) In vitro cell culture

Fibroblasts cultured from affected patients have provided evidence that enzymic deficiencies can be corrected by means of exogenous enzymes (Bach et al, 1972). Skin fibroblasts from Hurler’s patients (mucopolysaccharidosis I) were used to demonstrate that over 4-0% of purified a-.iduronidase from urine could be taken up from the surrounding medium into fibroblasts. This resulted in a 90% correction of the disease, even though only a fraction of the normal enzyme level was replaced. Human 3-glucuronidase taken up by deficient fibroblasts was found to be localised within the lysosome and about 58% of the activity remained after 19 days in culture (Lagunoff et al, 1973). These experiments demonstrated that macromolecules can gain access to intracellular sites when administered exogenously.

Hunter’s patients fibroblasts (failure to degrade dermatan and heparan sulphate) were grown in the presence of iduronate sulphatase from human urine (Bach et al, 1973). This was found to be the corrective factor for oversulphated areas of the mucopolysaccharide and was taken up by pinocytosis into lysosomes. There are "high" and "low" uptake forms of the same enzyme, depending on the presence or absence of a carbohydrate recognition marker (Brot et al, 1974-) . It was found that the uptake marker for 3-glucuronidase into fibro blasts was mannose 6-phosphate (Kaplan et al, 1977) and is a general marker for uptake of lysosomal glycosidases by human fibroblasts. Other carbohydrate recognition signals for pinocytosis into different cell types include galactose for hepatocytes (Ashwell and Morrell, 1974-), mannose al->6 for aveolar macrophages (Stahl et al, 1978), 3-galactose for kidney (Teichberg et al, 1975). Effective delivery of lysosomal enzymes across cell membranes occurs by receptor mediated processes and this has been exploited in the delivery of enzymes in vivo in liposomes and other carriers and by the direct perfusion of free enzyme (see Sections B and C). ) -

In the replacement of ai-antitrypsin deficiency, where the enzyme is required in the circulation, the most sialated isoenzyme is used so that terminal galactose residues that might otherwise signal its fast removal from the plasma into hepatocytes are disguised.

Thus fibroblasts grown in cell culture have led to diagnosis and attempted therapy of enzyme diseases. Although many genetic defects are expressed in cultures of fibroblasts, the cultured fibroblast may not represent the phenotype of the tissue in which the lesion occurs. In some cases differentiating tissue is needed to see the genetic defect at early stages of development. Prenatal diagnosis by growing amniotic cells in culture is a useful early warning technique that has recently become important.

Other cell types have been used for in vitro models of enzyme diseases. A macrophage model system was used to study- possible therapy of Gaucher’s disease with exogenous enzyme (Dale and Beutoer, 1981).

Macrophages were loaded with glucocerebroside by incubating them with glucocerebroside-loaded liposomes or with glucocerebroside precipitated with serum albumin. Gaucher’s patients macrophages could metabolise the gluco cerebroside just as efficiently as normal cells even though they had 2-1+% of normal 8-glucocerebrosidase activity, when administered in liposomes. Glucocerebroside-albumin particles were metabolised less in Gaucher’s disease macrophages and so these were used for chomic feeding of macrophages. This model of Gaucher's disease has not been studied in long-term and, although it is known that macrophages will incorporate exogenous glucocerebrosidase (Dale et al, 1979), the effect that will have on this model system is unknown.

Even though the in vitro mechanism of exogenous enzyme uptake into cells is important, animal model systems give far more valuable information about the interaction of enzymes with the immune system, plasma proteins and the reticuloendothelial system, each of which plays a very powerful role in modifying and eliminating foreign material. 16

(ii) In vivo animal models

The fate and action of enzymes administered to humans is extremely difficult to follow, and the benefit over the long term is hard to determine. In lysosomal storage diseases, measurement of urine or blood levels of substrate or metabolites can give an indication of the action of the enzyme in tissues. More accurate data can and should be obtained from animal models that are homologues of the human disease. Dose and duration of action of the enzyme and the result of enzyme modification and encapsulation can be followed.

An extensive compendium of animal models which occur spontaneously has been documented by Cornelius (1969). Animal models should be a single gene defect corresponding to the human gene in the human disease. Most of these models have been used for identifying the biochemical defect, for example, mannosidosis in cattle (Philips et al, 1974)» tut few have been exploited for therapeutic use. Mice are being constantly screened for biochemical variants, mutations that cause changes in enzyme activity or concentrations or with an affect on electrophoretic mobility (Bulfield, 1981). In this study, the two animal models used are both homologous to the human condition. In histidinaemia the genetically deficient enzyme is histidine ammonia lyase, histidase, the first enzyme in the major pathway of histidine metabolism. (Wright et al, 1982). In hyperprolinaemia the genetically deficient enzyme is mitochondrial proline oxidase (Blake et al, 1976). These models will be discussed in much greater detail in Chapters 2 and 3*

Induced models of a storage disease or enzymic lesion can be artificially obtained by either administering a large amount of the substrate that accumulates in the human condition or by administering an inhibitor that will specifically act on the enzyme that is defective in the genetic condition. An example of the former is iron overloading by injection of iron citrate or iron dextron to induce a model of idiopathic haemachromatosis (Richmond et al, 1972). Problems with this kind of model are that it may be difficult to direct the loading of the substrate to the correct subcellular localiz - 17 -

ation and that the endogenous enzyme activity might be so great that it is almost impossible to create a chronic situation in an otherwise normal animal. An example of enzyme lesion to induce an animal model is administration of a-methyl phenylalanine or p-chlorophenylalanine to inhibit phenylalanine hydroxylase for a model of phenyl ketonuria (Greengard et al, 1976; Del Valle et al, 1978). This model again is not very satisfactory since the inhibitors are toxic to many other systems and depress other enzymes beside phenylalanine hydroxylase. The duration, of the effect is not constant for each animal and the in utero effects of elevated maternal phenylalanine on the foetus cannot be mimicked. Another model that has been developed to create Gaucher Type I disease in animals was by inhibition of 3-glucosidase (Gregoriadis et al, 1982). The inhibitor, conduritol 3-epoxide, was injected into mice in liposomes to direct most of the inhibitor to the liver and spleen. Administration of the inhibitor in sphingomyelin liposomes retained it in the circulation and kept the inhibition of 3-glucosidase high in the organs over a long period of time.

Other ingenious methods of following the fate of the administered enzyme have been developed without the need for animal models of the disease. Achord et al (1977) infused human placental

3-glucuronidase into normal rats and were able to distinguish exogenous from endogenous enzyme by heating organ extracts to 6$°C. This selectively inactivated the rat enzyme and allowed the uptake and distribution of human enzyme to be followed.

This differential thermal inactivation has been exploited with bovine 3“glucuronidase infused into mice (Thorpe et al, 1974). C3H/HeJ mice with low 3-glucuronidase activity were administered bovine 3“glucuronidase which is thermolabile at 60°C. The mouse enzyme is stable at this temperature. The tissue levels of the administered bovine enzyme were thus measured by assaying before and after rapid heating at 60°C. Normal cats were used by Rattazzi et al (1980) to infuse human placental @-D-N acetylglucosaminidase (3-hexo saminidase) . Single radial immunodiffusion was used to .18

distinguish the feline enzyme from the human enzyme.

Animal models exist for human thalassemia (mouse), Gaucher’s type 2 (dog), mucopolysaccharosis I (cat), glycogenosis II (cat) and many more. Work in progress concerning detection and screening for new animal models and a complete compendium of inherited metabolic diseases is given by Desnick et al (1981) 3. FREE ENZYME THERAPY IN VIVO

The first report of in vivo enzyme replacement was with an enzyme rich extract from Aspergillus niger as the source of acid maltase (a-l,4-> glucosidase in Type II glycogen storage disease (Pompe's disease) (Baudhuin et al, 1964.). When the extract was injected into mice, the a-glucosidase was rapidly taken up by the liver and about 30% of the activity

appeared in the liver after k hours. They treated a Pompe's disease patient with extracts of A.niger over a three day period, after which she died. Although the activity in the liver at autopsy approached that of acid a-glucosidase in normal liver, the enzyme taken up either did not reach the glycogen-containing vacuoles, or if it did, was unable to display activity because the size of the vacuoles before and after treatment was unaltered. The experiment was repeated with another Pompe's disease child. Lauer et al (1968) found 25% of the infused activity in the liver but again no decrease in tissue glycogen was observed. A more intensive treatment again gave no long term clinical improvement (Hug and Shubert, 1967; 1968). Other attempts at Pompe's disease treatment have used human placental a-glucosidase (De Barsy et al, 1973).

Another lysosomal storage disease where therapy has been attempted is Fabry's disease (deficiency of a-galactosidase and accumulation of ceramidetrihexoside). Plasma, from normal subjects, which contains a-galactosidase, was infused into Fabry's patients (Mapes et al, 1970). The infused activity in the blood was enhanced 20-30 times after 6 hours and the ceramidetrihexoside level declined to 50% after 10 days. The enhancement of activity may be in some part due to a change in pH optimum of the enzyme in vivo. Purified human placental a-galactosidase has been administered to Fabry's patients (Brady et al, 1973). There was no effect on glycolipid accumulated in liver and plasma, but these workers also found an enhancement of enzyme activity in vivo. Infusion of fresh plasma by the same group had no effect.

Hunter's and Hurler's patients (mucopolysaccharidoses I and II) 20 have been treated with infused plasma (Di Farrante et al, 1971). The patients showed clinical improvement for a few weeks and the ability to degrade dermatan and heparan sulphate was increased. An infusion of leukocytes showed even greater effects. San Filippo patients treated with plasma showed increased urinary excretion of glycosaminoglycans (mucopolysaccharidosis III, deficiency of heparan N-sulphatase or N-acetyl glucosaminidase and build up of heparan and dermatan sulphate) (Dean et al, 1973). Other groups have found no effect of plasma infusions in this disease (De Kaban et al, 1972).

Another attempted therapy for a lysosomal storage disease, metachromatic leukodystrophy, by infusion of arylsulphatase A prepared from human urine was attempted by Austin (1967). The patient showed no improvement and the uptake into tissues was not investigated. Arylsulphatase A from bovine brain was infused into a patient and the activity in the plasma disappeared after three hours (Greene et al, 1969). The enzyme did not cross the blood brain barrier and could not be found in the central nervous system (CNS) and there was no clinical improvement.

Gaucher*s disease (mucolipidosis I) is most amenable to treatment because the CNS is not affected and the reticulo endothelial cells, in which the accumulated substrate is stored, are accessible to exogenous enzyme. A large scale purification from human placenta of glucocerebrosidase was developed (Furbish et al, 1977) and extensive trials on Gaucher’s patients have been carried out (Brady et al, 1974-)* The injected enzyme was cleared from the blood with a half-life of 20 minutes and 26% appeared in the liver. Liver levels returned to normal after three days. Infusion bimonthly over two years has led to an improvement in two patients (Brady et al, 1981). Most of the enzyme was taken up into hepatocytes because this enzyme had several terminal galactose residues. Since Kupffer cells are the main site of storage of glucocerebrosides, the enzyme was sequentially cleaved to remove sialic acid, galactose and N-acetyl gluco samine to produce mannose terminal carbohydrate chains on 21 the glycoprotein. This caused an 8-fold increase of transfer the enzyme to non-parenchymal cells (Steer et al, 1978).

Massive amounts of human 8-glucosidase have been infused into rats so that it was possible to measure the increase in the activity over the normal endogenous level. The enzyme appeared in both parenchymal and Kupffer cells (Furbish et al, 1978).

Of course, the most clinically applicable enzyme replacement therapy is infusion of the serine proteases, Factors VIII, IX and X missing in Haemophilia A and B. Lysis of clots in thrombosis is another use. Streptokinase from Streptococci and urokinase from urine are administered to promote lysis in deep vein thrombosis. They activate plasminogen by cleaving it to plasmin (Kakkar et al, 1975).

Other means of enzyme therapy in vivo have been by allograft (transplantation of organs or cells). Skin grafts have been used for fibroblast transplantation for Hunter’s disease (Dean et al, 1976). This depends on finding a histocompatible donor, but has been shown to be successful in one case in degrading glycosaminoglycans that have accumulated in tissues. Fibroblasts have also been injected in a case of Hunter’s disease. Transplantation of hepatocytes and pancreatic islets have not met with much success as yet, because of the inability to prevent rejection of transplanted cells (Najarian et al, 1977). Kidney transplantation has been attempted in cystinosis (Mahoney et al, 1970), in Gaucher’s disease (Groth et ai, 1971), but although the kidneys were not rejected in most of these attempts, there was no clinical improvement in the condition or enzyme level. - 22 -

1. COMPLICATIONS IN ENZYME REPLACEMENT THERAPY

A source of enzyme must be developed. It must be delivered to the cell in which it is to function or it must remain in the plasma to function there. It must be stable in the cell or in the plasma and interact with its substrate long enough to produce clinical benefits.

(i) Source

Even if it is of isogenic origin, the enzyme may be antigenic since isoenzyme forms may have different antigenic determinants. Microorganisms are the most practical source as they can be manipulated in culture to give high production, for example, by enrichment culture techniques where the organism is grown on the substrate as its sole carbon and nitrogen source. Replacement of lysosomal enzymes in humans or animals may not be conducive to bacterial enzyme treatment because the bacterial form may lack the glycoprotein determinant responsible for efficient incorporation into lysosomes. Major sources of enzyme for human therapy have been placenta, plasma and urine, and development of high affinity purification techniques has enabled these to be purified in large amounts. The production of large amounts of some enzymes for replacement purposes has to await recombinant DNA technology.

(ii) Delivery

Enzymes must be able to be delivered to specific cells and subcellular compartments or be retained in the plasma. In some diseases brain, muscle, heart, liver or kidney can be affected and so exogenous enzyme must find a way to penetrate through the blood-brain barrier, through continuous or fenestrated endothelial cells, or through barriers that have been evolved to be impassable to any foreign material. The results of the last two decades have shown the majority of enzyme is cleared to cells whose function is to clear foreign materials, even when high uptake receptor marker forms are used. The most successful applications of exogenous enzyme therapy have been where action in the serum or reticuloendo thelial cells is required. The form of enzyme used needs to have a high affinity for the substrate, no inhibition by the 23

product and give an irreversible reaction under physiological conditions. For example, Escherichia coli glutaminase, which could act as an antineoplastic agent, has a pH optimum of 5 and no activity at physiological pH (Holcenberg et al, 1973). Glutaminase B from E .coli is allosterically regulated by carboxylic acids, divalent cations and adenine nucleotides and would have little activity in vivo. Many enzymes require cofactors for activity. When tyrosine phenol lyase was administered to inhibit growth of Bl6 melanoma tissue, the cofactor, pyridoxal phosphate, was rapidly lost. Enzyme carriers can be used to administer protein plus cofactor together, with the cofactor bound to the carrier to prevent its loss (see Section B).

(iii) Stability

The enzyme must be stable in the plasma and on entry to cells. Snyder et al (1974-) investigated ways to stabilize human a-galactosidase and, by treating it with hexamethylene diisocyanate (a bifunctional cross-linking reagent), increased its stability to heat and protease attack. Gluteraldehyde has been extensively used to stabilize enzymes in vivo (Gregoriadis, 1974-)- Acinetobacter glutaminase-asparaginase was chemically linked by succinylation and glycosylation to peptides from human fibrin and y-globulin to prolong its half life by decreasing their isoelectric point (Holcenberg et al, 1975) . It has been found that increasing the isoelectric point causes increased uptake into cells.

(iv) Immunological Effects

Immunological complications of administering foreign enzymes to patients or animal models depends on the type of genetic enzyme deficiency. An antibody raised against the purified foreign enzyme may or may not react with material from an affected patient. When the genetically deficient enzyme is completely absent, there will be no cross-reacting material (CRM negative). When the enzyme deficiency is based upon the presence of a structurally altered protein, there may be antigenic determinants which are common with the purified foreign enzyme (CRM positive). In this case, the immune response against the foreign enzyme might be decreased. 2h

Many enzymes exist in a polymorphic state and therefore, even when the donor and recipient have normal enzyme activities, they may have different isoenzymes and thus an immune response would be provoked in vivo. Proteins with only small structural differences (one amino acid substitution) when infused, can cause an immune response. For example, porcine and human insulin differ in one amino acid, and infusion of porcine insulin leads to an immune response in patients (Root et al, 1972). Most enzyme deficiencies are CRM positive, but almost all allogenic enzymes will induce an immune response. By screening several sources of enzyme, it may be possible to obtain one that has good kinetics for clearance and low immunogenicity. Promotion of enzyme uptake or longevity can be performed by attention to carbohydrate-chain composition. Tolerance may be developed to particular forms of an enzyme and this depends on the strain of animal, the immunocompetence of the host, the nature and dose of the antigen and the route of injection (Weigle, 1973).

The immune response may activate a humoral or cell mediated action or both. Antibody-antigen complexes are frequently rapidly eliminated from the body, thus hampering enzyme replacement. The reaction after repeated administration of antigen can lead to hypersensitivity, including anaphylaxis, allergy and serum sickness, each of which ;is life-threatening. All recent attempts at enzyme replacement have used a non- immunogenic, biodegradable carrier (see Section B) to try and minimise the unwanted side effects of enzyme administration 25

5. ENZYME THERAPY FOR AMINO ACID DEPLETION IN VIVO

The work described in this thesis is one of the first in vivo attempts at amino acid depletion in 2 inborn errors in metabolism that are aminoacidopathies. Enzymes have been used quite extensively as drugs in cancer chemotherapy to deprivecellsofessential aminoacids> deplete folate, to act as fibrinoly tic and defibrinating agents in thrombosis as well as in replacement therapy for other inherited enzyme deficiencies.

The use of asparaginase for cancer chemotherapy is well documented because of the dependence of certain neoplasms on extracellular asparagine for their survival. The amino acid must be continually depleted by parenteral administration of the enzyme. About two thirds of acute lymphocytic leukaemia patients have achieved complete remission when treated with asparaginase (Ottengen et -al, 1967). Some of the patients developed hypersensitivity reactions to the enzyme preparation. Asparaginase is sometimes contaminated with glutaminase activity and this has an antitumour effect in some tumours which have a slow rate of synthesis of glutamine (Levintow, 1954)• Acinetobacter glutaminase- asparaginase (AGA) and E ,coli asparaginase have been compared for their effects on plasma and tissue levels of amino acids, ammonia and glutamyl transferase activity. Free asparagine was reduced equally by both enzymes and the AGA produced a partial depression of glutamine concentrations in muscle, liver, intestine and spleen. Plasma glutamate increased 100 times but kidney was the only tissue in which the product rose (Holcenberg, 1975). Amino acid degradative enzymes purified from different sources show different kinetic and biological properties in vivo. Glutaminase-asparaginase purified from Pseudomanas had a much longer half-life than AGA and demonstrated antineoplastic properties with leukaemias that the Acinetobacter enzyme was not effective against (Roberts et al, 1976). Novel enzymes capable of depleting histidine, tryptophan and glutamine/asparagine have been purified from soil organisms (Roberts et al, 1979). These have been administered to mice bearing ascites carcinomas 26

and the effect on tumour growth studied. Both the histidine-degrading enzyme, histidase, and the tryptophan degrading enzyme, indolyl-3-alkane-a-hydroxylase, depleted amino acids in the plasma and tissues and had antineoplastic activity against a number of mouse tumours.

Arginase has been postulated as an inhibitor of tumour growth in vivo (Bach et al, 1965). The enzyme, purified from ox liver and used against Walker 256 carcinoma, was successful at retarding the growth of the tumour. However lower concentrations of the enzyme seemed to work better than higher concentrations. Phenylalanine ammonia lyase from Rhodotonilia glutinis has been used to deplete phenyl alanine in L5178Y lymphoblastic leukaemia in mice (Abel et al, 1973).

Other ways to use enzymes as anticancer agents is to degrade vitamins or cofactors such as folic acid. Several attempts to inhibit tumour growth have used carboxypeptidase G1 in vivo and in vitro to deplete folate (Berlino et al, 1971) . 27

6. THERAPY FOR METAL DEPLETION

Metal storage diseases are inborn errors in metabolism affecting the metabolism or transport of metals. In Wilson’s disease, biliary excretion of copper and incorporation into ceruloplasmin are both severely impaired. Effective treatment is with D-penicillamine. In Menkes’ disease there is defective intestinal absorption and accumulation in the mucosal cells of copper. There is a homologous strain of mouse with mottled hair (M_o mutant) that is amodel for this disease. Treatment for humans has not been found and death usually occurs before the age of two years. Idiopathic haemochromatosis is another metal storage disease, with enhanced absorption of iron from the gut. Excessive quantities of iron are stored in the liver and in other organs as ferritin and as insoluble haemosiderin and these impair the functions of the cells. Death is by cardiac failure unless iron is removed by treatment with iron chelators. In thalassemia major and other refractory anaemias with ineffective erythropoiesis, intestinal absorption is also enhanced and iron overload is usually made much worse by multiple blood transfusions. The storage iron concentrations are 50 to 100 times the normal figure in. liver and pancreas. The liver shows fibrosis and lobular disruption. The most common method of treatment is by venesection. Chelating agents, of which the most specific for iron is desferrioxamine, are used but the rate of iron removal is much less than by venesection unless they are infused every day. Desferrioxamine is toxic to non-affected cells and is rapidly broken down in the circulation. It is possible to arrest the accumulation of iron and the progression of hepatic fibrosis in thalassemia patients with the prolonged adminis tration of desf errioxamine (Barry et al, 1974.) but it is a very expensive therapy. The toxicity of the iron is not only because of its concentration in tissues, but also because of its cellular distribution. Many new chelating agents have been developed to try and introduce them into the subcellular sites of pathology (Dawson et al, 1980). Entrapment of chelating agents in liposomes and erythrocyte ghosts has been effective at removing an increased amount of iron from liver over the free drug (see Sections C and D). 28

SECTION B

ENZYME CARRIERS FOR REPLACEMENT THERAPY

Many synthetic and biological materials have been used to immobilize or protect enzymes for in vivo administration. Industrial use of immobilized enzyme for continuous catalytic processes has caused the development of supports and semi- permeable membranes, a few of which have been adapted for medical use. Other carriers that have been investigated include lipid vesicles called liposomes, erythrocyte ghosts and other biological cells. This section deals with carriers which have been used in enzyme therapy in vivo and excludes liposomes and erythrocyte ghosts, which will be dealt with in Sections C and D respectively.

Immobilization techniques include the cross-linking of enzymes within or on the surface of carriers, the covalent attachment to and the encapsulation within carriers. The advantages of enzyme replacement using a carrier is that it can be tailored for individual diseases to enable the enzyme to be either stabilized or protected in the circulation and it can possibly be targetted using the carrier to the site of pathology, thus promoting its therapeutic effects. The targetting of drugs has been a goal since the turn of the century when Paul Ehrlich developed the concept of nThe Magic Bullet” (address bo the German Chemical Society, 1909). 1. ANTIBODIES

The most obvious carrier for specific enzyme targetting to cells is the antibody. Cell specific antibodies can be conjugated to the enzyme using a variety of cross- linking reagents. The cross-linking reaction can allow control of the size of the enzyme carrier complex without affecting the activity of the enzyme. Antibodies have been used as carriers of enzymes for cell specific cytotoxicity. Glucose oxidase was coupled to anti-trinitrophenyl (anti-TNP) antibodies to destroy a TNP-substituted cell line (Philpott et al, 1973)- The complex bound to the cells and the enzyme catalysed the oxidation of glucose to gluconic acid with the formation of hydrogen peroxide. Lactoperoxidase and potassium iodide were subsequently added, which used the hydrogen peroxide to iodinate membrane proteins. It was found to be a very efficient way of killing cells. This method has also been used to kill bacteria in vitro (Knowles et al, 1973). Conjugates between glucose oxidase and antitumour antibodies have been shown to exert antibody dependent toxicity against tumour cell lines in vitro and in vivo (Shearer et al, 1974-) . When tested in vivo there was both higher tumour localisation of glucose-oxidase-antitumour antibody than the enzyme alone and, when tumour cells were isolated from tumour-bearing animals, there was a greater invvitro sensitivity to lactoperoxidase dependent iodination.

Enzyme delivery to cells for replacement therapy with antibody- enzyme-albumin conjugates has been attempted by Poznansky and Bherdwaj (1981) using a-1,4-“glucosidase-albumin-anti- hepatocyte antibody complexes. Antisera produced in rabbits against isolated liver hepatocytes were conjugated to a-1,4- glucosidase-albumin with glutaraldehyde. It was found that the ratio of 125I enzyme uptake into hepatocytes over Kupffer cells was increased using this method, than with using enzyme-albumin alone. Since the reticuloendothelial cells

(Kupffer cells) are the usual s i t e of uptake of foreign protein, this experiment demonstrates that the distribution of enzyme can be altered by antibody targetting. There was no evidence that the complex was internalized. 2 . RECEPTOR MEDIATED TARGETTING

In 1968, it was discovered that ceruloplasmin from which the terminal sialic acid had been removed was cleared more quickly from the circulation into the parenchymal cells than sialyated ceruloplasmin (Morrell et al, 1968). The galactose residues exposed by desialyating this glycoprotein reacted with galactose receptors on hepatocytes. Since then numerous other sugar recognition receptors have been found on other tissues, for example skin fibroblasts have mannose 6-phosphate and low density lipoprotein receptors, heart and kidney have 3-galactose receptors and lung macrophages have mannose and 3-galactose receptors. This discovery has been exploited to target many enzymes and drugs to hepatocytes, fibroblasts and other selected cells. Desialyation of human glucocerebrosidase causes a preferential uptake into hepatocytes over Kupffer cells, whereas the sialyated form is taken up into both types of cell (Furbish et al, 1978). This receptor uptake mechanism can also be exploited by selection of an isoenzyme with the required uptake characteristic (Fiddler and Desnick, 1977). When

bovine hepatic, renal and /splenic-■ 3-glucuronidase was administered to 3-glucuronidase-deficient mice, the splenic isoenzyme was retained longer in the circulation and, when it was taken up into liver, it remained three times longer than the hepatic or renal form of the enzyme. This was also found to be true for N-acetyl 3-D-glucosaminidase, where the epididymal form was cleared more slowly than the liver lysosomal form (Stahl et al, 1976). These facts should be considered when looking for a source for enzyme replacement. Proteins and glycoproteins can be attached to specific carbohydrate residues for targetting (neoglycoproteins) but these receptor mediated events are only desirable for enzymes that are required in the lysosomal apparatus. Large molecules only escape from lysosome when they rupture, normally when the cell dies. Macromolecules which are to act outside the lysosome must be directed there in the first place. Liposomes became popular vesicles for this purpose because it was thought liposome-cell fusion could deliver enzymes to the cytoplasm of cells (see Section C). 3. ARTIFICIAL CELLS

Synthetic ultra thin spherical polymer membranes have been used to form art.ifical 'cells’, including cellulose nitrate, polystyrene, cellulose acetate and silicone rubber. The pioneering work on microcapsule formation was by Chang (1964). Since then improved methodology has made them applicable to several inborn errors of metabolism. A full account of the preparation of microcapsules is given by Chang (1976). Many enzymes have been encapsulated, including glucose oxidase, 3-fructofuranosidase, a-glucosidase, asparaginase, catalase, uricase and urease. Microcapsules have not only been administered parenterally , but also orally, in local implantations and in extracorporeal shunt chambers, to decrease elevated substrates in the blood. Urease-loaded microcapsules have been used to convert 80$ of blood urea to ammonia in 90 minutes in dogs (Chang, 1966). Micro capsules have been very successful in the acatalasaemic mouse model to replace deficient blood catalase (Chang and Poznansky, 1968). Acatalasaemic mice were administered sodium perborate (a substrate for catalase) and then treated with microencapsulated catalase, either intraperitoneally or in an extracorporeal shunt. The extracorporeal shunt proved better at both removing the substrate and avoiding the toxic effects of microcapsule accumulation in the peritoneum. When microencapsulated urease in an extra corporeal shunt chamber was intermittently perfused with blood, the effect of different compositions of microcapsules on platelet and leukocyte levels was studied (Chang et al, 1968).

Heparin-complexed c o l l o d i o n microcapsules had the least effect on both. Activated charcoal has been effective in removing metabolites or toxins from perfused blood, but only with a significant reduction of platelet levels. For this reason, charcoal was microencapsulated to avoid this problem (Chang et al, 1978).

The reversal of hepatic coma in galactosamine-induced fluminant hepatic failure has been treated by haemoperfusion through an extracorporeal shunt containing microencapsulated charcoal. Encapsulation of asparaginase for treatment of asparagine-dependent tumours has been attempted by intraperitoneal injection of the microencapsulated enzyme (Chong and Chang, 1974.) . This maintained asparaginase levels in the body for longer than the free enzyme and depleted asparagine levels for longer. Tyrosinase has been microencapsulated and its effect against metabolic disorders like hepatic coma, caused by the elev ation of false neuro transmitters, studied (Chang et al, 1981). Artificial cells have the capability of containing multi-enzyme systems, for sequential operation and recycling of cofactors, For example, the entrapment of hexokinase and pyruvate kinase for the continuous conversion of glucose to glucose 6-phosphate and phosphoenol pyruvate into pyruvate and the recycling of ATP (Chang et al, 1979)* Artificial cells containing alcohol dehydrogenase and malate dehydrogenase can recycle NADH. A multienzyme system for conversion of urea to glutamate has been microencapsulated (Cousineau and Chang, 1977). A modification of the above techniques is the immobilization of cofactors on dextran to retain them in the artifical cell (Grunwald and Chang, 1979). Recently lipid-polymer membrane artifical cells have been used to retain small cofactors and solutes (Yu and Chang, 1981). When alcohol dehydrogenase, ADP, a—ketoglutarate, MgCl^t KC1 and NADP or NADPH were encapsulated in lipid polymer capsules, external ammonia and ethanol could cross the membrane and be converted to glutamate and acetaldehyde respectively, while the cofactors were retained internally. ALBUMIN

The linkage of enzymes to albumin as a carrier has a number of potential advantages. Albumin is non-toxic, lacks antigenicity, has a slow catabolism and confers stability to the enzyme in the circulation. The action of uricase cross-linked to homologous albumin was investigated in an animal model of hyperuriceaemia (Poznansky, 1979). The complex was more heat stable and more resistant to proteolytic attack in vitro, had a longer half-life in vivo (26 hours as opposed to 4- hours for the native enzyme) and was more effective at lowering plasma uric acid levels than the free enzyme. The immunogenicity of the complex was investigated by injecting either rabbit or dog albumin complexed to dog-liver uricase into rabbits. Rabbit- albumin-uricase complexes elicited no immune response in rabbits. Dog albumin-uricase complexes in rabbits elicited an immune response against the dog albumin but not against the uricase. Therefore albumin could mask the antigenic sites on the enzyme. When superoxide dismutase was conjugated to albumin, its plasma half-life clearance was decreased from 6 minutes to 15 hours and it effectively inhibited carrageenan-induced pawoedema (Wong et al, 1979). Phenylalanine hydroxylase and asparagine have been linked to albumin (Poznansky and Cleland, 1980). To some extent, albumin can be targetted to reticuloendothelial cells by denaturing it so that it forms aggregates. Albumin has been targetted to hepatocytes using antibodies (Section B.l. Poznansky and Bherdwaj, 1981)..

Anticancer drugs have been conjugated to albumin, but these do not seem to offer an advantage over the free drug in prolonging the survival of tumour inoculated mice (Chu and Whiteley, 1977). Asparaginase-albumin conjugates have been used to treat mouse 6 C3HED lymphosarcoma to try and combat the severe immunological reactivity to the enzyme found in 40% of human trials (Poznansky et al, 1981). The major problems were overcome by cross-linking of the asparaginase- albumin polymer to a monoclonal antibody directed against a tumour specific antigen. 3K

5. NANOPARTICLES AND MICROSPHERES

Nanoparticles may be useful for sustained release of enzyme and also can be target ed to the reticuloendothelial system. They are 200-500 nanometers in diameter and molecules of the drug or enzyme are evenly dispersed through the solid matrix. They are made from proteins or celluloses, for example human serum albumin, ethylcellulose, casein or gelatin, by coecervation. A solution of the macromolecule and active ingredient is made and desolvated by adding a solvent- competing solvent such as sodium sulphate or alcohol. By controlling the desolvation process, colloidal-size particles rather than large aggregates can be formed. The enzyme phenylalanine ammonia lyase has been entrapped in gelatin capsules, enteric coated and orally administered to humans (Hoskins et al, 1980).

The development of microspheres of human serum albumin has been used as a reliable agent for measurement of the circulation in man. They are taken up into the reticulo endothelial system and can be used to target enzymes to this area in diseases affecting the reticuloendothelial system. The drug 6-mercaptopurine has been encapsulated in albumin microspheres by homogenisation in dimethyl formermide and cotton seed oil, followed by heating. This carrier system would only be suitable for enzymes stable to heat (Kramer and Burnstein, 1976). Recently, Kreuter (1983) has used polycyanoacrylate to form nanoparticles and has been using them as "tissue glue" in surgery. When these particles were attached to methotrexate, they halved the size of human bone cancer. SECTION C

LIPOSOMES

1. FORMATION

Bangham was the first to show that phospholipids dispersed in water formed multilayered vesicles or liposomes (Bangham, 1963). Liposomes are smectic mesophases, layer lattices of alternating, closed, bimolecular sheets intercalated by aqueous spaces. They have been used both as model membrane systems and as vesicles for delivery of therapeutic drugs to cells. The study of the process of membrane fusion, the action of anaesthetics, the reconstitution of membrane proteins as well as the introduction into cells of foreign molecules have all exploited the liposome as a tool for developing the understanding of these problems. Liposomes are made of bio degradable, natural membrane molecules and their composition can be altered for different purposes. The can either act as a drug depot or deliver molecules to cells, mainly the reticuloendothelial system in vivo.

Multilamellar liposomes (MLV) are formed spontaneously on the addition of water to dried phospholipid in a round- bottomed flask. Alteration of the time of hydration and conditions of agitation determines the encapsulation efficiency and the thickness of the lipid film. They are heterogeneous in size, ranging from 0.1 to 10pm in diameter with a low aqueous volume (about 2-4- litres per mole phospholipid). Small unilamellar vesicles (SUV) are formed by sonication of MLV. They range in size depending on the length of sonication, from 25nra to lOOnm, and are a clear suspension. They can encapsulate about 0.2 to 1.0 litres per mole of lipid, and molecules above 4.0,000 MW are only encapsulated with difficulty, but they are much more homogeneous in size than MLV. Reverse phase vesicles (REV) are formed by dissolving the dried material. After sonication, a homogeneous emulsion is formed and the solvent is removed by rotary evaporation. This method gives a very high encapsulation, 20-60% of added material being encapsulated in the aqueous phase. The vesicles are uni- or oligolamellar and are heterogeneous in size from 0.1 to 10urn. They can be made homogeneous in size by extrusion through polycarbonate membranes (Szoka and Papahadjopoulous, 1978). - 36 -

Another technique of liposome formation is to infuse the lipid in organic solvent into the aqueous phase to form unilamellar vesicles. Batzri and Korn (1973) used ethanol as the organic solvent, whereas Deamer and Bangham (1976) used ether. A low entrapment of solute in the aqueous phase is obtained, but the captured volume is about 10-15 litres/ mole lipid. Detergent removal for formation of liposomes by- dispersing the lipids in detergent and then dialysis to remove it was developed mainly for membrane reconstitution experiments (Kagawa and Racker, 1971). Recently it has been demonstrated that enzymatic formation of bilayers from lysophosphatidylcholine and oleoyl CoA will encapsulate microsomal proteins (Deamer and Boatman, 1980). Another method of forming liposomes is by making cochleate cylinders with acidic phospho lipids. Firstly SUV are formed and fused by the addition of calcium into a cylinder. When EDTA is added, large enclosed spherical liposomes are formed. These are termed large uni lamellar vesicles (LUV) (Papahadjopoulous and Vail,1978). Liposomes have also been produced using a French Press (Hamilton et al, 1980).

The size, charge and properties of liposomes can be altered by changes in the lipid composition. The major component is phosphatidylcholine. For maximum stability in vivo, an equi molar proportion of cholesterol and phospholipid is required (see 6). Cationic liposomes are obtained by addition of a basic lipid such as stearylamine, while anionic liposomes are obtained by addition of acidic lipids such as phosphatidylserine, phosphatidylglycerol, phosphatidic acid or dicetyl- phosphate. Any other phospholipid will form liposomes, except phosphatidylethenolamine alone. The transition temperature (T ) of the phospholipid will also govern their properties. Egg phosphatidylcholine is a mixture of fatty acid chain lengths and degree of chain saturation with a Tc of -15°C. Dipalrnitoylphosphatidylcholine (DPPC) (Cl6:0) has a Tc of /.1°C, distearoylphosphatidylcholine (DSPC) (C18:o) a Tc of 55°C and sphingomyelin (SM) a Tfi of 32 C. Thus, at physiological temperatures, DPPC and DSPC liposomes will be "solid" while egg PC and SM liposomes will be "fluid".

Both lipophilic and lipophobic material can be encapsulated in liposomes. Recognition markers for cellular uptake and membrane proteins can be located in the lipid phase. 2 . CHARACTERISTICS OF LIPOSOMES

(i ) In vitro

Uptake of liposomes by cells takes place by two general mechanisms. They are either endocytosed into the lysosomes of cells or they fuse with the plasma membrane. Many experiment have been performed over recent years to distinguish between these two using different compositions and sizes of liposomes. Endocytosis is divided into two, absorptive or phagocytic uptake for large particles like viruses and fluid or pinocytotic uptake for smaller vesicles like lipoproteins, colloids and immune complexes. It is almost impossible to discriminate between these except by kinetic analysis (Silverstein et al, 1977). Phagocytosis is sensitive to inhibitors of oxidative phosphorylation and glycolysis and there should be an increased uptake with increased temperature (Steinman et al, 1974-)- There is a critical thermal temperature below which phagocytosis of large particles cannot occur. This is about 18-21°C. There is no critical thermal temperature below which pinocytosis ceases. The vacuole into which the particle is phagocytosed is called a phagosome. These can be either smooth or coated with short bristles that protrude into the cytoplasm. Pinocytotic vesicles are formed by fusion of membrane folds and invaginations of spherical or tubular structures. Most of these vesicles fuse with lysosomes in the cell or they may traverse the cell as in the case of vascular endothelium. Some endocytic vacuoles remain intra cellular but do not fuse with lysosomes.

The other mechanism of liposome uptake is fusion. The lipid bilayer of the liposome may be inserted into the plasma membrane with the release of the liposome contents into the cytoplasm. This process increases linearly between 0 and 18-20°C, followed by a more rapid linear uptake above this temperature (Poste and Papahadjopoulo s, 1976). It has been very difficult to discriminate between fusion and endocytosis, for several reasons. In fusion, liposomal lipid may be incorporated into the plasmalemma, but this is not definite evidence that endocytosis has not taken place because phago lysosome lipids may be recycled into the membrane (Tulkens 38 et al, 1977). Markers taken up from liposomes that are found in the cytosol may have been passed on through the lysosomal apparatus. Numerous uptake experiments with liposomes of different compositions into cell lines have been performed.

Chinese hamster lung cells have been used to study vesicle cell fusion because they do not engage in phagocytosis. When these cells were incubated with uni- and multi-lamellar 3 liposomes containing H inulin and the phospholipid marked 1 j with phosphatidylcholine, it was found that vesicle-cell fusion occurred at 37°C, while at low temperatures or when the cells were depleted of energy stores, lipid exchange between the liposomes and the plasma membrane occurred (Huang and Pagano, 1975)- Using mouse 3T3/Balb c cells, liposomes were shown to be taken up by endocytosis and fusion (Poste and Papahadjo poulo s, 1976). The non-endocytotic pathway predominated with negative fluid vesicles at 37°C, but neutral fluid and negative solid were taken up by endo cytosis. The uptake of solid negative vesicles was inhibited by 80-90$ by inhibitors of cellular energy metabolism but uptake of fluid vesicles by- only 30-4-0$. These results suggested that mechanism of uptake was dependent on the lipid composition of the liposomes. When cyclic AMP was entrapped in vesicles and incubated with cells, it produced a significantly greater inhibition of cell growth than equivalent concentrations of exogenous free cyclic AMP. This suggested that active cyclic AMP was released from vesicles into the cytoplasm by fusion (Papahadjopouios. et al, 1974-) . Solid liposomes containing cyclic AMP did not induce alterations in growth of cells. But these results may have been a concentration effect alone, the liposomes providing a higher concentration of entrapped solute immediately outside the cells. When the uptake of liposomes into Acanthamoeba castellanii was investigated, it was found that the vesicles were endocytosed by or fused with the plasma membrane according to their lipid composition (Batzi and Korn, 1975)* Egg phosphatidylcholine SUVs were endocytosed at 28°C, with the equal uptake of the phospholipid bilayer and contents of the internal aqueous space. But DPPC SUV and MLV were taken up by fusion, as demonstrated by the fact that only 40$ of the aqueous phase - yj - was internalized. Fusion has been induced between liposomes and cells by using lysophosphatidylcholine as a constituent of the vesicles (Dunham et al, 1977). In cells not capable of phagocytosis, it has been shown possible to introduce peroxidase-laden liposomes into the cytosol by using the "fusogen" lysophosphatidylcholine in the lipid bilayer of multilamellar liposomes (Weissman et al, 1977). The peroxidase could only be taken up when added in liposomes and not as free enzyme. On the other hand, when peroxidase-laden liposomes were coated with IgM and incubated with phagocytes, the lipo somes were found only in lysosomes, being internalized by means of the immunoglobulin-F receptor complex (Weissman e_t al, 1975). Uptake of liposomes by peritoneal macrophages also showed phagocytosis into cytoplasmic vacuoles (Mattenberger- Kreber et al, 1976).

The association or adsorption of liposomes to cells by inter action with cell surface components has been shown (Magie and Miller, 1972). They found that cationic MLV were attached instantaneously to cells, but there was little absorption of anionic MLV. A model system of Chinese hamster V79 fibroblasts was used to study the adhesion of liposomes (Pagano and Takeidi, 1977). Below their phase transition temperature, vesicles made from DPPC or DMPC, absorbed to the surface of the cells. The adhesion increased with decreasing temperature. But cells that were trypsinized before incubation with vesicles showed no temperature dependence of vesicle uptake. When these cells were incubated with isotopically asymmetric vesicles, uptake of exogenous lipids was found to be directly proportional to the amount of radioactivity present in the outer leaflet of the vesicle (Sandra and Pagano, 1979). This showed that only the outermost lipid layer became associated with the cell during vesicle-cell lipid exchange. Fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine showed low temperature exchange of lipids to the plasma membrane of the recipient cells (Struck and Pagano, 1980). The mechanisms of uptake and their criteria have been given by Pagano and Weinstein (1978). Wh en SUV containing the fluorescent dye, 6 carboxyfluorescein, were incubated with human lymphocytes, the fluorescence was distributed widely throughout each cell (Weinstein et al, 1977). Uptake of liposomes into leukocytes 3 haS been evaluated using liposomal markers of H phosphatidyl- - 40 - choline and inulin and cytochemistry (Finkelstein et al, 1980).

(ii) In vivo

When liposomes are administered to animals they are removed from the circulation into the tissues, mainly the reticulo endothelial cells, according to their size, lipid composition charge, and route of administration. The majority (60-85$) of liposomes injected localize in the liver and spleen (Gregoriadis and Ryman, 1972) with only limited accumulation in other organs, even when the above parameters are changed. Clearance from the circulation is faster the larger the liposome (Juliano and Stamp, 1975) and anionic vesicles are cleared more quickly than cationic (Gregoriadis and Neerunjun, 1974-) • When liposomes are exposed to blood they acquire serum proteins which either enhance or inhibit uptake by the reticulo endothelial cells and circulating monocytes. (See Section C (6)). Liposome targetting has become more realistic over recent years. The fact that extravasation only occurs in sinusoidal capillaries has meant that efforts are now directed towards targetting to liver, spleen and bone marrow as well as to cells in the circulation (Poste, 1982). Three types of capillary are found in the body, depending on the type of endothelial cells lining it, sinusoidal, continuous and fenestrated. The latter two have underlying basement membranes and so it is very difficult for the liposomes to escape from them.

The pharmokinetics of drug and enzyme removal is changed by entrapment in liposomes (Juliano and Stamp, 1978). Liposomes are unstable in the serum and several investigations following dual labels have shown that the kinetics of clearance of the liposomal aqueous marker follows more closely that of free markers than liposomal lipid markers (Kimelberg and Mayhew, 1975). Many investigations into serum interaction with lipo somes have been performed to discover if liposomes are delivered intact to tissues (see Section G (6)). Most of the in vivo behaviour of encapsulated drugs have involved the use of antitumour drugs (Kaye and Richardson, 1979)* These investig ations have shown that the pharmokinetics of the encapsulated drug followed that of the liposome carrier. The half-life of clearance of free daunorabicin was about 2 minutes, while the half-life of liposome-encapsulated drug was 2 hours (Juliano and Stamp, 1978). It was also shown that liposome- encapsulated cytosine arabinoside persisted in the tissues for longer periods than the free drug. Intraperitoneal administration of liposomes leads to a slower sequestration of intact liposomes into the tissues. Other routes of admin istration have included intramuscular, subcutaneous and local injections into heart, brain, testes and tumour.

Clearance of liposomes from plasma following intravenous injection of neutral or negatively charged liposomes is biphasic, a rapid initial clearance, followed by a slower phase (Gregoriadis and Neerunjun, 1974.). The rate of liver accumulation of liposomes is proportional to the rate of plasma clearance, and the charge on the liposomes affects the rate of clearance into organs other than liver (Kimelberg, 1976). In the liver, Kupffer cells have a lower affinity for small rather than large liposomes. Liposomes that are to escape uptake into tissues must be stable, non-antigenic and must possess some of the features of an erythrocyte to have a long circulatory half-life. This is very useful for sustained release of drugs.

Liposome disposition in vivo has been studied by Abra and Hunt (1981). After 2 hours the negatively charged liposomes were predominantly found in the reticuloendothelial system. There was a saturation of uptake with increasing lipid dose for both large and small liposomes. By adjustment of liposomal lipid dose, the percentage of dose in the blood could be varied 733-fold, in the spleen 9-fold and liver 4,-fold. Multilamellar vesicles of large size were more stable in vivo than those of medium size which in turn were more stable than those of small size formed by the French press technique. The effect of a high intravenous dose of liposomes on the ability of mouse tissue to take up or bind a second intravenous dose of liposomes was studied (Abra et al, 1982). When the two doses were separated by one hour, the entrapped marker was found to be 29-fold increased in the circulation and 6-fold depressed in the liver. When the two doses were separated by 24- hours, the first dose had little effect on the disposition of the second dose. This reversible blockade could be exploited for increasing the circulation time of liposomes. The blockade of the reticuloendothelial system has also been investigated by administering dextran sulphate or carbon particles (Souhami et al, 1981). Liposomes were labelled with cholesterol, phosphatidylcholine or ^ mT in 1 / c the bilayer and C inulin in the aqueous phase. The rate of uptake of MLV of all charges into liver was only depressed by 25-50% after blockade, whereas sheep red blood cell uptake was depressed 80-90%. This suggested that some mechanism other than phagocytosis was responsible for normal uptake of liposomes by liver and that blockade was not of great value in changing the distribution of liposomes in vivo.

Reverse phase liposomes were used to blockade, rat liver (Kao and Juliano, 1981). The second dose of reverse phase liposomes was cleared slower by the liver, but there was no great alteration in tissue distribution in vivo. Portacaval shunts have been used to attempt to alter liposome distribution (Freise et al, 1980). The in vivo uptake of % methotrexate containing cholesterol liposomes into parenchymal (PC) and non-parenchymal cells (NPC) in the liver was investigated. One hour after injection, NPC had 2-3 times more liposome bound radioactivity than PC, whereas after six hours the ratio was inverted. In animals with a portacaval shunt, the PC contained 3-4- times more liposomal radioactivity per cell than NPC one hour after injection, although the total uptake by the liver was not diminished. 3. ENZYME DELIVERY IN LIPOSOMES IN VITRO

The first enzyme to be entrapped in liposomes for a therapeutic use was Aspergillus niger amyloglucosidase. There was about a 4- to 6.5$ entrapment which could only be measured after disruption of the liposome integrity (Gregoriadis et al, 1971). In 1970, lysozyme had been entrapped in liposomes as a model for lysosome sequestration of enzymes and to investigate latency of enzymes (Sessa and Weissman, 1970). A model storage disease was used to investigate the uptake of enzyme containing liposomes (Gregoriadis and Buckland, 1973). Exposure to sucrose of mouse peritoneal macrophages and Chinese hamster fibroblasts, both lacking invertase, led to accumulation of sucrose in their lysosomes. Incubation of these cells with both free invertase and liposomally entrapped invertase caused degradation of the sucrose. The introduction of hexosaminidase A into Tay Sachs leukocytes was performed by means of liposomes (Cohen et al, 1976). The liposomes were coated with aggregated immunoglobulin to provide a better endocytic stimulus. Significantly more hexosaminidase A was incorporated when enzyme was presented to the cells in liposomes coated with aggregated immunoglobulin (2.5 mu in 10 minutes) compared with liposomes coated with native IgG (0.21 mu in 10 minutes). It was shown that peroxidase could be taken up into peroxidase deficient phagocytes from smooth dogfish using MLV (Weissman et al, 1975). The enzyme was taken up more avidly when presented as aggregated IgM-coated liposomes than native IgM, with over 50$ being incorporated in the first hour compared to 1$ of the free enzyme.

In 1974, the uptake of amyloglucosidase was investigated into fibroblasts from patients with Pompe’s disease (Roerdink and Scherphof, 1974). They found a 50$ decrease in glycogen content from the initial value after incubation with liposomes. The molecules a 1-antitrypsin and soya bean trypsin inhibitor have been entrapped in anionic multilamellar liposomes and the entrapment was shown to be proportional to the anionic surface charge in the lipid bilayer (Finklestein and Weissman, 1978). There was minimal adsorption to negative vesicles, but extensive electrostatic binding to positive vesicles. Liposomes containing horse radish peroxidase have been incubated with three human non-phagocytic cell lines in vitro (Weissman et al, 1978). Less than lmu of enzyme was taken up if negatively charged liposomes were added, but uptake could be enhanced by the addition of 1 mole $ lijsophosphatidylcholine.

Ultrastructural cytochemistry revealed that peroxidase was within liposomes in the cytosol of the cultured cells 15 to 90 minutes after liposome-cell fusion. In 1975 Fishman and Citri entrapped 12$ of added L-asparaginase in liposomes and this led to an increase in stability and protection against proteolytic attack by trypsin. Many enzymes have been reconstituted in liposomes, including ATPase (Kleeman and McConnell, 1976), and cytochrome oxidase (Eytan et al, 1975). Cystine has been removed from cultured cystinotic cells in culture by administration of reducing agents entrapped in liposomes (De Brohun-Bulter et al, 1978). After 2 hours, cystine content of cells was reduced more by agents in liposomes than free agents. The most effective reducing agent was cystamine MEA in liposomes, but these results could not be repeated when serum was removed from the culture medium. This indicates that the serum may have had some disruptive effect on the liposomes and the liposomes only serve to hold a high concentration of the agent at the cell surface. When urease was entrapped in egg phosphatidylcholine liposomes, the entrapment procedure led to a change in the apparent Michaelis constant (Kapp) from 68mM to l67mM. The maximum velocity did not change (Maderia, 1977). The entrapped enzyme was less sensitive to the pH of the external medium than free enzyme and seemed to be more stable. The free enzyme had an activation energy of 7.5kcal/mol above and below 30°C while the entrapped enzyme had an activation energy of 17.5kcal/mol below 30°C and 8.9kcal/mol above 30°C. (3-Galactosidase from feline liver was entrapped in liposomes and incubated with feline gangliosidosis GM1 fibroblasts (Reynolds et al, 1978). The efficacy of the therapy was evaluated by labelling the fibroblasts with galactose. Treatment with unencapsulated 3-galactosidase had no effect. Liposomally entrapped enzyme cleared 80$ of "*"^C galactose- labelled galactopeptides and increased intracellular activity to 70$ of normal. 4-5

Entrapment of a-amylaso in liposomes to investigate vesicle fusion with the membrane of Acanthamoeba castellanii was performed by Batrzi and Korn (1975)* The degradation of IP glutamic dehydrogenase entrapped in SUV by cultured mouse Balb c/3TC fibroblasts was followed by Hernandez-Yago et al (1980). They found an increase in label in lysosomes with increased chase time, suggesting a meroautophagic mechanism for the segregation of cytosol proteins. The mechanism of preparation of liposomes was found to have an effect on the stability of enzymes in liposomes (Kurosaki et al, 1981). A novel mechanism for estimation of enzyme within liposomes was developed using single radial immuno diffusion and quantitative immunoelectrophoresis (Dobre and Motas, 1980). The stability of elastase in reverse phase liposomes (REV) was investigated compared to SUV. (Kurosaki et al, 1981). REV encapsulated more enzyme and imparted more stability than SUV. K. ENZYME DELIVERY IN LIPOSOMES IN VIV0 3 The fate of liposomally entrapped H amyloglucosidase in rats was the first in vivo investigation of a liposome- entrapped enzyme (Gregoriadis and Ryman, 1972). Uptake of radioactivity was a maximum of 56% after fifteen minutes, after which it was extensively catabolised. Subcellular fraction ation showed a predominance of the label in the mitochondrial/ lysosomal fraction shortly after injection. When 3-fructofuranosidase-containing liposomes were injected into rats, 4.5% of the activity was found in the liver after six hours (Gregoriadis and Ryman, 1972). When lysosomes were made denser by the injection of Triton or dextran, the administered enzyme activity after liver fractionation moved along a FicoEL density gradient in a similar manner to the lysosomal markers. A model for a 11,303 omal storage condition was produced by administration of dextran to rats (Colley and Ryman, 1976). The enzyme dextranase, which is absent in rats, was entrapped in sonicated liposomes and administered to dextran-treated animals. The liposomally-entrapped enzyme caused a decrease in radioactivity in the liver of the same magnitude as free dextranase. This indicated that some of the liposomally- entrapped enzyme may have been released into the circulation. Another model of a storage condition was induced by depriving rats of zinc. This decreased a-mannosidase levels in liver, lung, intestine, kidney, spleen and serum by about 50% (Patel and Ryman, 1974-) • Administration of liposomally- entrapped a-mannosidase caused an increase in enzyme activity in the liver and spleen lysosomes. Entrapment of invertase in liposomes with GM1 ganglioside entrapped in the lipid bilayer increased the rate of uptake of liposomes into liver (Surolia and Bacchhawat, 1977).

Treatment of humans with enzymes entrapped in liposomes has also been carried out. A Pompe’s patient (deficiency of lysosomal a-glucosidase) was treated with the Aspergillus niger enzyme in liposomes (Tyrrell et al, 1976). The patient died before any long-term trial could be made, but liver glycogen levels did seem to be decreased at autopsy. A long term study of 3-glucosidase in liposomes to Gaucher’s disease patients has been made (Belchetz, 1977). There was a general - 4-7 - improvement in health, an improvement in reticuloendothelial system function, but no decrease in the size of the liver.

The in vivo subcellular distribution of 3-glucuronidase infused into mice was followed (Steger and Desnick, 1977) . About 50-80$ of the injected dose accumulated in liver and was stable for 4-8 hr, and depleted by 11 days. Enzyme- loaded liposomes were cleared more rapidly by the liver and remained there longer than buffer-loaded liposomes. The enzyme must have been partially exposed, showing an increased affinity of the liposomes for the liver. When negative liposomes were used, about 70$ of the enzyme appeared in the lysosomes between 1 and 14-4- hours after injection. With positive liposomes, only 50$ was found in the liver, with 20-30$ from 1 to 4- days after injection. The positive liposomes seemed to disrupt the integrity of the lysosomes.

The penetration of tissues by liposome-associated glucose oxidase was investigated by Dapergolas et al (1976). Free glucose oxidase, when given intravenously into rats, decreased blood glucose levels by more than 90$ in 15 minutes, with only a minimal effect over 24- hours. Glucose oxidase in phosphatidyl inositol liposomes caused a blood glucose level decrease of 70$ over 4- hours.

It has been claimed that 3-galactosidase ( ^ I-labelled) can be located in the brain after intravenous injection in liposomes (Takada et al, 1982). Another group has administered horse-radish peroxidase in liposomes to rats and detected the presence of the enzyme in the nucleus supra opticus, where the blood brain barrier is well developed (Yagi et al, 1982). The passage across the blood brain barrier of intact liposomes seems most unlikely, the above experiments may demonstrate that radioactive label ( I) or peroxidase alone can penetrate into the brain.

Neuraminidase from Clostridium perfringens has been entrapped in liposomes and administered to rats (Gregoriadis, 1974-) . With the free enzyme about 7-10$ was found in the liver and none in the lysosomal fraction. With the liposomally - /f8 -

entrapped enzyme 20-26% was found in the liver, of which 60-69$ was in the lysosomal fraction. The presence of non-entrapped neuraminidase in blood led to the desialyation of plasma and to a decrease in concentration or total removal from the circulation of some of the plasma glycoproteins. Entrapment in liposomes prevented this.

When liposomally entrapped asparaginase was administered to mice with 6L3 HED lymphoma cells, a higher amount of enzyme was needed to provide tumour regression than when free asparaginase was administered (Neerunjun and Gregoriadis, 1976). It was assumed, but not demonstrated, that asparagine could cross the lipid bilayer. The entrapped asparaginase prevented anaphylactic shock. It is most likely that asparagine cannot cross the liposome membrane,(it is impermeable to erythrocyte membranes),and that the effect was caused by a slow release of asparaginase into the circulation.

The release of inulin and peroxidase from MLV has been studied in vitro by incubation with serum and it is possible for biological fluids to cause enough damage to liposomes to create holes big enough for enzymes to leak from them (Finklestein and Weissman, 1979). See Section C (6) for serum effects on liposomes. 5. CHELATION THERAPY WITH LIPOSOMES

Liposomes have been used for metal chelation therapy because most toxic metals accumulate in the reticuloendothelial cells, into which liposomes are readily sequestered. The protection of chelating agents against biological degradation in the circulation by liposomes causes increased delivery to the sites of pathology. Entrapment of chelating agents in liposomes means that uptake into organs where they may be toxic is minimised. The most useful iron chelating agent used so far has been desferrioxamine. When entrapped in liposomes, the rate of clearance from the circulation was increased ten fold over the non-encapsulated form (Rahman, 1981). Chelation of plutonium by encapsulation of ethylenediamine tetra acetic acid (EDTA) and diethylenetriamine penta acetic acid (DTPA) in liposomes was very successful when administered to mice (Rahman et al, 1973). Chelating agents in liposomes have also been used to mobilise mercury from kidneys and promote faecal excretion of colloid gold (Rahman and Rosenthal, 1973, and Rahman and Wright, 1975).

Liposome encapsulated desferrioxamine has been targetted to parenchymal cells of the liver by attachment of galactocerebroside to the lipid bilayer (Lau et al, 1981). It was found that for both normal and hypertransfused mice, liposome encapsulated desferrioxamine containing glycolipid removed more yFe ferr tin from mice parenchymal cells than liposomes without glycolipid, which had a higher affinity for Kupffer cell

An ionophore (A 23187) has been administered with DTPA in liposomes (Young et al, 1979) • DTPA has not been very success ful at iron chelation because it cannot cross cell membranes. Liposomes containing the ionophore and DTPA caused an excretion of 59 Fe from iron-overloaded mice, whereas DTPA alone had no effect. Desferrioxamine in its free form also does not cross cell membranes (Guilmette et al, 1978). When this chelating agent was labelled with 59 Fe and administered in liposomes, radioactivity was found in the liver demonstrating successful transport of the encapsulated drug across the cell membrane. Liposomes of small size were predominantly taken into parenchymal cells whereas the larger liposomes were found more in Kupffer cells (Rahman et al, 1982). 50

V/hen parenchymal cells were preferentially labelled with 59 Fe ferritin and Kupffer cells with 59 Fe damaged red blood cells, it was shown that unilamellar liposomes were more effective than inultilamellar at iron removal in mice given ferritin, whereas multilamellar liposomes were more effective for removal of iron in mice given damaged red blood cells. Both increased the urinary excretion of iron, but neither the faecal excretion of iron (Rahman, 1981). Liposomally entrapped chelating agents are therefore useful both for acting as a slow release depot for the drug and to deliver it to the storage site of the metal. See Chapter 4-. 51

6. PROBLEMS WITH LIPOSOMES IN VIVO

(i) Serum

Biological fluids have an extensive effect on liposome integrity in vivo and in vitro. Incubation of liposomes containing sucrose, inulin or albumin with blood or plasma caused rapid release of the entrapped marker (Zborowski et al, 1977) . It has been demonstrated that apolipoprotein subunits and intact high-density lipoproteins (HDL) can have a deleterious effect on liposomes. Apolipoprotein AI has been shown to be released from HDL and incorporated into the liposomal membrane causing it to dissolve (Tall and Small, 1978) . Also liposomal lipid has been shown to be taken up by HDL to form a lipid-enriched HDL particle (Scherphof et al, 1978). Albumin has been thought to have a damaging effect on liposomes(Zborowski et al, 1977) but this was later shown to be due to impurities in the albumin of apolipoprotein CII and Cl (Finkelstein and Weissman, 1979). Purified apolipoproteins interact with phospholipid vesicles to form disc shaped particles (Atkinson et al, 1976). An attempt at Isolation of a new particle from liposomes and HDL by isoelectric focusing has been made (Damen et al, 1981). Other workers have found that bovine HDL liposomal phosphatidylcholine was then trans ferred in a one-way process to pre-existing HDL (Jonas, 1979). A factor in plasma strongly stimulates the transfer of phospha tidylcholine between the liposomes and the lipoproteins (Damen et al, 1980). This is a protein in the non-lipoprotein fraction of plasma which acts with HDL to have the same effect on lipo somes as whole plasma. The nature of this factor is not yet known. When phospholipases were used to degrade liposomes, it was shown that only the outer leaflet of the bilayer was susceptible to phosphatidylcholine exchange with HDL (Wilschut et al, 1979). In vitro incubations of liposomes and plasma have shown that molecules as large as albumin can be lost by incorporation of plasma proteins into the membranes (Kimelberg, 1976). This suggests that more than just superficial damage is done, as indicated by Wilschut et al (1979).

After liposomal phosphatidylcholine has become associated with HDL, it has been demonstrated that the HDL interacts with the plasma membrane of hepatocyte with only the phosphatidyl choline being transferred to the cell (Scherphof et alt 1980). They postulate that this occurs by means of fusion, rather than endocytosis.

Other plasma proteins have been found to interact with lipo somes. a2-Macroglobulin in human plasma was the only protein found to be associated with washed liposomes (Black and Gregoriadis, 1976). The interaction of liposomes with human skin fibroblasts and mouse P815Y mastocytoma cells and the perfused rat liver was investigated (Tyrrell et al, 1977) . Albumin enhanced the exchange transfer of cholesterol between liposomes and cultured cells and a- and 3-globulin enhanced the uptake of anionic liposomes by the perfused rat liver. The 3-globulin fraction caused increased leakage of methotrexate from liposomes. It has been found that coating liposomes with protein decreases their capture by macrophages (Torchilin et al, 1980). There was a decrease in capture of albumin-coated liposomes by macrophages if albumin was present free in solution to saturate the binding sites on the cells. The release of 6-carboxyfluorescein from vesicles prepared by the French press procedure (see C, 1,) in the presence of serum gave an order of potency of disruption of liposomes by serum components of apolipoproteins Al and E greater than HDL and VDL which was greater than whole serum or plasma (Guo et al, 1980). A study on the interaction of apolipoprotein from human very low density lipoproteins (apo CIII) with egg phosphatidylcholine liposomes showed that the reactivity of single bilayered vesicles was greater than for multibilayered vesicles (Morrisett et al, 1977). In complexes formed by the association of apo CIII with single bilayer vesicles, the a helical content of the peptide backbone and the apolarity of the environment are greater than those observed in the complexes formed with MLV.

The lipid composition of liposomes directly affects their stability in plasma. The cholesterol content has a significant effect both on the leakage from liposomes and their half-life in the circulation. When 6-carboxyfluorescein was entrapped in liposomes, it was retained to a greater extent in liposomes with an equimolar phospholipid and cholesterol content (Kirby et al, 1980). The widely different half-lives of liposomes 53 of different compositions reflects the differences in bilayer permeability (Senior and Gregoriadis, 1982). The leakage of calcein from liposomes in the presence of serum was decreased by increasing the ratio of cholesterol incorporated (Allen and Cleland, 1980). The cholesterol stabilized liposomes by prevention of loss of phosphatidylcholine to HDL, probably by altering the packing of the phosphatidyl choline, so that apo HDL could not bind (Kirby et al, 1980). Clearance of SUV from the circulation is more rapid for unstable cholesterol-free or cholesterol-poor SUV than it is for cholesterol-rich SUV (Kirby and Gregoriadis, 1983). Recently it has been suggested that cholesterol has an inhibitory effect on uptake of liposomes by the reticuloendothelial system (Patel et al, 1983) . When inulin was entrapped in lipo somes, it was cleared more readily from the circulation in cholesterol-poor than in cholesterol-rich liposomes. Incorpor ation of cholesterol into liposomes makes them less susceptible to phospholipase action (Op den Kamp et al, 1975).

The transition temperature of the lipids in the bilayer will also affect the stability of liposomes in vivo. Lipids that are below their transition temperature at physiological temperature will make nsolid,r liposomes and therefore be more stable, whereas those which are above their transition temperature will be fluid and less stable to leakage of aqueous phase solutes. The kinetic uptake of sphingomyelin-cholesterol liposomes into liver has been estimated by y ray perturbed angular correlation (Hwang et al, 1980). They found that these liposomes had a very long half-life (16.5 hours) after intravenous injection into mice, and were taken up intact into liver. But Sherphof (1982) found that liposomes made of pure egg phosphatidylcholine or sphingomyelin were relatively unstable and lost substantial proportions of both solute and phospholipid, more so for SUV than MLV. DMPC liposomes showed interactions with HDL (Tall et al, 1978). The role of phase transition and phospholipid backbone in leakage was studied using the calcein dequenching method (Allen, 1981). As the proportion of DSPC (Tc=56°C) relative to egg phosphatidylcholine increased, the retention of liposome contents increased. Brain sphingomyelin also increased the retention of egg PC transferred to HDL, as did DOPC. At 37°C, the sphingomyelin is above its transition temperature but did not exchange because of the intermolecular hydrogen-bonding between the sphingosine backbone. Mayhew et al (1980) looked at the leakage of inulin and sucrose from PS/DSPC/DPPC lysosomes, and because these liposomes were near their phase transition temperature, they were highly susceptible to interactions with serum. The stability of liposomes to serum was increased when a dialkyl analogue of phosphatidylcholine was substituted for the diester form. It was found that after 1 to 3 days the radioactivity was 3 to 4- times higher with this modified lipid. It was semi-resistant to phospholipases and taken up by endocytosis (Deshrmihh et al, 1978).

(ii) Immunological Interactions

The adjuvant effect of liposomes has been investigated. When diphtheria toxin was entrapped in liposomes and repeatedly administered to mice, although the antibody titres agai'hst ‘ the toxin were not lower than in mice injected with free toxin, there was no allergic reaction (Gregoriadis and Allison, 1974)- The severe allergic reaction was prevented possibly by inhibiting the interaction of the antigen with its antibody. The immune response to bovine serum albumin in liposomes was investigated (Heath1et al, 1976; Heath, 1976). They found sphingomyelin lowered the immune response, compared to both positive and negative phosphatidylcholine liposomes which act as adjuvants. Introduction of cholesterol into liposomes reduces the immune response and the rate of digestion of liposomes by macrophages (Johson, 1975). Several lipids have been found to be antigenic in vivo. Antibodies against lipid A (Schuster et al, 1979) against phosphatidylserine,cardiolipin, phosphatidylglycerol and phosphatidic acid have been found (Alving, 1977). Liposomes containing N-dinitrophenyl (DNP) phosphatidylethanolamines have been used to study the roles of complement fixation (Kinsky and Nicolotti, 1977). The study of the cascade system of complement has made great use of liposomes. When a surface antigen is present and the corresponding antibodies, the full complement pathway is activated. Also the alternative pathway starting at C3 can be activated by the lipid membrane alone, containing phospha 55 tidylcholine, cholesterol and a mono-or dihexosylceramide or diglyceride (Cunningham et al» 1979).

The immune response to negatively charged liposomes loaded with bovine 3-glucuronidase was evaluated (Hudson et al, 1979). They found that liposomes did not protect the entrapped protein from immunological surveillance and rapid tissue clearance and degradation of the entrapped enzyme in sensitized mice was in part due to the processing of antigen-antibody complexes. A cellular response was elicited to the liposome vesicle itself by phagocytic cells of the reticuloendothelial system.

The stimulation of macrophages by immunomodulators entrapped in liposomes has been discussed by Poste and Fidler (1981).

(iii) Targetting

The direction of enzymes in liposomes to the specific site in the body from which they are absent was the original goal of carrier research. In many diseases, enzymes are absent from the CNS, from muscles, heart, kidney. Research to date shows that liposomes are only., able to escape from the circulation into the reticuloendothelial system with only minor amounts arriving at the parenchymal cells of the liver. Cell specific antibodies have been used to alter the tissue distribution of liposomes with very little success. Most antibody-liposome targetting has been performed in vitro. Antibodies attached to liposomes have been used to remove from the circulation toxic or undesirable material. Anti-digoxin antibodies were used to clear digoxin from the circulation for a therapy for dixogin overload (Campbell et al, 1980). Improvement in external scintigraphy by entrapment of a secondary antibody to a primary antibody used to detect tumours has proved successful at removing background radioactivity in the circulation (Barratt et al, 1983)•

Carbohydrate determinants included in the lipid bilayer have been used for liposome targetting. Cell surface carbohydrate binding proteins have been found on many cells (see Section B). The hepatic uptake of liposomes was increased only marginally by inclusion of asialoglycoprotein into liposomes (Gregoriadis - 56 - and Neerunjun, 1975). Several groups have used monosialo- gangliosides to try and increase uptake of drugs and enzymes into hepatocytes. Invertase was entrapped in monosialo- ganglioside liposomes (Surolia and Bacchhawatt, 1977), carboxyfluorescein in cerebroside liposomes (Hoekstra et al, 1980), bovine serum albumin in asialoglycophorin liposomes (Hildenbrandt and Aronson, 1980) and chromate in synthetic glycolipid liposomes (Wu et al, 1981) to try and increase the uptake of liposomes into hepatocytes or to increase their retention at the site of injection. 7. OTHER THERAPEUTIC USES

Liposomes have been used to entrap dozens of different anticancer drugs such as actinomycin D, cytosine arabinoside, metholrexate and vinblastine (Gregoriadis, 1980). They are especially active against ascites tumours by acting as a slow release depot for the drug intraperitoneally. They can act as a slow release depot in the circulation for solid tumours.

Liposomes have been used as a carrier for hormones, particularly insulin. When diabetic rats were fed insulin entrapped in MLV, a lowering of blood glucose was observed (Patel and Ryman, 1976). Liposomes have been used successfully for the treatment of leishmaniasis (Alving et al» 1978).

Treatment for myocardial infarction is a potential use of liposome encapsulated drugs. Multilamellar liposomes accumulate in the capillaries near the infarction (Caride and Zaret, 1977). Liposomes have been used to introduce RNA and DNA into cells (Fraley etal, 1979). 58

SECTION D

ERYTHROCYTE GHOSTS

Erythrocyte ghosts have been used as carriers for enzymes and drugs in vivo.either to target them to the liver and spleen or to prolong their circulatory half-life. They are ideal carriers, both because they are biodegradable and non-immunogenic, when prepared from the recipient’s own cells, or from a genetically identical individual, and because they are easily loaded with exogenous protein. The techniques used to lyse and reseal erythrocytes have been developed over recent years and include hypotonic dilution haemolysis, hypotonic dialysis haemolysis, electrical haemolysis and chemical perturbation. Each has advantages for different applications. 1. HYPOTONIC DILUTION HAEMOLYSIS

Erythrocytes swell on addition of a hypotonic medium and after attaining a certain volume (V^), they become permeable to haemoglobin (Hoffman, 1958). After hypotonic haemolysis, the cell retains osmotic properties characteristic of semi- permeable membranes. Erythrocytes haemolysed in water or hypotonic saline spontaneously recover their low permeability to sodium, potassium, glucose and sucrose. The sodium and potassium are equally distributed across the membrane but incubation at 37°C enables them to actively exclude sodium and retain potassium (Hoffman, I960). The size of the holes that are formed during haemolysis was investigated by Seeman (1967). He found that colloidal gold and ferritin could be entrapped and that the holes must be between 200 and 500 A0. The holes were only transiently opened at 0°C for about 15 to 20 seconds after the onset of haemolysis and then became permanently impermeable. Hypotonic haemolysis occurred at lower salt concentrations when haemolysis was carried out gradually than when done rapidly.

Three types of ghost are formed when erythrocytes are hypo- tonically haemolysed and then restored to isotonicity. Type I ghosts are those that reseal immediately after haemolysis. These cannot incorporate salt after being lysed, and on restoration of isotonicity, they shrink. Type II ghosts reseal after salt is added. Ghosts which do not reseal, even at 37°C, are called Type III ghosts (Bodeman and Passow, 1972). At 0°C, resealing is very slow even in isotonic medium and so for this reason, enzymes are entrapped in ghosts at this temperature and then ghosts are resealed at 37°C so that Type II ghosts predominate. Resealing is almost spontaneous in hypotonic solution at 37°C (Funder and Wieth, 1976). Both "pink” and "white" ghosts can be prepared by the dilution procedure (Taverna and Langdon, 1973). White ghosts are prepared by haemolysis in water or weak buffer and several washings and centrifugations to remove all the haemoglobin. "Pink" ghosts (retaining 20% of their haemoglobin) are formed by lysing erythrocytes in a 1:10 dilution hypotonic buffer or less and resealing by adding sufficient salt to restore 60 isotonicity (Steele and Kant, 1974.) .

The study of membranes in isolation has made use of inside out red cell membrane vesicles, whose outer faces are the cytoplasmic aspects of the parent membrane. They are formed by the budding of the plasma membrane of erythrocyte ghosts into their cytoplasmic spaces which occurs spontaneously when white cell ghosts are suspended in hypo osmotic buffer 5 and centrifuged for one hour at 10 xg. These endocytic vesicles are released by passage through a fine needle. Many membrane studies have been performed on this type of ghost (Glauert et al, 1963).

Erythrocytes are about 8ym in diameter and biconcave in shape. They have to pass through narrow capillaries and through the splenic cord where there are tiny slits 0.5-1.0pm wide. An important property of the cells is to remain deformable and flexible which is dependent not only on membrane properties and internal viscosity, but also on the energy level of the cell. Cyclic nucleotides play an important part in cell shape regulation and permeability (Yawata.. et al, 1976) . Studies with vinblastine and colchicine which alter the assembly of membrane proteins causing a conformational change in the erythrocyte, have shown that their effects can be- inhibited by cAMP and cGMP. Both completely prevent the cupping and sphering of the red cells induced by vinblastine. ATP is the direct precursor of cAMP so the level of this nucleotide will regulate cell behaviour in vivo. Palek et al (1974-) studied the discocyte-echinocyte transformations.associated with ATP, magnesium and calcium ion level in erythrocytes. A biconcave, cup-shape was seen with equimolar concentrations of ATP and magnesium ions, a flat discoid shape was given by either of these two alone and a spherical, spiculated shape when calcium ions were present. Glucose levels, also play an important part in erythrocyte viability in vivo. Ghosts retain their active transport of glucose, and flux through this falls to about one-third if red cell energy stores are depleted (Jacquez, 1983). Batt and Schachler (1973) reported that washing of erythrocytes twice with nine volumes of saline for 5 minutes reduced intracellular glucose from 4-mM to O.lmM. Therefore careful attention must be paid to intracellular metabolites during lysis and resealing of erythrocytes. 61

Humphreys and Ihler (1980) have shown that glutathione levels fall to 30$ when erythrocytes are lysed and resealed. In all erythrocyte ghost experiments in this work, care was taken to ensure that levels of glucose, ATP, magnesium ions and glutathione were supplemented in ghosts used for in vivo administration of enzyme.

Injured erythrocytes are removed by the reticuloendothelial system, where after sequestration and haemolysis in lysosomes, there is enzymic degradation in cytoplasm (Weed and Reed, 1966) The degree of damage to the cell determines whether it is taken up into the spleen or liver, which have different sensitivities for damaged cells. Severely damaged cells go to the liver after just one. passage through this organ, while the spleen removes less damaged cells. Cells taken up by the liver include those damaged by complement fixing iso antibodies, sulphydryl inhibitors, oxidant drugs, metallic cations or prolonged heating. Cells.taken into spleen include those damaged by non-complement fixing, incomplete antibodies, low doses of sulphydryl inhibitors or oxidant drugs and metallo protein complexes. These facts.have been used by investigators to target drugs and enzymes to one or other organ (see later). Normal red blood cells have a half-life in the circulation of 120 days. It is thought that as. they age they undergo gradual desialyation of membrane glycoproteins. The reversible discocyte to echinocyte morphology is controlled by means of these membrane lectins, and it. is thought that glycophorin dissociates from dimer to monomer to control the cytoskeletal movements. Neuraminidase treated cells are no longer able to undergo this transformation and the cells are no longer able to change shape and so are eliminated by the reticuloendothelial system.

The earliest reports of entrapment of exogenous molecules were by membrane workers in the 1950s. It was shown that the red cell membrane was permeable to dextran with molecular weights from 10,000 to 250,000 (Marsden and Ostteng, 1959) and ^3^1 albumin (Hoffman, 1958), as well as to small molecules like ATP, adenosine, cytidine, glucose 6-phosphate (Gourley, 1957). The hypotonic dilution method was the first to be used 62

to entrap enzymes in erythrocytes and to administer these enzymes in vivo. It was found that although membrane probes could detect no difference between the surface proteins of ghosts and normal erythrocytes (Cabanchink, 1975), they were sufficiently damaged after haemolysis to be removed quickly from the circulation. Also, only low amounts of exogenously added protein could be entrapped (between 0.2 and 10$ of that added before haemolysis). Therefore other techniques were developed to try and produce ghosts with different properties. These include electrical and hypotonic dialysis haemolysis. 2. ELECTRICAL HAEMOLYSIS

Erythrocytes can be haemolysed, without addition to a hypotonic 3 solution, by application of an electric pulse at 10 to 10^ kVcrn”^ for 1 to 50 useconds. The damage to the membrane occurs when there is about 1 volt across it (Sale and Hamilton, 1968). When bovine red cells were haemolysed at 300 V and then incubated with 131 I albumin, for 1 hour in isotonic conditions, the protein was entrapped inside the erythrocyte

(Zimmerman et al, 1975) • These cells retain about 5% of their haemoglobin. When urease was entrapped in erythrocytes by dielectric breakdown, about 20$ of. the added enzyme was loaded (Zimmerman et al, 1976). The advantages of this technique are that the preparation can be performed in isotonic solution and the ghosts can be prepared with various membrane permeability properties (Kinosita and Tsong, 1978). Larger pores are obtained either by using a higher field intensity, by increasing the pulse duration or by reducing the ionic strength of the pulsation medium. Even though "^C sucrose- dielectric breakdown ghosts were seen to have a long half-life of 18 days by Kinosita and Tsong (1978), all other workers have found very rapid clearance of these dielectrically prepared ghosts. Zimmerman et.al (1981) found a half-life of ten minutes of methotrexate loaded mouse cells. The 125 in vivo survival of I PVP loaded beagle dog dielectric ghosts did not exceed 2 hours (Chalmers et al, 1981).

Haemolysis of erythrocytes in isotonic solution can also be induced by a rapid (2ysec) temperature jump (0.05°C). Although haemoglobin is released by this method, only small molecules such as glucose can be entrapped (Tsong and Kingsley, 1975). 3. HYPOTONIC DIALYSIS HAEMOLYSIS

Improvement in encapsulation efficiency and in vivo survival characteristics was established by ghosts prepared by a hypotonic dialysis haemolysis technique. The technique of gradual haemolysis by dialysis was first performed by Seeman (1967). He compared the time course of entry of ferritin into erythrocytes prepared either by rapid lysis by dilution or slow lysis by dialysis against a hypotonic buffer. This method was developed later to load enzymes and to retain endogenous erythrocyte proteins and small molecules for better erythrocyte in vivo viability (Dale et al, 1977; De Loach and Ihler, 1977). The advantage of this technique is that small molecules that are lost during haemolysis, such as ATP and glucose, may be replaced in the dialysing buffer (Chalmers et al, 1980). These ghosts have much longer in vivo lifespans in a variety of animals (Chalmers et al, 1981). The cells are lysed by dialysing against a weak hypotonic buffer or against water for one to two hours depending on the haematcrLt. The cells are mixed by gentle rotation to ensure maximum entrapment and resealed by replacing the hypotonic buffer with isotonic medium. Over 50$ entrap ment of added enzyme activities have been obtained in this way and De Loach and Ihler (1977) suggested that enzymes that require detergent for solubilization could be entrapped in this way (such as $-glucosidase). An erythrocyte encapsu- lator dialyzer was developed to prepare large quantities of erythrocytes for use in large animals such as cows, horses and pigs. This gave usually about a 30$ encapsulation (De Loach et al, 1980) . - 65 -

/*. ERYTHROCYTE ENTRAPPED ENZYMES IN VITRO

Taverna and Langdon (1973), in showing that glucose uptake into erythrocyte ghosts was mediated by a membrane bound glucose carrier, incorporated glucose oxidase and catalase into npinkn ghosts prepared by the dilution method. Glucose oxidation was followed by oxygen consumption in an oxygen electrode. The rate determining step was the carrier- mediated diffusion of glucose into the cell .

When white ghosts were used to entrap glucose oxidase (Taverna and Langdon, 1973), glucose entered via a "leak", which allowed entry of glucose but not exit of glucose oxidase. The glucose and phosphate transport of ghosts has been studied by Mawby and Findlay (1978) who found that although the glucose transport system was identical in ghosts and normal erythacytes the phosphate-transport system did not appear to have survived the membrane isolation procedure unaltered.

The first enzymes entrapped in erythrocytes with a therapeutic application were 3-glucosidase and 3-glucuronidase (Ihler et al, 1973). This was performed by the dilution haemolysis method, where 20yl of cells were diluted with 200yl of enzyme solution and kept at 22°C for five minutes. Various aliquots of sodium chloride from 0 to 0.9$ were added and after 30 seconds, 5ml of 1.2$ NaCl added to reseal the membranes. Only 0.2$ of the added enzymes were encapsulated. They found that smaller proteins were preferentially encapsulated. A crude extract of 35 S labelled E.coli proteins predominantly in the 200,000 to 300,000 dalton range were added to haemolysed cells. Proteins encapsulated were predominantly 90,000 daltons (The two enzymes entrapped each have a MW 180,000).

The activity of an erythrocyte encapsulated enzyme was studied using pig liver uricase (Ihler et al, 1975)* The substrate, uric acid, is a small molecule that can enter the erythrocyte by passive diffusion and the rate of uptake was measured radioactively. It was found that the rate of degradation of uric acid by uricase entrapped in human erythrocyte was limited by the rate at which it could diffuse into the erythrocytes. 66

When excess enzyme was present within the erythrocyte, the rate of reaction was equal to the rate of entry. But encap sulation of this enzyme does not seem to affect its activity, and uricase loaded erythrocytes are potentially capable of removing as much uric acid as the human kidney.

Entrapment of a number of therapeutic agents in mouse, rat and human erythrocytes has shown that the amount of substance entrapped depended on the concentration of the substance in the lysing solution (Tyrrell and Ryman, 1976). They found that mouse and human erythrocytes were much easier to reseal than those of rat. Efficiencies of entrapment of different substances (albumin, methotrexate, 3-fructofuranosidase) ranged from 0.5 to 4--0% of the material added.

Adriaenssens et al (1976) entrapped bovine liver arginase in human erythrocytes prepared by the hypotonic dilution method and administered them to patients with arginase deficiency and to patients who were obligate heteiuzygotes for this condition. Activities were increased about 20-fold in hyperarginaemic patients erythrocytes in vivo. They suggested using the low urea-producing goat as an animal model to study the arginase-loaded ghost activity in vivo.

The improved method of dialysis haemolysis and resealing was first used to entrap the enzyme hexosaminidase B and 3-glucosidase (Dale et al, 1977). The incorporation of enzyme was found to be between 36 and 4-2$ of the added material, and optimal incorporation was found to be at 80$ haematocrit. Glucocerebrosidase was encapsulated by De Loach and Ihler (1977) using the hypotonic dialysis method.

The in vitro uptake of enzyme containing erythrocytes by phagocytic cells was demonstrated by De Loach et al (1979). Bone marrow macrophages were incubated with gluteraldehyde treated erythrocytes containing 3-galactosidase or alkaline phosphatase and resulted in uptake of 15-4-0% of the included enzyme within 4-hrs. The outline of erythrocytes was seen by electron microscopy in phagolysosomes, and internalized enzymes had a half-life of 15-30hrs. Monocytes from Gaucher’s disease patients have been used as a model to study the 67 efficacy of erythrocytes to deliver enzymes to cells in vitro (Dale et aly 1974-) . Erythrocytes were coated with anti-Rhesus IgG and these were found to be much more effective at delivering enzymes to cells than soluble enzyme or enzyme incorporated into liposomes. The intracellular half-life was at least 24- hours and microscope examination showed internalized erythrocytes. A method of introducing enzymes into the cytoplasm of fibroblasts in culture was attempted by inducing the fusion of erythrocytes containing bovine arginase with fibroblasts of mouse and human origin deficient in arginase using Sendai virus (Kruse et al, 1981). Thymidine kinase and 125 I bovine serum albumin have been loaded into erythrocytes and fused to a thymidine kinase deficient cell line using Sendai virus (Schlegar and Rechsteiner, 1975). Intact human erythrocytes and erythrocyte ghosts have been fused to form polyerythrocytes by a similar mechanism (Peretz et al, 1964.). This fusion technique has been used to transfer tRNA from yeast to mouse cells via erythrocyte ghosts (Kaltoff et al, 1976). Many workers have tried to incorporate viruses into erythrocyte ghosts. Rechsteiner (1975) found bacterophage T4- was too large, but Humphreys et al (1981) found that 0X174-> SVA, lambda and T7 could all be entrapped in erythrocytes.

Various methods of erythrocyte entrapment have been compared (Fiddler et al, 1980) . Six methods of hypotonic dilution exchange loading were used and one of chloropromazine-induced endocytosis to entrap inulin, H glucose and 3-glucuronidas Maximal incorporation was when the initial concentration of sodium chloride was reduced to 50-75mM. Entrapment methods requiring the greatest reduction in salt concentration resulted in the formation of echinocytes, whereas stomatocytes were observed after entrapment methods requiring less salt dilutions The in vitro morphology after loading should give a good indication of the in vivo fate of erythrocyte entrapped enzyme

The stability of enzymes is often increased by entrapment in erythrocytes because of the high protein environment (Humphreys and Ihler, 1980). Entrapment in the presence of glucose kept intracellular glutathione levels constant, and this protected 3-glucocerebrosidase by maintaining a reducing environment. 68

The half-life in vitro increased nearly 5-fold. Enzymes that may become inactivated by the high oxygen tension of erythrocytes should be sealed in the presence of glucose or glutathione.

Sprandel et al (1979) found that there was a much greater incorporation of Na into dialysis ghosts than into intact erythrocytes, another indication of an alteration in membrane components. This group also looked at the transport of substrates and metabolites into and out of ghosts prepared by the hypotonic dialysis method. Influx of phenylalanine and uric acid were closely similar in intact and erythrocyte ghosts. Low molecular weight organic acids were taken up at a closely similar rate in erythrocyte ghosts and normal erythrocytes (Hubbard et al, 1980). The morphology of the resealed dialysed erythrocyte ghosts was found to be affected by the preparation techniques used. When the cells were centrifuged at 100g, 60$ of the cells appeared as biconcave discs and 30$ as stomatocytes, whereas speeds of lOOOg caused more echinocytes and stomatocytes (Spraiidel et al, 1981). This group also compared the entrapment in hypotonic dilution, and dialysis ghosts and found a five times better entrapment in dialysis compared to dilution erythrocyte ghosts (Sprandel et al, 1979)* Resealed cells are smaller than normal cells. Ghosts with entrapped protein are smaller than ghosts which have been lysed and resealed without entrapment of protein. Hypotonic dilution cells are smaller than hypotonic dialysis cells. Unhaemolysed cells or cells with no entrapped protein can be removed using a Percoll or Ficoll gradient. The intact, unloaded erythrocytes band near the bottom, while the enzyme- filled erythrocytes band at the top, as has been shown for arginase entrapped ghosts (Kruse and Popjak, 1981).

The potential use of erythrocytes in the porphyrias has been suggested (Bustos et al, 1983)- Lead intoxication causes an increased concentration of 6-aminolaevulinic acid (ALA) by inhibiting ALA dehydratase. This model was used to investigate the in vitro correction of lead overloaded erythrocytes by incubation with erythrocyte ghosts containing 6-aminolaevulinic acid dehydratase. 5. ERYTHROCYTE ENTRAPPED ENZYMES IN VIVO

Erythrocyte ghosts, with entrapped enzymes or drugs, have been used either as long-circulating, slow release vesicles or as carriers that can be targetted to the erythro- phagocytic cells of the liver and spleen. In the former case, it is important that a depot of drug should provide a steady supply of the agent and not lyse and produce an overdose or be removed and produce no drug at all. In the latter case, erythrocytes as carriers for anti-cancer drugs for histiocytic medullary reticulosis, reticulum cell sarcoma or monocytic leukaemia has been suggested and in inborn errors affecting the reticuloendothelial system.

An anti-leukaemic enzyme, L-asparaginase, has been administere to monkeys entrapped in autologous dilution erythrocyte ghosts with a 20$ entrapment. The carrier had a half-life of 8 days and after this time, circulating asparaginase was 2-fold higher in animals injected with enzyme-loaded erythrocytes as compared with free enzyme. The in vivo lysis of the ghosts releasing the enzyme, caused asparagine levels to fall for 19 days (Updike et al, 1978). When dilution erythrocyte ghosts containing 3-fructofuronosidase, dextran and methotrexate Q O tw were labelled with Tc, about 90$ were found in the liver after 1 hour (Tyrrell and Ryman, 1976).

Gluteraldehyde, which renders erythrocytes resistant to turbulence-induced lysis and osmotic shock, was used to cross link dilution erythrocyte ghosts containing 3-galactosidase and 3-glucosidase and target them to the liver (De Loach et al 1977). In vitro, these erythrocytes lysed very quickly, while in vivo they were taken up by the spleen when treated with low concentrations of gluteraldehyde or by the liver when treated with high concentrations. Gluteraldehyde-treated- methotrexate-containing erythrocytes were administered to dogs and after 1 hour, 88$ of the drug had disappeared from the circulation, 50$ having appeared in the liver (De Loach and Barton, 1981). The concentration was kept at a higher level for longer in the liver and this could be useful for treating primary or metastatic hepatic malignancies. 70

Nucleated chicken erythrocytes were loaded with (3-palacto — Cl sidase and labelled with ^ Cr (Ang et alt 1977). Although the ghosts were supplemented with glucose and inosine, 4.0$ were lost in the first hour and only 5$ were present after 15 days. This was probably due to the hypotonic dilution method procedure used, in which haemoglobin and other large proteins were lost, thus reducing their in vivo viability. The in vivo survival of dialysis ghosts has been shown to be greater than for dilution ghosts in some animals (Sprandel et al, 1980). This group has made a study of rabbit carrier erythrocyte ghosts in vivo.(Sprandel et al, 1980) and found that rabbit erythrocyte ghosts at best have a half-life of 1 hour and are suitable only if uptake into the liver is required. Erythrocytes from different animals have a different, susceptibility to hypotonic haemolysis (De Loach and Barton, 1981). It was found that the viability of mouse erythrocytes depended on the inclusion of substrates for ATP synthesis, glucose, adenosine and magnesium, the half-life increasing from 4- hours to 4- days with a reduced early loss (Hubbard et al, 1980). Adenosine was a better substrate for ATP synthesis than inosine, and reduced early loss of dog carrier erythrocytes (Sprandel et al, 1980). When porcine carrier erythrocytes were prepared with added ATP, the half-life was not increased over cells prepared without added ATP, but in this case, the half-life was already 22 days with very little early loss (De Loach, 1983).

The survival of dialysis carrier erythrocytes containing sucrose in splenectomized calves was studied to see why the early removal of ghosts occurred (De Loach and Wagner, 1982). Splenectomized calf erythrocyte ghosts had a clearance rate of 50-75$ in the first hour compared to 10$-15$ of the erythrocyte ghosts from normal calves. Erythrocytes from splenectomized calves are more fragile and less resistant to the stress of dialysis encapsulation.

A babesicide (drug against Babesia infection) has been encapsulated in erythrocytes (De Loach et al, 1981). It was administered to cows. Most of the drug was in the free form after two days. 71

6. IMMUNOLOGICAL EFFECTS OF CARRIER ERYTHROCYTES IN VIVO

The immunologic effect of administering enzymes entrapped in erythrocytes has been determined by Fiddler et al (1977a). They used an animal model that had been developed earlier using a selective thermal inactivation assay of encapsulated bovine 3-glucuronidase injected into C3H/HeJ 3-glucuronidase deficient mice (Thorpe et al, 1975)* Nearly 71? of the free administered activity was removed to the liver after 2 hours, while erythrocyte entrapped activity was retained four times longer in the circulation than in the non-entrapped enzyme. When mice were sensitized intravenously with erythrocyte entrapped enzyme or empty erythrocyte ghosts (prepared by the direct dilution method) and then challenged with erythrocyte entrapped protein, there was no alteration in the time course clearance of the ghosts. When mice were sensitized subcutaneously, the time course of clearance was markedly altered. Mice sensitized with non-entrapped 3-glucuronidase subcutaneously did not have an altered tissue distribution of erythrocyte-entrapped enzyme, thus demonstrating the protection of protein from an antibody already present in the circulation. Repeated iv administration of erythrocyte- entrapped protein showed no antibody response by double diffusion or by passive haemaggiutination• 72

7. HUMAN ADMINISTRATION OF ERYTHROCYTE-ENTRAPPED ENZYMES AND CHELATING AGENTS

The first patient in which it was suggested that red cells could be loaded with enzyme for therapy was one suffering from adenosine deaminase deficiency (Polmar et al, 1976). Frozen, irradiated red blood cells from a normal donor were infused and rapidly restored immunocompetence. The exogenous adenosine deaminase facilitated the development of lymphocytosis. They suggested loading the patient's own erythrocytes to circum vent problems of hepatitis and the development of antibodies against minor blood group determinants. It has been suggested by Ihler (1979) that hypoxanthine-guanine phosphoribosyl transferase may be entrapped in erythrocytes to metabolise the plasma hypoxanthine in Lfcsch-Nyhan syndrome. The first administration of carrier erythrocytes to patients was to a Gaucher's patient (Beutter et al, 1977). In this disease glucocerebroside accumulates in phagocytic cells. 3-glucocerebrosidase, highly purified from human placenta, was entrapped in normal human erythrocytes by the dilution technique and given in five courses to the patient. It was difficult to appraise the efficacy of the treatment, but there was a decrease in liver size and increase in liver function. There was no sign of any secondary side effects. The entrapment of glucose 6-phosphate dehydrogenase in erythrocytes from humans genetically deficient in this enzyme was performed in vitro to normalise hexose monophosphate shunt activity (Morelli et al, 1979)- Erythrocyte entrapped glucose oxidase and catalase have been suggested for use in hyperglycaemia (Kitao and Hattori, 1980). Another human trial of erythrocyte ghosts involved the entrapment of desferrioxamine for iron-overloaded patients (Green et al, 1980). The red cell ghosts consistently increased urine iron excretion over free drug, especially in patients with only slightly elevated iron stores. Goldsmith et al (1979) prepared resealed ghosts containing human Factor IX and X, the serine proteases missing in Haemophilia B, but they did not try these in vivo.

Administration of bovine liver arginase in human erythrocytes to hyperarginaemic patients was suggested by Adriaenssen et al (1976) (see earlier). 8. CONCLUSION

Erythrocytes therefore are useful non-immunogenic carriers that have been used to deplete excess amino acids and other circulating metabolites in vivo, either by acting as depots to release the enzyme, as in asparaginase therapy (Updike, 1976), or by acting as circulating biodegradable metabolic reactors, where the excess metabolite can permeate the erythro cyte membrane and be degraded. This has been suggested for organic acid diseases such as primary hyperoxaluria type I where there is an accumulation of glyoxylic, oxalic and glycollic acids (Hubbard et al, 1980) . In these cases, the erythrocyte ghosts should be as much like normal cells as possible, with unaltered membrane structures and normal levels of haemoglobin, glycolytic enzymes, cofactors and energy supplies. These ghosts are best prepared by the hypotonic dialysis technique.

For the targetting of erythrocyte ghosts to liver and spleen in lysosomal storage diseases, such as Gaucher1s disease or iron-overload, erythrocyte ghosts that are rapidly removed from the circulation are required. Therefore non-viable erythrocytes, such as those produced by the hypotonic dilution technique are better suited to this purpose. When great quantitites of drug or enzyme are needed to be entrapped, the removal of haemoglobin to produce ’white’ or ’pink’ ghosts is desirable. IK

SECTION E

OBJECTIVES OF THE PROJECT

To investigate the removal of elevated amino acids from two models of inborn errors of metabolism - histidinaemia and hyperprolinaemia and to investigate the removal of iron from a rat model of haemochromatosis by:- Purification of sufficient quantities of the deficient enzymes, histidine ammonia lyase and proline oxidase, from a mammalian or bacterial source. Entrapment of histidine ammonia lyase, proline oxidase and a chelating agent, desferrioxamine, in liposomes or erythrocyte ghosts. Characterisation of the activity of the encapsulated enzymes. Adminstration of the encapsulated enzymes and chelating agents to the respective animal models. Characterisation of the fate and in vivo activity of the administered agents and carriers. CHAPTER 2

THE POSSIBLE USE OF LIPOSOMES AND ERYTHROCYTE GHOSTS FOR ENZYME THERAPY IN MURINE HISTIDINAEMIA SECTION A

INTRODUCTION

1. Histidinaemia in Man

Histidinaemia is a disorder of histidine metabolism, character ised by elevated levels of histidine in the plasma and urine of affected individuals (La Du, 1968). It is caused by the lack of histidine ammonia lyase (histidase) from the liver and skin. Histidase is the first enzyme in the major degradative pathway of histidine and catalyses the non-oxidative deamination of histidine to urocanic acid (Figure Al). Histidinaemia is inherited as an autosomal recessive trait. The exact incidence is unknown, since many cases go undetected, but it is a relatively rare metabolic disorder with an occurrence of about 1 in 20,000 births. Over half the histidinaemic patients that have been studied have a speech defect, which is felt to be related directly to the metabolic disease. It is not clear whether histidinaemia is generally associated with mental retardation because of the wide variation in the clinical features in patients. The normal levels of histidine in blood range from 0.3 to 2.6mg in 100ml. Blood histidine concen trations above 3mg per 100ml are considered histidinaemic. Following an oral loading dose of histidine, the highest concentrations in histidinaemic subjects have-reached 4-Omg per 100ml. The urine of patients shows a positive ferric chloride test (reacting with the imidazole pyruvic acid), and so they were originally mistaken for phenylketonurics. The imidazole derivatives of histidine, imidazole pyruvate, imidazole lactate and imidazole acetate, formed by transamination and deamination of histidine are also elevated in plasma and urine. In three cases where serotonin levels have been determined, the value in platelets were about half normal (Auerbach, 1962). It was suggested that elevated levels of histidine interfere with the biosynthesis of serotonin. Inhibition studies on 5-hydroxy L tryptophan carboxylyase have shown that imidazole derivatives of histidine significantly affect this enzyme (Small, 1970). H H ^ ^ H c c c cooh (Pipic.n Sypif'esisj C-C-C-COOH HC=C-C-C-COOH o-c— ‘• - - - H NH,

l 3 3 M t Ihyl Hijndinf HISTIOASE

Carnosme 3 Anserine Hitl idin i (ImidaioltalaninO uJ

M H Hiilidini HC=C-i-d-HM, Transaminase ! I HH

VH HC= =C-C-C-COOHI 1 M H 0 \ N. NH <3 /midaiei»ace'aid«P»d« <3 Imidatolsprepienic acid Formiminoglutomic acid (FIGLU) Fermyliteglwia'"'"*

Im idaiaiiactlic acid Imidatalsloclic acid

im id aiatiace iic acid ribeiidt

Figure A1 The metabolism of histidine. The vertical line separates the metabolites of histidine which are dependent on the presence of histidase from rhose which are not. (Auerbach et al, 1962). 78

Histidinaemia can be diagnosed during the neonatal period and screening programs include blood tests for elevated histidine. Treatment by dietary intake restriction leads to a fall in blood histidine levels and restoration of serotonin levels in platelets.

The maternal effect of histidine on the foetus has not been established in humans. In one case, an asymptomatic histidinaemic mother had four normal sons (Armstrong, 1975). In another study (Lyon et al, 1974-) a family of nine with 7 normal sibs and 2 histidinaemic was discovered. One of the histidinaemic females had 5 children, which were normal but had a slightly lower IQ than their parents. The possibility was considered that this might have been due to a maternal histidinaemic effect.

2. The Histidinaemic Mutant Mouse

A mouse model for histidinaemia was isolated from a wild mouse stock trapped in Peru (Kacser et al, 1973). During a routine screening of mice, high histidine levels were found in blood and urine. Histidine levels were at least ten fold higher than in normal mice. Measurement of the enzyme, histidase, showed that these animals had between 0 and 0.05ymol min~^g""^ liver histidase activity at 30°C, whereas normal mice had between 0.08 and 0.37umol min -1 g -1 liver. The condition was found to be inherited as an autosomal recessive and the affected allele was designated his.

(i) Biochemical Characteristics

Histidinaemic mice have high levels of histidine in urine, liver, plasma, brain and skin, and high concentrations of imidazole derivatives in urine (Table Al from Bulfield and Kacser, 1974-) •

A comparison of human and mouse data shows that the enzyme activity in the liver of both is low or absent. The histi dinaemic to normal ratio of plasma histidine is 28.7 in mouse and 5 to 25 in man. Both are autosomal recessives. It has been concluded that the mouse mutant is a homologue of the human disorder. 79

Table A1 Tissue levels of histidase, histidine and its imidazole derivatives

Liver and brain metabolites are expressed as pmol/g wet tissue. Plasma and urine metabolites are pmol/ml. All determinations were by column chromatography and ninhydrin or Pauly reaction. Histidase activity is expressed as pmol urocanate min ^g""^ wet weight at 30°C.

Tissue Histidinaemic (H) Normal (N) R =

LIVER Histidase 0.013 0.280 0.046 Histidine 20.9 0.880 23.7 ( Ac Histidine ( + lm pyruvate 0.111 0.0082 13.5 lm lactate 0.102 0.0069 14.8 lm acetate 0.017 <0.0044 > 3.7

URINE Histidine 0.86 0.04 21.5 ( Ac Histidine 0.11 ( + lm pyruvate 3.19 29.0 lm lactate 1.21 0.07 17.3 lm acetate 1.29 0.05 25.8

PLASMA Histidine 3.30 0.115 28.7 ( Ac Histidine 0.016 <0.0008 > ( + lm pyruvate 20.0 lm lactate 0. 012 <0.001 >12.0 lm acetate 0.008 <0.001 > 8.0

BRAIN Histidine 2.16 0.169 12.8 ( Ac Histidine ( + lm pyruvate 0.004-4- <0.0006 lm lactate 0.0204 <0.0007 > 7.3 lm acetate <0.004 <0.0006 >29.1 80

A comparison of the properties of histidase in the normal and histidinaemic mutant mouse has been carried out (Wright et al, 1982). They found that the residual 5# liver histidase activity in the mutant had markedly different properties from the majority of the normal liver histidase enzyme.

The enzymes from the normal and mutant mouse were partially purified by salt precipitation. It was found that all the mutant activity and 5$ of the normal histidase activity were precipitated by 4-OmM sodium chloride. The majority of the normal activity was precipitated at 200mM sodium chloride. There were no dissociable inhibitors of histidase present in the mutant extract but absent in the normal. The normal enzyme on native polyacrylamide gel electrophoresis showed a strong ultraviolet absorbing peak with an R^=0.24- for urocanate, but the mutant enzyme did not show this peak. The ability of crude liver extract from the mutant mice to inhibit the immunoprecipitation of normal histidase was examined. There was no cross-reacting material (CRM). This may mean that the mutant enzyme was either so structurally altered that it no longer reacted with the antibody to normal histidase or that there was no histidase present at all in the mutant mouse.

The mutational loss of the liver histidase also means a loss of the skin enzyme. Urocanate levels are lower in skin of histidinaemic mice. There is a developmental change in skin histidase. At birth in normal mice, there is low activity which increases up to 3 days. The activity then falls until there is very little 15 days after birth. It has been suggested (Wright et al, 1982) that there are two isoenzymes in skin, one of low and one of high activity. It is the major isoenzyme that loses its activity after 15 days, leaving only a low residual activity. In the mutant mouse, only the low activity isoenzyme is expressed. The presence of two iso- enzymic forms of histidase in the liver could explain the 5% residual activity in the mutant, which may be a low activity isoenzyme, with the major isoenzyme being absent. Alternatively the residual activity could be explained by a completely different enzyme having a low affinity for histidine. 81

(ii) Genetic Characteristics

Matings between histidinaemic mice produce exclusively histidinaemic offspring. Matings between known heterozygotes give a 3:1 ratio of normals to histidinaemics at the metabolite level. The his allele is recessive. When liver histidase activity is measured, heterozygous crosses show 1:2:1 segregation at the enzyme level, with heterozygotes having approximately half the enzyme activity compared with normal homozyqotes.

Heterozygous mice have normal histidine levels. This means that the flux through the pathway to glutamate is not at a ’’threshold” with 50$ of normal histidase activity. This is explained by the nature of multienzyme systems in vivo (Kacser and Burns, 1973). In his/his animals, where there is less than 5l of the normal activity, flux through the histidase pathway is still about 4-0$ of normal (Kacser et al, 1979a). The relationship between liver histidase activity and liver histidine content is shown in Figure A2. - Histidine concen trations are ’’buffered” down to histidase activities of approximately 0.1 units and only begin to significantly rise at values below this. The relationship between enzyme concentration and the phenotype of the individual has been studied in great detail (Kacser and Burns, 1980). This is briefly described by the fact that the movement of a metabolite through a pathway is dependent on the ’’sensitivity coefficient” of the enzyme in the pathway. The coefficient is defined as the fractional change in flux divided by the fractional change in enzyme causing the change in the flux.

Sensitivity coefficient = z = dF v dE F E E = enzyme concentration or activity F = flux through pathway

Therefore when the coefficient is small, considerable movements in enzyme levels result in only marginal changes in flux or metabolite levels. Most enzymes in vivo are complex multi enzyme systems. The vast majority have very low coefficients. Therefore not only do the histidine levels in histidinaemia cause a large mass action effect on the flux, but also the change in flux will be negligible down to very low enzyme eainhpbtenlvrhsiie andhistidinehistidaseliverbetween in Relationship itdnei mc. (Kacser etal, 1973). mice.histidinaemic Histidase activity (pinoles urocanate/min/g liver) 0.3 Figure A2 83 activities. The fact that 5$ histidase activity results in nearly 4.0$ of the normal flux means that enzyme replacement can be a viable proposition. It would only be necessary to replace a small percentage of the normal liver activity to return the flux of histidine to glutamate to normal.

(iii) Endogenous Teratogenesis in Maternal Histidinaemia in Mice

A behavioural abnormality was found in the histidinaemic mouse strain (Wallace, 1970). Genetic crosses and histidine supplementation experiments concluded that this was caused by high maternal histidine levels during the second week of pregnancy (Kacser et al, 1979b). Balance defective offspring were only detected when histidinaemic (his/his) females were mated to normal (+/+), heterozygous (his/+) or histidinaemic (his/his) males. Therefore it was the genotype of the females that was important, not the genotype of the offspring. Non-histidinaemic offspring could be balance defective.

Balance defective offspring had malformations of the inner ear. They were deaf, and circled with head tilting. Histo logical investigation showed abnormal otoliths, utriculus, sacculus, semi-circular canals and cochlea. Offspring in the same litter or even individual ears in one animal could be mildly or severely affected. There was never 100$ balance defective offspring in one litter and the number of affected could vary from litter to litter.

The elevated histidine or some metabolic product produces a teratogenic effect on the offspring in utero. The blood histidine level in heterozygous female mice has been raised by dietary means (Kacser et al, 1979b). When pregnant hetero zygotes, which would otherwise give birth to normal offspring, were fed on a histidine supplemented diet during the second week of gestation, 1/ balance defective offspring were obtained from 130 in 21 litters compared to none on unsupplemented diets (Kacser et al, 1977). When histidinaemic (his/his) mothers were supplemented with histidine the proportion of balance defective offspring remained the same. Therefore there is a ’saturation level1 over which increase in histidine in the maternal blood will not increase the number of affected 84 offspring. When histidinaemic mice were deprived of histidine in the diet, the number of affected offspring fell from 26$ to 3$.

When selection for balance defective offspring was relaxed, the penetrance of the defect fell from 80$ to 10$. Inter crossing a high penetrance stock with a low penetrance stock (80$ offspring affected compared to 10$), in order to look at the susceptibility to the teratogen, has shown that another genetic system may be involved (Burns, 1983).

3. The Physical and Chemical Characteristics of Histidase

(i) Mammalian Histidase

In mammals, the enzyme is found in liver and skin (Zannoni and La Du, 1963). The function in the skin is probably to produce urocanic acid for protection against ultraviolet light (Zenisek et al, 1955). Histidase from a number of sources has been studied, including rat, mouse, human and guinea pig. Each has slightly different properties in terms of molecular weight, subunit composition and Km value for histidine.

Histidase becomes present in the liver either just before or at the time of birth. Female rats have two to five times the male level of enzyme activity (Makoff and Baldridge, 1964). Several workers have reported that the developmental course is governed by several hormones and nutritional factors acting at strategic stages, including oestrogen, glucocorticoid and glucagon acting via cyclic AMP (Feigelson, 1968; Schirmer and Harper, 1970; Feigelson, 1973). Other hormones such as androgen and thyroxine suppress histidase activity (Feigelson, 1971; Neufeld et al,1971). Antibod ies against homogeneously purified histidase have demonstrated that elevations in hepatic histidase activity by administration in vivo of oestradiol, cortisol, glucagon or dibutyryl cAMP have been due to increases in histidase biosynthetic rates (Lamartiniere and Feigelson, 1977).

Skin histidase shows a markedly different developmental course. As the hepatic enzyme activity rises, there is a simultaneous decline in epidermal activity, also due to alteration in histidase synthetic rates (Bhargava and Feigelson, 1976). 85

V/hen the mechanism of synthesis of urocanic acid was investigated in guinea pig epidermis, it was found that the majority of the histidase activity was confined to the dead cells of the strateum corneum (Scott, 1981).

Histidase was first purified from guinea pig liver by Kato et al (1955). It was found that the enzyme activity was enhanced by cadmium and glutathione. The pH optimum was found to be at 8.7 with a K of 1.2mM for histidine. Purification of rat m liver histidase gave a molecular weight of 226,000, a K for histidine of 2.0mM and a pH optimum of 8.8-9*2 (Cornell and Villee, 1968) . The cofactor requirement of glutathione was shown, the activity being twice as high in its presence. Manganese and zinc activated the enzyme.

Histidase from female rat liver purified by Okamura et al (1974) to homogeneity showed a molecular weight of 190,000 and a sedimentation coefficient ($20 ^.) of 11.6. The iso electric point was pH 5*2 and the pH optimum between 8.8 and 9.0 with a K for histidine of 1.2mM. Parachloromercuri- m benzoate and EDTA inhibited enzyme activity.

The rat liver enzyme has also been purified by Brand and Harper (1976). They found a molecular weight of 200,000, a pH optimum of 8.5 and a Kffl for histidine of 0.5mM at pH 9.0. The quarternary structure of rat liver histidase has been suggested to be either a hexamer (Brand and Harper, 1975) or a trimer (Lamartiniere and Feigelson, 1975)• These conflicting results indicate that the liver enzyme may be subject to cleavage of a labile peptide bond.

The pH dependence of the enzyme reflects the decrease in catalytic activity due to the protonation of an ionisable group having pK 7.7 and deprotonation of an ionisable group having pK 9.5 (Brand and Harper, 1976). The mammalian enzyme has a dehydroalanine at its active site (Givot and Abeles, 1970). The enzyme phenylalanine ammonia lyase from Rhodotonila glutinis has a similar catalytic centre.

(ii) Bacterial Histidase

The majority of studies have been performed on the bacterial form of histidase. Histidase has been induced from a number 86 of bacteria, including Pseudomonas fluorescens (Frankfater and Fridovich, 1970), Achromobacter liquidum (Shibatani et al, 197$), Bacillus subtilis (Magasanik et al,1970 ) .

The enzyme has been extensively purified from Pseudomonas sp ATCC 11299b. The first purification was established by Tabor and Mehler (1955)- They found that activity depended on the presence of glutathione and that inhibition occurred with EDTA, cyanide and glycine. The pH optimum was found to be 9*5. The mechanism of action was investigated (Peterkofsky, 1962). The purified enzyme catalyzed two partial reactions, a hydrogen and a urocanic acid exchange into histidine, in addition to the irreversible deamination reaction. An amino-enzyme inter mediate was formed, which could either reversibly form histidine or irreversibly be degraded to ammonia and free enzyme. Kinetic studies have shown that there is no significant release of ammonia before urocanate.

+ His URO NH A A A

E > E.His E URO (NH,+) * e (n h 4+) * E amino- enzyme

When it was discovered that nitromethane inactivated the enzyme (Givot, 1969)» it was postulated that a dehydroalanine residue was present at the active site. This was also shown by Wickner (1969)•

There are two active sites per molecule and the turnover number at least 170 sec’Vmolar active site. The K m and the Vmax Pseudomonas enzyme varies according to the buffer employed. The increases above pH 8 and there are pKs at pH 7.9 and pH 10.2. The histidase from Pseudomonas is composed of 4- subunits (Rechler, 1969; Soutar and Hassall, 1969). A major band at molecular weight 55,000 and a minor one at 36,000 was found (Klee, 1970a). There seemed to be a labile chemical linkage. Rechler (1969) found the molecular weight of the whole enzyme to be 210,000 with 15-19 moles of half cystine per mole of enzyme. At pH 7 to 8 and at low histidine concen trations, the heat purified enzyme is inhibited by urocanate (K^ = 0.13mM). Complex kinetics occurred when the enzyme assay was conducted in the presence of EDTA, tyrosine and cysteine. 87

Klee (1970a) found that the Pseudomonas enzyme is held together by disulphide cross links into a mixture of polymeric species. The purification procedure was designed to isolate all the polymeric species. These had a series of sedimentation coefficients of 9 . 3S, 13.6S, 16.4S and 19S. Treatment of this enzyme with cysteine, dithiothreitol or mercaptoethanol converted it to the monomeric 9-3S species. The monomer had a molecular weight of 215>000 with four subunits of 55,000 molecular weight. The Pseudomonas enzyme has also been shown to be dependent on the addition of a divalent cation for optimal activity (Mn^+ , Fe^+, Zn^+ or Cd2+) (Klee, 1972). The reduced form of the enzyme is only slightly stimulated by Mn 2+ and the oxidised form of* the enzyme is not sensitive to EDTA. (Frankfater and Fridovich, 1970). Incubation of the reduced enzyme with histidine, protected against EDTA inhbition. The EDTA inhibition is not due to the removal of the metal ion but to a more specific effect of EDTA on the structure of the reduced enzyme. There is a relationship between the metal ion activation and the titratable -SH groups. The oxidised enzyme which contains no reactive -SH groups, binds metal very loosely. Metals bind much tighter when the enzyme is reduced.

Klee and Gladner (1972) isolated a cysteine-peptide at the active site. There were k moles of enzyme per mole of tetra- meric enzyme. Therefore there are four similar or identical polypeptide chains per monomer. Hassall and Soutar (1974-) showed that during polymerisation, the disulphide bonds are formed between identical regions of the enzyme, and that the cysteine residue involved is also the one required in the reduced state for full activity of the enzyme.

The amino acid composition of Pseudomonas histidase has been determined by Rokosu (1979) and by Soutar (1971).

4.. A Synopsis of This Study

This chapter investigates the possibility of histidase replacement in histidinaemic mice using liposomes and erythrocyte ghosts.

Firstly, a source of enzyme was developed to provide sufficient quantities of enzyme for replacement therapy. The mammalian 88

enzyme from rat liver was purified to near homogeneity. When it was demonstrated that the rat would not provide enough enzyme for therapy, a bacterial source was used. Histidase was induced from Pseudomonas ATCC 11,299b, using histidine as its sole carbon and nitrogen source. After purification, the bacterial enzyme was used for all encapsulation and in vivo studies. The properties of the purified bacterial enzyme were investigated.

Secondly, an investigation into suitable types of liposome preparation for maximum entrapment of histidase and for desirable in vivo characteristics was conducted. Liposomes, prepared in various ways and of different lipid compositions and sizes, were used to entrap the enzyme. The clearance kinetics of each of the liposome preparations from blood into liver was followed by entrapment of a non-biodegradable radio active polymer, radioiodinated polyvinyl pyrrolidone (^^1 PVP). After the in vivo characteristics of the carrier had been determined, histidase, entrapped in the most suitable liposome preparation, was administered to histidinaemic mice. Delivery of the enzyme to liver was monitored and the plasma levels of histidine measured. Because enzyme activity itself could not be followed in vivo, the fate of the enzyme was determined by radiolabelling it.

Thirdly, erythrocyte ghosts prepared by the hypotonic dialysis technique were used to entrap histidase. The activity of the entrapped enzyme was determined. Erythrocyte ghosts were made to resemble normal erythrocytes, to obtain a long circulatory- half-life, by supplementation of the ghosts with ATP (Hubbard et al, 1981) and glutathione. The characteristics of the erythrocyte ghosts in vivo were determined by encapsulation of 125 I PVP and following clearance from the blood. Histidase was entrapped in erythrocyte ghosts and administered to histidin aemic mice. The effect on blood histidine levels was measured.

This is the first attempt at enzyme replacement therapy in histidinaemia using liposomes and erythrocyte ghosts. In a previous study, histidase has been microencapsulated in poly (piperazine terephthalamide) capsules by in situ interfacial polymerisation (Wood et al, 1979). Although these showed some promise in reducing blood histidine levels after intraperitoneal administration to histidinaemic mice, only initial experiments were done and the preparation appeared to be toxic (Bacon and Kacser, unpublished work). 89

SECTION B

MATERIALS AND METHODS

MATERIALS

1. Animals

Histidinaemic mice were from a stock maintained in the Department of Genetics, University of Edinburgh. They were isolated from a partially inbred Peru strain by Wallace, Department of Genetics, University of Cambridge.

Mice used for other experiments were A2G strain. Rats used for liver histidase isolation were female WAG strain. These were provided by the Animal House, Charing Cross Hospital Medical School.

2. Bacteria

The bacteria used for histidase isolation were obtained from the American Type Culture Collection, Rockville, Maryland. The strain used was Pseudomonas ATGC 11,299b, and was received as a freeze-dried culture.

3. Reagents

Sepharose 2B and 6B, Sephadex G50 and G200, DEAE Sephadex and QAE Sephadex A50 were obtained from Pharmacia, Uppsala, Sweden. Radiochemicals listed below were from Amersham International:- Histidine

Iodine-125 PVP (125I) Injection All chemicals were analytical grade. Phenylalanine ammonia lyase from Rhodotorula glutinis was from P. L. Biochemicals, Inc. Chemicals from BDH/Hopkin and Williams included:-

Aluminium oxide, CAMARG M.F.C. (Brockman activity 1, alkaline 90 for column chromatography) Acrylamide NN1 methylenebisacrylamide NNN 'N1 totramethylethylenediamine Ammonium sulphate (Aristar) Potassium dihydrogen phosphate Dipotassium hydrogen phosphate Folin and Ciocalteu's phenol reagent. All other chemicals used were obtained from Sigma.

Lipid (phosphatidic acid) was obtained from Lipid Products. Bacteriological media were obtained from Oxoid. Silica gel for TLC was obtained from Merck. Carbowax 20m (polyethylene glycol) from Raymond A. Lamb. Chloroform, methanol, petroleum ether, acetic acid, acetone, and all other inorganic acids were obtained from May and Baker. Heparin was from the Pharmacy, Charing Cross Hospital.

4.. Apparatus

Fermentation of bacteria was carried out in a LH Engineering Type LHE 1/1000 laboratory fermentor. Sonication was performed on an MSE soniprep 150. Homogenisation was in a Braun Melsungen homogeniser. The spectrophotometer used was a Gilford 24-0 single beam.

Gamma counting was performed on a Packard Model 4.5-26 y counter. Gel electrophoresis was carried out in Shandon apparatus. Centrifuges used included MSE 18, superspeed 65 and Mistral 6L.

METHODS

1. Assays

(i) Histidase Assay

The non-oxidative deamination of histidine by histidase produces urocanate. The molar extinction coefficient of urocanate at 277nm is 18,800 M ~^cm ^ between pH 7.2 and 11. Histidine has very little absorption at this wavelength. Tie activity of histidase was measured by the increase in absorbance at 277nm (Tabor and Mehler, 1955). 91

II II

x c\ N NH, N NH T I NH, I I IIC c h 2c h n h 2cooh HC — CCH = CHCOOH

One unit of histidase catalyses the deamination of one nanomole of histidine per minute at 30°C and pH 9.0.

The assay mixture consisted of 1ml of the following (final concentrations)

0.2M Tris HC1 pH 9.0 3mM Glutathione pH 9.0 3mM Magnesium Chloride pH 9.0 Enzyme (2 units to 500 units).

After incubation at 30°C the reaction was started by addition of histidine, pH 9.0. Except where otherwise stated, the final concentration of histidine in the assay was 3mM. The rate of reaction was measured on a Gilford spectrophotometer.

Assays of histidine during purification were performed at pH 9.0 to inhibit the activity of urocanase, the enzyme that degrades urocanate to imidazolone prop ionic acid.

Assays of histidase after entrapment in liposomes and erythro cyte ghosts were performed at physiological conditions in phosphate buffered saline at pH 7.4- and at 37°C. This was in order to reflect the activity found in vivo. Histidase was preincubated with glutathione and magnesium ions prior to entrapment and so these activators were omitted from the assays. Glycerol was always removed from the stored enzyme before use by dialysis.

Assays on fractions of histidase obtained after column chromatography were performed by incubating a portion of the fraction with histidine, followed by precipitation of the protein with 10% trichloroacetic acid after ten minutes. The samples were then centrifuged at 3000 x g for 10 minutes and read at 277nm.

Other modifications of the assay included replacement of glutathione with dithiothreitol or mercaptoethyfcamine and replacement of magnesium chloride by cadmium chloride 92 for maximum activity.

All histidase units presented during its purification are measured at pH 9.0* All histidase units presented in this work associated with in vitro entrapment in liposomes and erythrocyte ghosts and in vivo administration are measured at pH 7.4-.

(ii) Phenylalanine Ammonia Lyase Assay

Phenylalanine ammonia lyase was assayed by the method of Hodgkins (1971). The formation of cinnamic acid was monitored by the appearance of optical absorbance at 290nm. One enzyme unit was defined as that amount of protein catalyzing the appearance of lumole of cinnamate per minute at 30°C. The assay was carried out in a volume of 3ml.

2.9ml 0.1M Tris HC1 pH 8.5 0.1ml 25mM phenylalanine pH 8.5. This was left for five minutes at 30°C. The enzyme was added and the increase in optical density at 290nm measured. The enzyme had a specific activity of 1.0 unit, /mg and an activity of 15 units/ml. The molar extinction coefficient for cinnamic acid is 10,000M '*‘cm ^ at 30°C, pH 8.5 (Zucker, 1965).

(iii) Pauly Assay for Histidine and its Imidazole Derivatives

The Pauly assay (Pauly, 1904-) was used to detect histidine, urocanate and its imidazole derivatives.

The assay consisted of coupling the imidazole compounds with diazotized sulphanilic acid at alkaline pH.

The Pauly reagent was made up as follows:-

A: one volume of sulphanilic acid, consisting of 9g sulphanilic acid in 90ml concentrated hydrochloric acid and 900ml of water. B: one volume of sodium nitrite 5% in water. C: two volumes of anhydrous sodium carbonate, 10$ in water.

Solution A was mixed with solution B in a 1 to 1 volume to volume ratio and left to stand for exactly five minutes at 93 room temperaturc. It was then cooled in ice to below 20°C for 15 minutes. Two volumes of solution C were added. This reagent was used immediately.

3.6ml of Pauly reagent was added to 0.4. ml of sample. The solution was left for fifteen minutes exactly. Histidine gave a yellow-orange colour. Other imidazoles yielded red and brown colours (Tabor, 1957). The optical density was measured at 520nm.

Automated Pauly analysis was performed on blood samples after separation of the imidazole compounds by ion-exchange chromatography on Dowex (see (14-) Amino Acid Analysis). The eluent was reacted with the Pauly reagents and passed through a spectrophotometer with a 4-4-Onm filter. The peaks were recorded on a chart recorder.

(iv) Protein Assay (Lowry method)

The reagents for this assay consisted of:

A: 2% (w/v) sodium carbonate in 0.1M NaOH. B: 0.5$ cupric sulphate pentahydrate in 1% (w/v) sodium potassium tartrate. G: (fresh) 50ml A mixed with 1.0ml B.

D: Folin and Ciocalteu1 s phenol reagent (BDH) diluted 1:1 with water and stored at 4-°C.

The protein solution in 1.0ml was mixed with 5.0ml C and allowed to stand for exactly ten minutes. Then 0.5ml of D was added with thorough mixing. The blue colouration was allowed to develop for 30 minutes to 2 hours and the optical density at 750nm read on a Corning Spectrophotometer 256. A standard curve was constructed using bovine serum albumin (Lowry et al, 1951).

(v). Protein Assay (Coomassie method)

A more rapid protein assay was required for some experiments (Bradford, 1976). lOOmg Coomassie Brilliant Blue G250 was dissolved in 50ml 95% ethanol. To this solution, 100ml 85% (w/v) phosphoric acid was added, and the volume was diluted to 1 litre. Final concentrations in the reagent were 0.01% 94.

(w/v) Coomassie Brilliant Blue G250, 4-.7$ (w/v) ethanol and 8.5$ (w/v) phorphoric acid. Protein solution containing 10 to lOOpg protein in a volume up to 0.1ml was placed in tubes and 5ml of the protein reagent added. The contents were mixed and the absorbance at 595nm was measured after 2 minutes and before 1 hour in 3ml cuvettes. A blank consisted of the appropriate buffer and $ml of protein reagent.

(vi) Phosphorus Assay

A number of methods have been developed for assaying organic phosphorus (Pelitou, 1978; Fiske and Suborrow, 1925; Morrison, 196/). In this study phosphorus,for liposome measurement and in the assay of glucose 6-phosphatase,was measured by the ANSA reagent of Fiske and Suborrow (1925).

The reagents for this assay consisted of:- A: Ammonium molybdate 2.5$ in 5N (w/w) stored at room temperature. B: l-amino-2-naphthol-4--suphonic acid (ANSA) reagent. 0.5g ANSA was added to 195ml sodium bisulphite 4-0$ (w/w) and the mixture swirled. Then 5ml 20$ sodium bisulphite was added and the mixture shaken. Further 5ml aliquots were added until a clear solution had been obtained. This was stored at 0-4-°C.

To 4--3ml of deproteinized sample, digested by heating in perchloric acid, 0.5ml of reagent A was added followed by 0.2ml reagent B. The optical density was read at 680nm. A phosphorus standard curve was performed between 0 and 80yg using potassium dihydrogen phosphate.

(vii) Cholesterol Assay

Cholesterol was assayed in liposomes by the method of Rudel and Morris (1973). A: Ferric chloride 84-Omg in glacial acetic acid made up to 1 litre. B: Cone, sulphuric acid.

The test sample of liposomes was diluted with chloroform to 95

between 0 and 60ug in 0.1ml. To 0.3ml sample was added 3ml of reagent A and mixed. Then 2ml of reagent B was added and the tube mixed. It was left in the dark for 4-5 minutes. The optical absorbance at 560nm was determined. A standard curve using a cholesterol solution was constructed between 0 and 60ug/0.1ml. Free and liposomal cholesterol gave similar calibration curves.

(viii) Subcellular Fractionation Assays

After subcellular fractionation, markers for the various fractions consisted of 3-glucosidase for the lysosomes, lactate dehydrogenase for the cytoplasm, succinate dehydro genase for the mitochondria, and glucose 6-phosphatase for the microsomal fractions. B-Glucosidase was assayed by the method described by Borcoach. et al (1961). Briefly, a synthetic substrate p-nitrophenyl 3-glucoside was dissolved at 100pg/ml in 0.2M citrate buffer pH 4.8. 0.2ml of substrate was added to 1ml enzyme (O-lOOpg) in 0.2M citrate. After 1 hour at 37°C, the reaction was. stopped by addition of 2.0ml 0.2M borate buffer pH 9.8. The tubes were centrifuged and read at 400nm. Blanks consisted of adding borate buffer before the tissue. Lactate Dehydrogenase was measured by the decrease in optical density at 340nm due to the utilisation of NADH in the presence of pyruvate. Sodium pyruvate (25mg/10ml) was made up in phosphate buffer pH 7.0. 0.1ml pyruvate was added to 50|_il 12mM NADH and made up to 3ml with phosphate buffer. The reaction was started by addition of 20ul sample and the optical density followed at 340nm (Bergmeyer, 1963). Succinate Dehydrogenase was measured using a synthetic hydrogen acceptor, INT, 2-(p-iodophenyl)-3-(p-nitrophenyl)-51-phenyltetrazolium chloride (Nachlas et al, I960). Using 0.2M sodium succinate pH 7.4 as substrate, the sample was incubated at 37°C for 20 minutes in the presence of the electron acceptor before stopping the reaction by the addition of 10% TCA. After centrifugation, the optical density was read at 490nm. Glucose 6-Phosphatase was estimated by the method of Hers (1964) which is based on the release of inorganic phosphate from glucose 6-phosphate. To correct for the hydrolysis of the 96 substrate by unspecific phosphatases, it was necessary to run a control experiment in which the specific glucose 6- phosphatase had been inactivated by incubation at pH 5.0 and deduct this value from that obtained at pH 6.5. The assay was therefore performed at pH 6.5 in citrate buffer with glucose 6-phosphate as substrate at 30°C for five minutes and again at pH 5.0 in acetate buffer with the same substrate. The inorganic phosphate released was determined by the ANSA method (see (vi)).

2. Purification of Phosphatidylcholine from Egg Yolk

This method is a modification by Dawson (1958) of the method of Lea et al (1955) and Hanahan (1957).

I Crude Extract

Six egg yolks and 250ml acetone were placed in a 1 litre beaker and thoroughly mixed with a glass rod. The solution was then filtered through Whatman filter paper using a 2 litre Buchner flask under vacuum. The precipitate was resuspended in a further 250ml volume of acetone and again, the supernatant was removed by filtering. This was repeated until all the coloured impurities were removed.

The lipid was extracted from the precipitate by addition of a 1:1 chloroform:methanol solution (250ml). The supernatant n was removed from the precipitate using a Buchner funnel. This was repeated twice. The supernatant was retained each time. The supernatants were combined and extraction on the precipitate repeated until the supernatant became clear. The supernatants were then evaporated at 4-0°C to a damp mass. 50ml of petroleum ether (60°-80°) was added to dissolve the phospholipid. Acetone (200ml) was added to reprecipitate the phospholipid, the free fatty acids remaining in the petroleum ether. After being left in the deep-freeze overnight, the supernatant was removed. The precipitate was dissolved in chloroform:methanol 1:1 and stored at -20°C under nitrogen. This is crude egg phospholipid. 97

II Alumina Purification

Alumina (320g) was activated by drying in a hot oven overnight. It was then washed three times in chloroform:methanol 1:1 and packed into a 4-»2x50cm column.

The crude egg phospholipid was placed on the column in a volume of about 20ml. The first 200ml eluted from the column was discarded and the next 4.00ml collected. The alumina column retained all coloured impurities and any amino lipids such as phosphatidylethanolamine. The eluent whs rotary evaporated and redissolved in 20ml chloroform:methanol 68:32.

III Silicic Acid Purification

Silicic acid (220g) was washed three times in chloroform: methanol 68:32 and packed into a 4-»5x50cm column. A filter paper was placed on top of the column and the sample loaded. The first 800ml that eluted were discarded and contained coloured impurities. The next five litres through the column were collected. The eluent was then rotary evaporated to dryness and redissolved in chloroform to a concentration of lg/lOOml. The silicic acid column retains lysoleuthin.

Dittmer reagent (Dittmer and Lester, 1964-) was used to detect phospholipids from the column. The column fractions were spotted on filter paper and dried. On adding the reagent, phosphate esters were visualised as blue spots.

3. Thin Layer Chromatography

Thin layer plates were made by shaking 30g silica gel G with 60ml distilled water for 90 seconds. The gel was spread on clear glass 20x20cm plates at a thickness of 250urn. The plates were activated at 180°C for 3 hours.

Phosphatidylcholine, prepared as in (3) and other phospholipids were run on TLC plates to investigate their purity. The solvent used was 69 parts chloroform to 27 parts methanol to 1.5 parts ammonia (0.88) to 2.1 parts water. The spots were developed in iodine vapour. The phospholipids give yellow spots with values of the following:- 98

Lysopho sphatidylcholine 0.21 Phosphatidylcholine 0.39 S phingomyelin 0.29 Phosphatidylethanolamine 0.37 Cerebroside 0.78 Cardiolipin 0.92 Phosphatidic acid 0.10

4-• Preparation of Liposomes

(i) Multilamellar Vesicles MLV

MLV were prepared by the method of Gregoriadis and Ryman (l972a) Lipid in chloroform was added to a round-bottomed flask and the chloroform removed by rotary evaporation under low pressure. An aqueous solution, containing the material to be entrapped was added and the flask shaken to remove the lipid from the glass. The liposomes were left to swell for 2 to 4- hours. Free material (not entrapped) was removed by centrifugation at 100,000xg for 30 minutes. The liposome pellet was washed and resuspended in 5mM phosphate buffer pH 7.4-.

The usual composition of liposomes used were phosphatidyl choline and cholesterol, with the addition of phosphatidic acid for anionic liposomes and stearylamine for cationic liposomes. (The molar ratios of lipid used for liposomes are given with the results of each experiment).

Latency of enzyme entrapped in multilamellar liposomes was investigated by incubation with 0.2$ Triton X100. The disrupted liposomes were passed through a Sepharose 6B column to remove the detergent when histidase was entrapped. The column (1.5x20cm) was equilibrated with 3mM phosphate buffer pH 7.4-* Fractions of 1ml were collected and the eluent, after concentration with carbowax, was assayed for histidase activity.

Multilamellar vesicles were visualised under the electron microscope (Figure Bl).

(ii) Sonicated Liposomes

Sonicated liposomes were prepared as for multilamellar liposomes (above). After allowing the lipid to swell for 99

Figure. B1 Electronmicrograph of multilamellar vesicles. Magnification x 87,000. 1cm = 115nm. 100

2 to 4- hours, the liposomes were sonicated on ice with an MSE Soniprep 150 ultrasonic disintegrator. This consisted of 23KHz titanium probe (9.5mm) and liposomes were sonicated for a total of five minutes in thirty second bursts (to allow cooling) at an amplitude of 14- microns peak to peak, in a volume of less than 4-ml.

Free enzyme was separated from entrapped by gel filtration through Sepharose 6B. The column was equilibrated with 5mM phosphate buffer pH 7.4-* The liposomes were eluted in the void volume, while free enzyme was retained until near the end of the included volume. The liposome peak was concentrated using carbowax.

Latency of entrapped enzyme was demonstrated by incubation of the concentrated liposomes after gel chromatography with 0.2% Triton X100. The detergent was removed by gel filtration through Sepharose 6B. The lipid-detergent eluted in the void volume, while the liberated enzyme eluted in the same fractions as the free enzyme in previous passage through the column.

These liposomes were not unilamellar when visualised under the electron microscope. They were a heterogeneous population of vesicles, but smaller than multilamellar vesicles (Figure B2)

(iii) Reverse Phase Vesicles REV

Reverse phase vesicles were prepared by the method developed by Szoka and Papahadajopoulos (1978). These encapsulate a high percentage of the initial aqueous phase and have a high aqueous space to lipid ratio. The lipids (phosphatidylcholine, cholesterol and phosphatidic acid or sphingomyelin and cholesterol) were dissolved first in diethylether (see Results for amounts used) . The aqueous phase containing the enzyme to be entrapped was added to this phospholipid/solvent mixture. The ratio of aqueous phase to organic solvent was 1:3. The preparation was sonicated for a total of three minutes until a homogeneous emulsion was formed. This was performed on ice with 30 second bursts of sonication and 30 seconds cooling between. The organic solvent was removed by rotary evaporation under low vacuum, followed by a high vacuum. This was to prevent frothing. The inverse micelles formed by sonication 101

Figure B2 Electronmicrograph of sonicated liposomes. Magnification x 87,000. 1cm = 115nm. 102

collapsed into a gel state with a bilayer structure. Additional buffer (0.5ml) was added to form a homogeneous suspension of liposomes.

Free enzyme was separated from entrapped by gel chromatography using Sepharose 2B liposomes with entrapped enzyme eluted in the void volume. Liposomes were concentrated with carbowax.

Latency of the entrapped enzyme was demonstrated by incubation with 0.2$ Triton X100, and subsequent passage through Sepharose 2B to remove the detergent.

When sphingomyelin (bovine brain) was used in reverse phase liposomes, rotary evaporation and sonication were carried out above its transition temperature (32°C).

Electron micrographs of reverse phase liposomes showed a heterogeneous population which were uni- or oligo-lamellar (Figure B3). They ranged in size from 200-10,OOOnm.

(iv) Double Emulsion Technique (Battelle)

Liposomes were prepared as described by Fr^kjcer et_al (1982) . This was from a recent patent (Battelle Memorial Institute, 1979)* The lipids distearoylphosphatidylcholine (DSPC) and cholesterol (2:1) were dispersed in dibutyl etherrcyclo hexane 1:1. An aqueous solution containing histidase, 30-4-Opl/ml organic solvent, was added, and reverse micelles were formed by brief sonication (6 x 30 second bursts). The reverse micelles, containing aqueous marker in the internal space of the micelles were dispersed in lOmM tris HC1 buffer, pH 7.4- in a volume ratio of 1:10. The organic solvent was removed by rotary evaporation. The liposome suspension was passed through a Sepharose 6B column equilibrated with lOmM tris HC1 buffer, pH 7.4-. The liposomes eluted from the column in the void volume were concentrated using carbowax. The latency of entrapped enzyme was determined by incubation of the liposomes with 0.2$ Triton X100 and removal of the detergent by gel filtration.

All procedures with DSPC-containing liposomes were carried out at 55°C, which is above the transition temperature of this lipid, but below the inactivation temperature of histidase.

These liposomes are small reverse phase vesicles with a large 103

Figure B3 Electronmicrograph of reverse phase liposomes. Magnification x 87,000. 1cm = 115nm. 104

Figure B4 Electronmicrograph of liposomes made by the Batelle method. Magnification x 87,000. lcm = 115nm. 105 entrapment capacity (Figure Bl) . They are about 80-200nm in diameter.

5. Efflux of Histidine from Liposomes

Efflux of histidine from liposomes was demonstrated by the method of Klein et al (1971). Phosphatidylcholine- cholesterol (10:8) and sphingomyelin-cholesterol (3:2) liposomes were made as prev iously described by the reverse phase evaporation procedure. Histidine (3mM) containing lOyCi 1/^C histidine in a volume of 1ml was added as the aqueous phase. After liposome formation, free histidine was removed by gel chromatography on Sephadex G50 (1.5x20cm). Liposomes, eluted in the void volume (2ml), were placed immediately into a dialysis bag and dialysed against 10ml 5mM phosphate buffer pH 7.5 in a shaking water bath at 37°C. lOOpl aliquots of the dialysis fluid were taken at intervals and the radioactivity present determined by scintillation counting.

In another experiment, plasma was added to the liposomes in the dialysis bag at a concentration of 33%. This plasma had been obtained from cardiac puncture of mice, erythrocytes and buffy coat removed and stored at 1°C overnight.

6. Preparation of Erythrocyte Ghosts

Hypotonic dialysis erythrocyte ghosts were used to entrap histidase. The characteristics of the preparation of these ghosts had been extensively investigated (Hubbard et al, 1980b). They were prepared by the method of Hubbard et al (1981) which included the supplementation of erythrocyte ghosts with glucose, adenosine and magnesium ions to ensure an ATP level and in vivo viability analogous to that of normal intact erythrocytes. Glutathione was also added to ensure full reduction of histidase and other erythrocyte proteins.

Mice were lightly anaesthetised with ether and bled by cardiac puncture using a heparinised syringe. Blood was placed in a heparinised tube and centrifuged at 1°C at l,000xg for ten minutes. The plasma and buffy coat were removed by aspiration. The erythrocytes were washed twice in isotonic phosphate buffered saline pH 7.1. 106

Electron microscopy demonstrated that murine erythrocytes appeared less shrunken and more as stomatocytes or spherocytes when prepared at an osmolality of 200m0sm. Great attention was paid to the concentration of isotonic phosphate buffered saline pH 7.4- and the above osmolality was used for all preparations of murine erythrocyte ghosts. Human erythrocyte ghosts had an optimum osmolality of 250 to 300 milliosmoles. This probably reflects the greater osmotic fragility of murine erythrocyte ghosts.

Washed erythrocytes (2-5ml) were placed in a dialysis bag (■^/32M) with the material to be entrapped at a 70% haematocrit. The bag was attached to a glass rod and placed in a 150ml bottle containing 5mM phosphate buffer pH 7.4- with 4-mM adenosine, 5mM glucose, 4-mM magnesium chloride and 3mM glutathione. It was turned on end for 90 minutes at 4-°C (Dale et al, 1977; Hubbard et al, 1981). This resulted in haemolysis of the erythrocytes. The dialysis bag was removed and placed in a bottle containing isotonic phosphate buffered saline pH 7.4- with adenosine, glucose, Mg 2+ and glutathione as above and rotated at 37°C for one hour. This allowed resealing and reannealing of the erythrocyte membrane. The erythrocyte ghosts were collected by centrifugation at 500xg for fifteen minutes. They were washed twice with isotonic buffer at low centrifugation speed (Hubbard et al, 1981). Erythrocyte ghosts were kept on ice and always administered to mice within a couple of hours of formation.

Erythrocyte ghosts from mice were slightly spherocytic in shape (Figure B5). Normal mouse erythrocytes are shown for comparison (Figure B6).

7. Estimation of Histidase Entrapped in Erythrocyte Ghosts

Histidase was entrapped in erythrocyte ghosts as in (6). Estimation of the total entrapped enzyme activity was performed by lysing the cells with distilled water and sonication for five seconds. Aliquots of the lysed cells were incubated with 3mM histidine pH 7.4- for various periods of time and the reaction stopped by the precipitation of protein with 10% TCA. After cooling to 4°C for twenty minutes, the samples were centrifuged, the supernatants removed and the optical absorbance at 277nm read. 107

Figure B5 Electronmicrograph of murine erythrocyte ghosts. 1cm = 5.5um« 108 .109

A sorption of histidasc to the outer membrane of erythrocyte ghosts was tested for by addition of the enzyme to preformed empty ghosts.

The production of urocanate by intact erythrocyte ghosts containing histidase was measured by incubation of the cells with various concentrations of isotonic histidine pH 7.k . At certain time points, the cells were washed once with ice cold phosphate buffered saline, pH 7.7- (isotonic) and the supernatant removed for optical absorbance measurement at 277nm. The pelleted cells were lysed with distilled water and by sonication and the protein precipitated with 10% TCA. The supernatant, representing the intracellular medium, was removed and read at 277nm. Urocanate in the incubation and intracellular media was also quantitated by automated Pauly analysis.

8. Influx of 1^'C histidine into Erythrocyte Ghosts

Mouse erythrocyte ghosts were made by the preceding method. Empty ghosts (no histidase entrapped) were suspended at 50% haematocrit in phosphate buffered saline pH 7.4 (isotonic) containing various concentrations of histidine and O.lyCi ■^C histidine. The samples were incubated in a shaking water bath at 37°C. At time intervals, 0.2ml aliquots were removed and added to 1ml ice cold phosphate buffered saline. The sample was spun at 15»000g for 20 seconds in an Eppendorf Microcentrifuge. The supernatant was removed and the radio activity determined in a scintillation counter. The pellet was resuspended in 1ml distilled water at %-°C and the cells lysed by sonication for 10 seconds. The protein was precipitated by addition of 200pl 10% TCA and kept on ice for 20 minutes. The precipitate was pelleted by centrifugation and the super natant removed for scintillation counting.

Results were calculated in terms of a distribution ratio, where:-

PPM found in intracellular medium Distribution Ratio PPM found in extracellular medium

Efflux of 1/^C histidine from erythrocyte ghosts was determined by preincubation with isotonic histidine pH 7.4- containing O.lpCi histidine for three hours. The ghosts were washed 110

and resuspended at a 50% haematocrit in isotonic phosphate buffered saline (pH 7./*.). At time intervals, 0.2ml aliquots were removed and added to 1ml ice cold phosphate buffered saline pH 7.A. The cells were spun, and the supernatant and intracellular activity was determined as before.

9. Iodination of Histidase

Iodination of histidase was performed by the chloramine T method of Hunter and Greenwood (1962). Histidase (2-5ug) was placed in a small glass vial in 20ul 50mM phosphate buffer pH 7.5. Sodium 125-iodide (lOOmCi/ml) was added (20ql), followed immediately by 20yl 5mg/ml chloramine T (sodium toluene-p-sulphorichloroamide). This was stirred for 30 seconds before addition of lOOul 1.2mg/ml sodium metabisulphite, followed by 820ql 2.0mg/ml potassium iodide. The reaction mixture was dialysed overnight against 5 litres of 50mM phosphate buffer, pH 7.5, to remove most of the free radio active iodine.

The solution was applied to a Sephadex G50 (0.9x100cm) equilibrated with 50mM phosphate buffer pH 7.5. Fractions of 1ml were collected and lOpl of each were counted for radio activity .

The 125 I directly substitutes onto tyrosyl residues in the histidase protein. It was eluted within the included volume of the column, in the same position as non-radioactive histidase. Free iodine eluted in the total column volume.

10. Polyacrylamide Disc Gel Electrophoresis

Non-denaturing disc gel electrophoresis was performed on purified histidase using the method of Davis (1964.) • The small pore gel only was used for electrophoretic separation. The reagents used were:- A: Acrylamide 22.2g NN' Methylenebisacrylamide 0.6g Water to 100ml Stored at 0°c.

B: Ammonium persulphate (fresh) 0.15g in 10ml water. .1.11

C: Sodium dihydrogen phosphate (l.OM) 28.5ml Disodium hydrogen phosphate (0.5M) 14.5.0ml V/ater to 100ml.

D: NNN'N’ Tetramethylethylenediamine (TEMED). E: Reservoir buffer, pH 8.3 Trisma base 6.0g Glycine 28.8g Water to 1 litre.

All solutions were degassed before use. Polyacrylamide gels were made to 7.5% acrylamide. This consisted of 6.75ml solution A, 1.0ml solution B, 4-.0ml solution C and 20yl solution D made up to 20ml with water.

The samples were prepared by addition of 25ug of protein to an equal volume of 10% glycerol, 0.05% bromophenol blue, ImM mercaptoethanol made up in solution E. The sample (50ul) was applied to the top of the polyacrylamide gel (5mmx7cm). The upper and lower reservoirs were filled with a ten times dilution of solution E. Each tube was run at 2mA at 4-°C until the bromophenol blue dye front reached 1cm from the bottom of the tube. The gels were syringed from the tube and stained overnight in 0.25% Coomassie Brilliant Blue G250 in methanol: acetic acidrwater 5:1:5. The gels were destained in 7.5% acetic acid and 5% methanol for two days.

Gels were scanned at 595nm using the linear transporter attachment with the Gilford 24.0 spectrophotometer.

The peaks obtained are expressed in terms of values where:

r _ distance moved by protein distance moved by bromophenol blue dye front

11. Sub-Cellular Fractionation of Mouse Liver

Sub-cellular fractionation was performed by the method of March and Gourlay (1971). Mouse livers were excised, weighed and homogenised in 0.25M sucrose pH 7.4- at 0°C. (EDTA was omitted as this interferes with histidase activity). A 33% homogenate was normally prepared, except when histidase activity in histidinaemic mice was investigated, when a 50% homogenate was used. Livers were homogenised by five passes of a Teflon pestle after mincing the tissue with scissors. 112

Differential centrifugation was used to separate the organelles rather than density-gradient centrifugation which would result in a more distinct separation of the fractions. This was not considered necessary for the purposes of this investigation. This resulted in good separation of nuclear, cytoplasmic and microsomal fractions but lysosomes and mitochondria were incompletely resolved. The scheme of centrifugation forces used is shown in Figure B7.

The isolation of each fraction was demonstrated by assaying for enzymes found specifically in one subcellular organelle. Lysosomes were detected by the enzyme 3-glucosidase, mitochondria by succinate dehydrogenase, cytoplasm by lactate dehydrogenase and the microsomal fraction by glucose 6-phosphatase. Details of the assays for each of these enzymes are given in Section B(l).

12. Growth of Pseudomonas

All procedures were carried out using sterile technique.

The bacterium, Pseudomonas sp. ATCC 11299b, was received as a freeze-dried culture. It was grown overnight in nutrient broth at 30°C.

The bacteria were streaked onto sterile agar plates, with medium composition of 0.15$ dipotassium hydrogen phosphate, 0.05$ potassium dihydrogen phosphate, 0.02$ magnesium sulphate, 0.2$ histidine monohydrochloride and 0.1% oxoid Yeast Extract. The plates were incubated at 30°C overnight.

Single colonies from the agar plates were transferred to agar slopes containing the histidine medium. These were incubated at 30°C overnight and then stored at 0-4°C.

When bacteria were required to inoculate growth flasks, a slope was incubated overnight at 30°C to accelerate growth. The bacteria were subcultured onto fresh histidine medium every four months.

The bacterium is a sub-species of Pseudomonas fluorescens. It is an aerobic, flagellated Gram-negative rod. It is a common member of soil and water microflora and it is non- pathogenic. It was first studied by Tabor and Hayaishi (1952). 113

Figure B7

Centrifugation procedure for subcellular fractionation of mouse liver.

Liver homogenate

10,200xg.min vK Nuclear sediment \k supernatant

33 ,OOOxg.min

i r Heavy mitochondrial \y sediment supernatant

490,OOOxg.min

\X supernatant

V Lysosomal sediment/ 3,000,OOOxg.min Light mitochondrial fraction. These can be separated by density gradient centrifugation Final supernatant (cytoplasmic fraction)

r Micro somal sediment 114

Histidase was induced by using histidine as its principal carbon and nitrogen source.

Growth of bacteria in shake flasks for inoculation of a 10 litre laboratory fermentor was carried out in a rotary shaker at high speed and 30°C in the histidine medium described above. Growth of the bacteria in the fermentor was in the same medium with an aeration of 18 litre air/min and vigorous stirring (770 rev/min) at 30°C. The bacteria were harvested in late logarithmic or early stationary phase.

13. Animal Experiments

Animals were lightly anaesthetised with diethyl ether before administration of liposomes or erythrocyte ghosts via the lateral tail vein. Blood samples at time intervals from histidinaemic mice were obtained by cutting l/8n from the tail and collecting 20-60yl in a heparinised capillary tube. Blood for erythrocyte ghosts preparation was obtained by exposure of the heart after heavy anaesthetic followed by cardiac puncture.

Erythrocyte ghosts were prepared from the pooled blood of several A2G mice for administration to other A2G mice. Since this is an inbred strain, the genotype of each mouse is identical. Erythrocyte ghosts administered to histidinaemic mice were prepared from other closely related histidinaemic mice. Because the colony is only partially inbred, genotypes may not be identical. In these experiments it was assumed that the blood groups in all the histidinaemic mice were similar.

14.. Amino Acid Analysis

Amino acid analysis of blood samples was carried out on an extensively modified Locarte analyser (Burns et al, 1965). Amino acids were eluted using a salt and pH gradient from pH 2.8 to pH 5.65. Ninhydrin derivatised samples were read at 570nm and 4-4-Onm (proline).

Blood samples from histidinaemic mice for amino acid analysis were prepared by lysis of a 20ul sample with 200pl water. The protein was precipitated with 50pl 30% 5 sulphosalicyclic acid. All amino acids were determined relative to a 0.05umole standard of L-a-amino-3-guanidino propionic acid (AGPA). 15. Scintillation Counting

A toluene-based scintillation fluid was used consisting of 0.25g dimethyl POPOP (l,4.-bis Q 2( 4--methyl-5-phenyl-oxazolyl)] benzene) and 15g of butyl PBD (2-(4.,-tert-butylphenyl)-5- ( /<."-biphenylyl)-1,3» 4-oxadiazole ) in 2.5 litres of scintillation grade toluene. Samples from lOOql to 1ml were counted in 10ml of the above fluid.

16. Preparation of Column Resins

Sephadex was prepared by swelling in excess of the appropriate buffer overnight and degassing or by using a boiling water bath for three hours.

Sepharose was prepared by degassing in the appropriate buffer.

DEAE Sephadex and QAE Sephadex were prepared by swelling overnight in buffer of the correct pH. The resins were then washed extensively using a Biichner flask. The conductivity was checked to ensure the resin was the same as the equilibrating buffer.

All columns were poured . and used at 4-°C.

17. Expression of Experimental Results

The entrapment of material inside liposomes is expressed as the percentage of the initial material added in their preparation Entrapment of histidase in liposomes was determined by incubation with detergent followed by removal of detergent by gel filtration The activity of these disrupted liposomes was both the activity entrapped inside the lipid bilayer and that associated with the outside of the bilayer. Association of the enzyme with the outer layer was determined by incubation of the enzyme with preswollen liposomes, followed by disruption with detergent. The true entrapment within the bilayer was then determined by subtracting the activity found associated with preswoller liposomes from the activity found when enzyme was added to dried lipid. This is the "latent” enzyme activity.

Entrapment in erythrocyte ghosts was determined after compensation for the percentage of cells recovered after resealing. For example, if 50% of the initial volume of erythrocytes were recovered after resealing and if 10$ of the 116

initial added material was found entrapped, then the overall % entrapment is 20%,

% material found in ghosts x % cell recovery = % material added

Results are expressed as a mean of two to four values with standard errors where applicable.

In the calculation of the distribution of substances in vivo the following values were used:-

Mouse blood volumes whole blood 77.8ml/lcg body weight plasma 4-8.8ml/kg body weight erythrocyte 29.0ml/kg body weight

Organ plasma volumes kidney 250yl/g tissue liver 350pl/g tissue lung 4-OOyl/g tissue spleen 190jjl/g tissue 117

SECTION C

HISTIDASE PURIFICATION

Although the mammalian model of histidinaemia is murine, purification of histidase from mouse would not necessarily be advantageous for enzyme replacement therapy. This enzyme is confined to liver and skin and so histidase from any source appearing in the circulation may be antigenic. Species or strain differences in histidase could result in isoenzyme forms that are more favourable for encapsulation in liposomes or erythrocyte ghosts and that are more stable, have better characteristics when administered in vivo and that are easily isolated. Purification of histidase was initially attempted from rat liver. After demonstration that this source did not have a high enough yield for isolating the quantities needed for enzyme replacement, histidase was purified from a bacterial source.

Histidase has been purified from few mammalian sources including mouse, guinea pig and rat, but the characteristics of each have not been compared. The bacterial forms of histidase from Bacillus and Pseudomonas have been purified extensively. Comparison of the bacterial and mammalian forms have shown that each has a dehydroalanine in the active site and depends on reduced thiol groups for activity. Although it has been established that the bacterial form of the enzyme is a tetramer, the enzyme from rat liver is either a hexamer or a trimer. The bacterial enzyme has a wider pH optimum and this may result in a greater activity at physiological pH.

1. Purification of Mammalian Histidase

Histidase was purified from fresh female rat livers according to the method of Okamura et al (1974-), with modifications according to the method of Brand and Harper (1976) and Cornell and Villee (1968).

All procedures were carried out at 0-4-°C, It was found that freezing and thawing of liver tissue inactivated the enzyme and so all preparations were carried out using fresh liver. 118

Female animals have higher levels of histidase than male animals, probably due bo oestrogen inducibility of histidase biosynthetic rates (Lamartinere and Feigelson, 1977). Female WAG rats were used.

Step I . Crude Extract

Rats were killed by cervical dislocation and the livers excised immediately and weighed (148.2g). Pooled livers were homogenised in 3 volumes of 50mM potassium phosphate buffer pH 7.4- with a Braun-Melsungen homogeniser at a speed of 1,000 rpm. Cells were disrupted by three passes of the homogeniser.

The homogenate was centrifuged at 15,000xg for 20 minutes. The supernatant was removed and the pellet washed with a few millilitres of buffer. The resuspended pellet was centrifuged again, the supernatant removed and added to the supernatant from the first centrifugation. This was the crude extract. A portion was retained for enzyme and protein assay.

Step II. Heat Treatment

The supernatant (390ml) from Step I was placed in a water bath at 50°C and after equilibration, it was left at this temperature for 2 minutes. It was immediately plunged into an ice bath. It was centrifuged at 15,000xg for twenty minutes to remove precipitated protein. The pellet was resuspended in 50mM phosphate buffer pH 7.4 before being recentrifuged. The supernatants were combined and the pellet was discarded.

Step III. Ammonium Sulphate Fractionation

The supernatant from Step II (375ml) was brought to 33% saturation of ammonium sulphate by addition of the solid with continuous stirring. The amount of ammonium sulphate needed to reach this saturation was calculated using the nonogram from Data for Biochemical Research (Dawson et alf 19&9)• The solution was readjusted to pH 7.4 by addition of a few drops of a 10% ammonium hydroxide solution. Because of the high salt concentration, the pH was finely adjusted by measuring a dilution of a small aliquot of the enzyme solution. The solution was left for one hour on ice with stirring before 119

centrifugation at 15,000xg for 20 minutes. A small amount of precipitate was formed. This was the 33$ pellet. The

supernatant was brought to 55% saturation ammonium sulphate by addition of the solid and the pH was adjusted to pH 7. A by addition of ammonium hydroxide. After one hour, the precipitate was collected by centrifugation at 15»000xg.

This formed the 33%-55% pellet.

Both the 33% pellet and the 33$-55$ pellet were resuspended in a minimum amount of 20mM potassium phosphate buffer pH 7.4-. The resuspended pellets and the final supernatant were assayed for histidase activity (Table Cl).

Step IV. DEAE Sephadex Ion-Exchange Chromatography

A DEAE Sephadex A50 anion exchange column (4-xlOcm) was prepared and equilibrated with 20mM potassium phosphate buffer pH 7.4-.

The 3o-55% ammonium sulphate pellet from Step III was dialysed overnight against 20mM potassium phosphate buffer pH 7.4-, with four changes of dialysing buffer. The conductivity of the solution was measured to ensure that it was below that of the ion-exchange column eluent. The enzyme was washed onto the column with one litre of 20mM potassium phosphate pH 7.4« The eluent was assayed to ensure that no enzyme was washed through. When the optical density at 280nm of the eluent had decreased to nearly zero, a stepwise salt gradient was passed through the column. One litre of 20mM potassium phosphate lOOmM sodium chloride pH 7.4- was washed through the column, followed by one litre of 20mM potassium phosphate 200mM sodium chloride pH 7.4« Fractions of 4--8ml were collected. The flow rate was lml/min. Each fraction was assayed for protein and histidase activity. Over 50$ of the applied histidase activity was eluted in one peak after washing with 200mM NaCl. The elution profile of histidase from the ion exchange column is given in Figure Cl. Fractions 26 to 35 were pooled.

Step V . Second Ammonium Sulphate Fractionation

Solid ammonium sulphate was added slowly with stirring to the pooled fractions from Step IV to give a 4-0$ saturation. After 120

Table Cl

Step III ammonium sulphate fractionation of rat liver histidase. The enzyme was brought to 33% saturation with ammonium sulphate and the precipitated protein removed

(33% pellet). The supernatant was brought to 35% ammonium

sulphate saturation. The precipitated fraction (33% - 55% pellet) and the supernatant (55% supernatant) were assayed.

Fraction % Total Histidase Activity

33% pellet 6% 33% - 55% pellet 60% 55% supernatant 23%

% recovery 89% Histidase activity (units/fraction) EESpae o-xhne chromatographyion-exchange of ratDEAE Sephadex oue a /.8ml. wasvolume ie itds. nye waseluted Enzyme 200mMwith NaCl liverhistidase. n 0M hsht bfe p 7/ se et. Fraction (see pH 7./ buffer text). phosphate 20mMin iue Cl Figure Fraction numberFraction 121 0 - - 0 rti (opticalProtein Histidase (units/ activity density 280nm) fraction)

Optical density 280nm 122 the pH had been adjusted, this was stirred on ice for one hour. It was centrifuged at 15,000xg for twenty minutes. The pellet was resuspended in a small volume of 20mM potassium phosphate buffer pH 7. A and recentrifuged. The supernatant was added to the first supernatant and the pellet was discarded. The supernatant was brought to 60% ammonium sulphate saturation, the pH corrected and left for one hour. The material that precipitated after centrifugation at 15,000xg was resuspended in 3ml of 20mM potassium phosphate buffer pH 7,A- This was dialysed against the same buffer, with four changes, overnight.

Step VI. QAE Sephadex Ion-Exchange Chromatography

A column (4-xlOcm) of QAE Sephadex A50 was prepared, equilibrated with 20mM potassium phosphate pH 7.4-. The material from Step V was washed onto the column with the same buffer. When the optical density of the eluent at 280nm was near zero, a linear gradient was applied to the column between 0 and 500mM sodium chloride. The linear gradient was made using a three-way peristaltic pump with 250ml of 20mM potassium phosphate buffer pH 7 . A and 250ml of 20mM potassium phosphate buffer with 500mM sodium chloride pH 7.4-.

Figure C2 shows the elution profile from the QAE ion-exchange column. Histidase was eluted approaching the end of the salt gradient. Fractions of 4-.2ml were collected.

Step VII. Concentration and Storage

The enzyme was concentrated using an Amicon ultrafiltration unit under nitrogen pressure with a PM30 Diaflo membrane. This membrane retains molecules above 30,000 molecular weight. It was discovered that freeze-drying or freezing and thawing the enzyme caused considerable loss of activity. The enzyme was always stored at 0 to 4-°C with the addition of 30% glycerol and 3mM glutathione. It was found that the enzyme was not stable, the activity slowly declining to zero after six weeks. Other workers have found that storage in certain buffers causes inactivation (Brand and Harper, 1976). A typical purific ation scheme for rat liver histidase is shown in Table 2. 2. Polyacrylamide Gel Electrophoresis of Rat Liver Histidase

Analytical polyacrylamide gel electrophoresis, by the method of Davis (1964.) was performed at 0-4-°C on the histidase from Histidase Activity (Units/Fraction) A ehdxinecag chromatographyofion-exchangerat QAE liverSephadex hsht p 7./. pHphosphate chloride sodium in 20mM $00mM 0 andpotassiumbetween elutedwithalinear wasgradient Enzyme histidase. iue C2Figure 13 - 123 - Histidase (units/ activity

Optical Density (280nm) Table C2

A typical purification scheme for rat liver histidase is summarised below. This purification was from 14-8.2g fresh female rat liver. Histidase units represent nanomoles of urocanate produced per minute at 30°C, pH 9.0. The specific activity is given as units enzyme activity per mg of protein.

Purification Step Volume Protein Units Specific 1 Purification (ml) (mg) Activity Activity Yield Fold (units/mg protein)

Crude Extract 390 12,4-80 43,440 3.48 100 1

Heat Treatment 37$ 1,100 23,098 16.49 53 4.7 Ammonium Sulphate 12 250 13,557 54 31 15.5 33-55? DEAE Sephadex $7 83.2 6,000 73 14 20 Ammonium sulphate 3 30 3,370 112 8 32.2 10-60$

QAE Sephadex 2.2 4.4- 1,362 309 3.2 88.8 125 the final stage of purification. After staining with 0.25% Coornassic blue, the gel was scanned at 595nm. Figure C3 shows the profile of the peaks. It can be seen that the enzyme is nearly homogeneous with the histidase peak at O.4.4. relative to the dye front.

An SDS polyacrylamide gel on which reduced histidase was run is shown in Figure C4-. This dissociated the enzyme into subunits, which were determined to have a major band of 50-60,000 daltons relative to a protein standard marker gel. Smaller molecular weight proteins can be seen, which indicated either small molecular weight subunits or fragments.

3. Characterisation of Liver Histidase Activity

A study of the activity of rat liver histidase after purification showed that thiol reagents were required for maximum activity, while detergents were inhibitory (Table C3)- Glutathione proved essential for maximum activity after storage of the enzyme. The effect of detergent was investigated in order to assess whether latent histidase activity after entrapment in liposomes could be determined by disruption of the liposomes with Triton X100. These results showed that detergent inhibited histidase in concentrations needed for liposome disruption. Consequently, in Section D, detergent was removed from all enzyme samples before assay by gel filtration. Other methods that have been used to remove Triton X100 have been by Bio-Beads SM2 (Holloway, 1973)•

The optimum pH of purified rat liver histidase was found to be pH 9 (Figure C5). The assay was performed in Tris HCI buffer and the pH decreased by the dropwise addition of 0.1M HCI. It can be seen that less than 50% of the maximum activity is expressed at physiological pH 7.4-* (The bacterial enzyme had a similar pH curve).

A study on crude mouse liver homogenate was performed in order to investigate product inhibition. Crude mouse liver was homogenised in 0.24-M sucrose and centrifuged at 100,000x:g for 30 minutes. The supernatant was then assayed at pH 9.0 for histidase in the presence of increasing concentrations of 1.26

Figure C3

optical density 595nm

Polyacrylamide gel electrophoresis of native purified rat liver histidase (7.5% gel). The gel was stained with 0.25% Coomassie blue and scanned at 595nm. The main peak is at 0.4-4- 127

Figure C4- SDS polyacrylamide gel of reduced rat liver histidase. It shows a major subunit band at 50-60,000 daltons relative to a protein standard marker gel. Smaller molecular weight material is present. 128

Table C3

The effect of thiols and detergent on the activity of purified rat liver histidase. Results are expressed as a percentage of the maximum rate found with any of the conditions shown below. The assay was performed at pH 9.0 with 3niM histidine in a volume of 1ml.

Addition to Assay % Maximum

None 74- 3mM Glutathione 100 3mM Dithiothreitol 87

0.2% Triton X100 9 % Maximum Activity iue C5 Figure fet f sa H n h activity of purified on the assaypH of rat Effect ie hsiae Te ufr pH adjusted bufferwas The by histidase.liver dropwise addition of 0.1M HC1. 0.1M of addition dropwise ----- 1 I------— . 75 . 85 . 9-5 9.0 8.5 8.0 7.5 7.0 ------129 1 ------pH 1 ------1 ------1 130 urocanate. The effect of urocanate on mouse liver histidase activity is given in Figure C6, which is a Lineweaver-Burk plot of the inverse of the velocity against the inverse of the substrate concentration in the presence of increasing urocanate concentrations. It can be seen that urocanate is a competitive inhibitor of mouse liver histidase. It appears that liver histidase activity decreases at high substrate concentrations.

The properties of mammalian liver histidase and comparisons of the rat and mouse liver enzymes wer e not the province of this research and so investigations were not continued in this direction. If time had permitted, these very interesting findings would have been developed further.

U • Growth of Pseudomonas ATCC 11,299b

A strain of Pseudomonas ATCC 11,299b was used for induction of the enzyme histidase (Section B(12)). The bacteria were grown on histidine as t h e i i sole carbon and nitrogen source.

The ■. grow th of bacteria in a 10 litre laboratory fermentor was initiated with a 2% inoculum of bacteria grown in shake flasks. Bacterial growth had entered stationary phase after 15 hours. This enabled fermentation to take place overnight (Figure C7) . Histidase production was determined during the exponential growth phase by removing aliquots of bacteria and lysing the cells by sonication. The production of histidase was closely associated with the growth of the bacteria (Figure C7).

After growth the cells were separated from the medium by centrifugation at 2,000 rpm in a 6 x 1 litre centrifuge for 2 hours. Bacteria were stored at 0*~4-°C because histidase activity was irreversibly lost on freezing and thawing.

Wet weight of bacteria was normally 20g per 10 litres.

5 . Purification of Bacterial Histidase

The histidase from bacteria was purified by a modification of the procedure of Klee (1970a). The procedure was carried out in a shortened form to provide a large but not homogeneous histidase activity units/g liver obe eirclpo f mouseofliverplothistidase reciprocal Double o te sa. sa i TrisHC1pH 9.0 inat 30°C Assay tothe assay. ocnrto. feto dig 50yMaddingof urocanate Effect concentration. againsthistidine (units/gliver)activity iue C6Figure 131 Optical Density HOnm 1.2 1.6. 2.0 0.1 0.8. iue 7 rwho suooa i aof in litre10 of Growth clofPseudomonashistidase Production ‘fermentor. 07Figure ece atr 5 hours. 15 after reached olwd the offollowed growth thebacteria, □ -- □ Histidase activityHistidase atra growthBacterial (opticaldensity (units/50ml) 14-Onm) Using aUsing □ 2% nclm stationary inoculum, phase r -60 -50 .40 .30 • 10 20 70

Histidase Activity (Units/50ml) wa s ely 133 supply of histidase for replacement therapy.

All procedures were carried out at 0-4.°C.

Step I . Crude Extract

The bacteria were suspended in three volumes of 50mM potassium phosphate buffer pH 7.4- containing ImM 2-mercaptoethanol and PMSF (50mg dissolved in 1ml isoproylalcohol per litre of buffer) added just prior to use.

The cells were disrupted using an ice cold French pressure cell at 11,000 to 12,000 psi. Cells were in 50ml batches. The cells were passed through two times. Bacterial cell wall debris and non-.disrupted cells were removed by centri fugation at 10,000xg for 20 minutes. The pellet was resuspended in 50mM potassium phosphate buffer pH 7-4- and recentrifuged. The supernatant was added to the supernatant from the first centrifugation and the pellet was discarded. A small aliquot of the supernatant was retained for protein and enzyme assay.

Step II. Ammonium Sulphate Precipitation

The supernatant from Step I was brought to 30$ ammonium sulphate saturation by addition of a volume of a stock 90$ ammonium sulphate solution pH 7.0. The solution was stirred for one hour and then centrifuged at 20,000xg for 10 minutes. The pellet was resuspended in a small quantity of 50mM potassium phosphate buffer pH 7.4- and recentrifuged. The supernatants from the first and second centrifugations were combined. The pellet, after assaying, was discarded. The supernatant was brought to 60$ ammonium sulphate saturation and, after one hour, the precipitate was collected by centrifugation. Over 50$ of the activity was found to be precipitated between 30$ and 60$ ammonium sulphate fractionation. The 60$ supernatant was found to contain nearly 20$ of the activity.

Step III. DEAE Sephadex Ion-Exchange Chromatography

(DEAE cellulose or DEAE sepharose were occasionally used as alternative ion exchange resins). 131

DEAE Sephadex A50, swollen in 20mM potassium phosphate buffer pH 7.1, was poured into a column 10x5cm. The column was equilibrated with 20mM phosphate buffer with 60mM sodium chloride pH 7.1.

The 30% to 60% pellet from Step III was diluted with the above buffer to a point where the conductivity was the same as that of the column eluate. The enzyme was washed through the column at a speed of 5ml per minute. The eluate was assayed to ensure that no enzyme was washed through. A stepwise gradient of sodium chloride was used to elute the enzyme activity. One litre of 20mM potassium phosphate buffer lOOmM sodium chloride pH 7.1 was passed through the column followed by one litre 20mM potassium phosphate buffer 200mM sodium chloride pH 7.1. Enzyme activity was eluted when the salt concentration was raised to 300mM. The profile of this is given in Figure C8. Further protein could be removed from the ion-exchange column with 500mM sodium chloride. Fractions of 5ml were collected. Those forming the first peak of activity from the 300mM salt wash were pooled.

Step IV. Sephadex .G2Q0. Gel Filtration

The pooled peak from Step IV was concentrated using an Amicon ultrafiltration unit with a PM30 membrane. The concentrated protein solution (5-10ml) was applied to a Sephadex G200 column (2.5x60cm) equilibrated with 20mM potassium phosphate pH 7.1 60mM sodium chloride. Fractions of 2ml were collected, the majority of enzyme eluting between fractions 25 and 31 (Figure C9) . The pooled peak fraction was concentrated by Amicon high pressure ultrafiltration.

Step V . Storage

The bacterial enzyme was stored at 0-l°C after it was found that freezing and thawing caused considerable loss of activity. The enzyme was stored in 30% glycerol, in the presence of 3mM glutathione, under nitrogen.

Table Cl shows a summary of a typical purification procedure, starting with about 80g wet weight of bacteria. Histidase Activity (Units/Fraction) suooa itds. itds a eluted Histidasewas300mM with histidase.Pseudomonas al n 0M oasu hsht buffer pH 7.4-*phosphate 20mM potassium in NaCl EESpae o-xhne chromatography ofpurifiedion-exchange Sephadex DEAE iue C8Figure

Optical Density • 280nm ehdxG0 gl itain ofPseudomonas filtration histidase. gelG200 Sephadex h ezm a eue ih 20mM potassium phosphatewith eluted enzyme Thewas H ., 60mM 7.1,pHNaCl. Protein (Optical Density 280nm) iue C9Figure 136 -

Histidase Activity (Units/Fraction) Table C4

Summary of the purification procedure of Pseudomonas histidase. Initial bacterial weight w about 80g (wet weight). Histidase units represent nanomoles of urocanate produced per minu at 30°C, pH 9.0. Specific activity = units per mg protein.

Purification Step Protein Histidase Specific Yield Purifica (mg) Activity Activity % Fold Units in Total

I Crude Extract 3654 782,000 214 100 1

II Ammonium Sulphate 891 410,000 460 52 2.2 35$-60$

III DEAE Sephadex 201 200,000 991 26 4.0 ion-exchange

IV Sephadex G200 46.6 98,000 2104 12.5 9.8 138

6. Polyacrylamide Gel Electrophoresis of Bacterial Histidase

An analytical polyacrylamide gel, by the method of Davis (196^) was performed on the purified enzyme. The gel was stained with 0.25% Coomassie blue and scanned at 595nm (Figure C 10) . There are two major peaks, one at R^O.AA and one at R^, = 0.66. Both these are histidase, the higher molecular weight one being similar to that observed with rat liver histidase. The lower molecular weight form may be a monomeric species of histidase.

7. Kinetic Studies on Pseudomonas Histidase

Kinetic studies were performed at pH 7.A to reflect activity that may be found in vivo.

When low concentrations of histidase were incubated with concentrations of histidine between 0.1 and lOmM, the initial rate of reaction increased with increasing histidine concentration. When histidine concentrations were above lOmM the initial rate of reaction began to decrease with increasing histidine concentrations. This is represented in a Lineweaver Burk plot (Figure Cll) . At low histidine concentrations, the K m was estimated to be 1.25mM.

At low histidine and histidase concentrations, the rate of production of urocanate with time, as measured by increased absorption at 277 nm, was linear. When histidase concentrations in the assay were increased, or when high histidine concentrations were used, the rate of reaction was initially linear but decreased with time. This indicated that product inhibition was occurring. In order to investigat the concentration effect of urocanate on histidase, the enzyme assay was performed in the presence of increasing concentrations of urocanate (Figure C12).

At low histidine concentrations (O.lmM, 0.5mM), the decrease in the initial rate of product formation by histidase was directly proportional to the concentration of urocanate added to the assay medium. This implied that urocanate was acting as a competitive inhibitor of histidase. Figure C13 estimated the K^ for urocanate as 0.26mM at low histidine concentrations 139

Figure CIO

Optical density 595nm

weight Polyacrylamide gel electrophoresis of native purified Pseudo monas histidase (7.5# gel). The gel was stained with 0.25# Coomassie blue and scanned at 595nrn. The two peaks are at 0 .UK and 0.66. QhistidineJ mM

Double reciprocal plot of Pseudomonas histidase activity against histidine concentration. At low histidine concen trations the was estimated to be 1.2AmM. At high histidine concentrations, the initial rate of reaction decreases with increasing histidine concentrations. - u i - 0.008

Figure C12

0.007

0.006

0.005

-p •H •H > 0 •H •P -P O O U 0.004 <$ P. 0 tiO CQ g cd X* m •H -P -p •H m £ •H 0.003 _ _

0.002

a 200yM 0.001 a 150UM □ 125uM • 100uM o 0

— i------1— 0.01 0.03 0.1 1 I Histidine! mM Double reciprocal plot of Pseudomonas histidase activity against histidine concentration. Effect of increasing urocanate on histidase activity at high histidine concentrations. This graph shows substrate and product inhibition of histidase. Histidine . o rcnt ws 0.26mM. was forurocanateK. histidase of against activityPseudomonas Reciprocal C13Figure rcnt cnetain Uoaae actedas competitive Urocanate a concentration.urocanatc niio o hsiae t o histidine concentrations. atlow histidase ofinhibitor 1 142

100 150 200 250 Urocanate (\jM) 143

However, at high hist idine concentrations (lOmM and

30mM ) r the decrease in the initial rate of histidase activity wa s not dire ctly proportional to the concentration of urocanat e added to the assay medium.

8. Inhibition of Pseudomonas Histidase by ATP

The effect of ATP on histidase was investigated because entrapment of the enzyme in erythrocytes would provide an environment rich in this nucleotide.triphosphate. ATP levels in normal erythrocytes are approximately lmM (Surgenor, 1974-’) • The ATP used for this study was the dibarium form from equine muscle. This was solubilised in potassium sulphate and neutralised with 0.1M NaOK. When histidase was incubated with increasing concentrations of ATP, the activity decreased linearly (Figure C14) . Histidase is known to be inhibited by other phosphates, such as pyrophosphate buffers (Hug and Roth, 1968).

9. Radioiodination of Pseudomonas Histidase

The radiolabelling of histidase with 125-iodiae was chosen in order to give a high specific radioactivity for following the in vivo distribution of small quantities of the enzyme. The elution profile of the iodinated enzyme indicated that it is eluted in the same position as non-labelled enzyme . Histidase Activity (units/rag protein) T cnetain Te sa a performedwas assay 3mM The with concentration.ATP was investigated by addition ofvarious concentrations addition by of investigated was The effect of ATP on the activity of activity the Pseudomonas on of histidase effectATPThe itdn a p 90 n 30 9*0and pH athistidine decreasedlinearlywith increasin ofhistidaseThe activity h nurlsdncetd triphosphate to the mediumnucleotide assaythe neutralised iue CIAFigure .1 4 4 M ATP uM ° q . g - U 5 -

SECTION D

ENTRAPMENT AND ACTIVITY OF HISTIDASE IN LIPOSOMES

The criteria for enzyme entrapment in liposomes have been discussed by Finkelstein and Weissman (1978). The liposome associated enzyme must be able to be resolved from the free enzyme by exclusion chromatography, or by differential centrifugation, and the liposome activity must be shown to be latent. The latter criterion is only true if the substrate cannot diffuse through the liposomal membrane. They also suggest that increased entrapment of the enzyme should occur with increased surface charge on the liposomal membrane. This would not hold true when an electronegative enzyme is entrapped in positively charged vesicles because electrostatic attraction may cause apparent increased association of the enzyme with the liposomes.- Entrapment in negatively charged vesicles may cause decreased association because of electro static repulsion. Demonstration of increments in the entrap ment in negatively charged liposomes of horseradish peroxidase (pl = 7.2) and hexosaminidase A (pI = 5.4-)> both anionic at pH 7.4- (liposome pH) have suggested that these enzymes are probably not entrapped as a result of nonspecific, electrostatic interactions (Cohen et al, 1976; Weissman et al, 1975). Pseudomonas histidase has ionisable groups at pH 7.9 and pH 10.2. It would therefore be anionic at physiological pH.

In order to assess which type of liposome gave the highest entrapment of histidase, a variety of vesicles were made with different lipid compositions, different charges and different sizes.

1. Entrapment and Latency of Histidase in Liposomes

For each of the conditions given below, liposomes were constructed with 25mg lipid and 1ml histidase containing 0.25-0.5mg protein (2,000 units/mg).

(i) Multilamellar liposomes MLV

Anionic multilamellar vesicles were constructed from phosphatidylcholine-cholesterol-phosphatidic acid in a - 14.6 -

molar ratio of 10:2:1 respectively. Entrapment of histidase in these liposomes was 1$ of the added enzyme (Table Dl). Association of the enzyme with preformed liposomes was also found to be 1$ of the added activity. Since the entrapment value includes enzyme which is associated with the outside, it can be concluded that there was no overall entrapment of histidase within the liposome bilayers.

Cationic liposomes were constructed from phosphatidylcholine-cholesterol-stearylamine in a molar ratio of 10:2:1 respectively. Entrapped histidase activity was 60$ of that added to the preparation. Addition of enzyme to preformed liposomes demonstrated a 57$ association with the outside. This indicated that there was a strong electrostatic attraction between the anionic enzyme and cationic liposomes. The difference between the entrapped and associated values may indicate that 3$ of the added activity was truely sequestered within the bilayer, but it is more likely that this was due to a concentration effect. The association value was enzyme exposed to preswollen lipid, while the entrapment value was enzyme exposed to dried lipid.

Entrapment of histidase in neutral liposomes (phosphatidyl choline-cholesterol 10:2) was negligible.

(ii) Sonicated Liposomes

The entrapment and association of histidase with sonicated liposomes of neutral and negative charge was negligible (Table Dl).

(iii) Reverse Phase Vesicles REV

The entrapment of histidase (31»000 units) in REVs, constructed from phosphatidylcholine-cholesterol-phosphatidic acid 10:8:1, was investigated. cholesterol oleate was included in the lipid phase as a liposome marker. The elution profile for separation of liposomally entrapped histidase from free enzyme by Sepharose 2B chromatography is given in Figure D1A. The elution profile after disruption of the liposomes containing histidase with detergent is given in Figure DIB. Table D1

Entrapment and absorption of histidase in and onto liposomes of various compositions and charges

In each case, 25mg of lipid was used to construct the liposomes. 1ml of histidase was added, containing 0.25 to 0.50mg protein (2,000 units/mg). Entrapment and association are expressed as percentages of the initial enzyme activity added.

Type of Liposome Composition Molar Ratio Charge Entrapped Associated of Lipid on Liposome Activitv Activitv i % AVI

(i) Multilamellar PC:Ch:PA 10:2:1 Anionic 1% 1% (MLV) PC:Ch:SA 10:2:1 Cationic 60% 572 PC: Ch 10:2 Neutral 0.52 0.52

(ii) Sonicated PC:Ch:PA 10:2:1 Anionic <0.5% <0.52 PC: Ch 10:2 Neutral <0.5% <0.5%

(iii) Reverse Phase PC:Ch:PA 10:8:1 Anionic 5% 1% Vesicles (REV) PC: Ch 10:8 Neutral u% 1% O V SM: Ch 3:2 N eutral <0.5% DSPC:Ch 2:1 N eutral <0.52 <0.52 (Battelle) —i i— o Cholesterol Oleate (DPM/lOpl from each fraction released from the liposomes was ehrs 2 crmtgah i soni iueDA The peak firstis included liposomes with shown chromatography is 2BinSepharoseFigureD1A. 1^+C (31,000 (phosphatidylcholine entrappedinHistidasenegative reverse phaseunits) was vesicles - fte de aeil Lpsms from first peakwerethe Liposomes disruptedwith. addedmaterial.0.25ofthe TritonX100. hs. h euinpoie o separation of elution enzyme The forfree enzymeprofile from byliposomally-entrapped phase. hlseo-hshtdcai 081. ui cholesterol oleate includedin the was lipid e 5uCi H cholesterol-phosphatidicacid10:8:1). Triton hlseo oet. Enzyme cholesterol oleate. FigureD1A 00 l X was removed rm h ezm b scn psae hog ehrs 2. h enzyme The second by from the enzymea passage through 2B.Sepharose 5% ciiy soitd ihte iooe i dmntae t be to demonstrated is liposomes the with associated activity or the added activity. —o o— itds ciiy (units/fraction)HistidaseActivity 14,C A Cholesterol Oleate Figure DIB Figure CholesterolOleate 1;1 Histidase Activity (units/fraction) The first elution profile shows that cholesterol oleate eluted in two peaks. The liposomes were eluted in the void volume. The second radioactive peak was cholesterol not included in the liposome bilayer. The liposomes were con structed with a very high percentage cholesterol (4.0mole %) . Of this, about 80% was recovered in the liposome peak. Histidase activity in the first profile could be demonstrated in both the void volume and in a peak near Vrp. The majority of the enzyme eluted in the second peak. About 1$ activity was demonstrable in the liposome peak without lysis of the liposomes.

The liposomes from the first peak were lysed with 0.2$ Triton X100. The disrupted liposomes were passed again through a Sepharose 2B column (Figure DIB). The enzyme eluted in a broad peak near V^. This contained approximately 5% of the added enzyme activity. This value included the activity that was demonstrated by intact liposomes. Therefore, the actual activity retained within the aqueous phase was kl of the added activity (1250 units). Alternatively, it could be said that the activity demonstrated by histidase in the presence of intact liposomes was less than that exhibited by the same amount of enzyme in the absence of liposomes. This was investi gated by addition of histidase to preswollen liposomes. There was no decrease in histidase in the presence of liposomes.

Therefore it was very likely that h% histidase demonstrated above was truly entrapped within the aqueous phase of the liposomes.

The entrapment of histidase in reverse phase vesicles of various compositions is shown in Table Dl. Sphingomyelin- cholesterol liposomes show very poor histidase entrapment. This may have been due to the prolonged exposure above 32°C (transition temperature of sphingomyelin).

Reverse phase liposomes formed by the Batelle method with distearoylphosphatidylcholine (DSPC) and cholesterol showed no histidase activity. The procedure for entrapment in this type of liposome is not suitable for enzymes, even though they have a large aqueous volume, because only 30-X0ul aqueous phase is added for inverse micelle procedure. They 150 were investigated because of their stability in vivo.

Negative phosphatidylcholine-cholesterol-phosphatidic acid reverse phase liposomes proved the most successful liposomes for entrapment of histidase. The ensuing experiment with histidinaemic mice used this type of liposome. 2. Passage of Histidine Through the Liposome Membrane The diffusion of histidine through the liposome membrane was determined in order to assess whether liposomes can act as metabolic compartments for histidine degradation in the circulation. The possible protection of histidase in lipo somes while decreasing plasma histidine levels would depend on the ability of the substrate to enter the vesicles and the product to leave. The passage of histidine across the liposomal membrane was measured by its efflux. There was a 35$ entrapment of 1^'C histidine inside reverse phase liposomes constructed from either distearoylphosphatidylcholine-cholesterol (2:1) or from sphingomyelin-cholesterol (3:2). Liposomes were incubated in the presence or absence of 33$ mouse plasma. Efflux of 1^'C histidine from distearoylphosphatidylcholine- cholesterol liposomes occurred at less than 2.0 p moles/hr (Figure D2). The distribution ratio of histidine outside the liposomes to histidine inside was 0.03 at the beginning of the incubation. This was probably due to a small amount of free histidine not removed by gel filtration. Incubation of liposomes with 33$ plasma seemed to increase the efflux of histidine. Efflux of histidine from sphingomyelin-cholesterol lipo somes occurred at a slightly faster rate than for egg phosphatidylcholine-cholesterol liposomes (Figure D3). Efflux was more than 7 p moles/hr with a distribution ratio of 0.06 after one hour. Plasma had little effect. The passage of histidine through liposomes of this composition was investigated because both have long circulatory half-lives (Section E) and it was intended to use these in histidinaemic mice for plasma histidine depletion. After it was discovered that both sphingomyelin and DSPC reverse phase vesicles had a low entrapment for histidase and that histidine influx was almost negligible, this objective was abandoned. 151

Figure D2

choline-cholesterol (2:1) reverse phase liposomes over four hours. 3mM histidine labelled with lOuCi histidine was entrapped. 2ml liposomes or 2ml liposomes with 1ml mouse plasma were placed in dialysis bags and dialysed against 10ml 5mM phosphate buffer pH 7./+ at 37°C. 100pl aliquots of the dialysis fluid were taken at time intervals and the radio activity determined by scintillation counting. Figure D3

Plasma 152

omes over f ive hours, es were pla ced in a 7.1 at 37° C. lOOul termined by scintill- 153

SECTION E

DISTRIBUTION OF LIPOSOMES IN MICE

Before studies on the distribution of liposomes containing histidase were performed, the characteristics of liposomes themselves in vivo were investigated. The iodinated polymer polyvinyl pyrrolidone (PVP) was used as an inert non-biodegradable aqueous phase marker for liposomes in vivo. It is not readily taken into cells and it is cleared quickly by the kidneys. It is therefore useful in examining the sites of uptake of liposomes. When released from liposomes in the tissues, it is only slowly metabolised. When released from liposomes in the circulation, it is quickly excreted.

The distribution of reverse phase liposomes of various compositions was determined in order to compare the circulation times and the sites of deposition. These experiments were performed in the mouse because this animal is the model for histidinaemia.

Liposomes containing protein have different sizes and in vivo characteristicsfrom liposomes containing inert molecules, such as PVP (MW 4-0*000) . After assessment of the distribution 125 of liposomes containing I PVP, the composition of liposomes with the best characteristics for histidase replacement were 125 used to entrap and administer I histidase.

The use of liposomes for enzyme replacement therapy can be divided into two main aims. Firstly, liposomes can be used to target the enzyme to the organ of pathology. In the case of histidinaemia, the enzyme, histidase,is absent from the hepatocytic cytosol. Since liposomes are readily taken up by the liver, albeit in the majority by the reticulo endothelial system, there is no need to organ target liposome- entrapped enzyme. Secondly, liposome size and lipid composition can be optimised for prolonged circulation. These may act as metabolic compartments or slow release depots for the enzyme. In Section D2, it was shown that histidine cannot pass through the lipid membrane and so long circulating liposomes could only be useful as slow release depots for the enzyme when disrupted by plasma proteins. 1. Liposome-Entrapped 125 I PVP in vivo

(i) Distribution of ^ ^ 1 pvp pn mjLCe

125 The fate of intravenously injected I PVP was followed in A2G mice (Table El). The half-life in the circulation was between 1 and 2 hours (Figure El). There was very little uptake into liver and spleen. 125 (ii) Distribution of I PVP entrapped in Reverse Phase Vesicles in Mice

The fate of 125 I PVP entrapped in anionic reverse phase liposomes (PC:Ch:PA, 10:8:1) was investigated. 16$ of the added 125 I PVP was entrapped. The distribution of the liposomes in A2G mice after various times is given in Table 22 and the clearance from the blood is given in Figure El. Nearly 50$ of the injected dose is found in the liver after five minutes, increasing to 70$ after twenty minutes. The level of radioactivity associated with the liver then falls to 38$ 125 by 3 hours. Since I PVP remains in cells into which it is taken up, release of radioactivity from the liver with time indicates that liposomes may be only loosely associated with "I p c extracellular surfaces (Section H). Clearance of PVP entrapped in liposom&Sfrom blood shows a half-life of less than five minutes. 125 (iii) Distribution of I PVP entrapped in Sphingomyelin- Cholesterol Reverse Phase Liposomes

The distribution of 125 I PVP entrapped in sphingomyelin- cholesterol (3:2) reverse phase liposomes showed a slow uptake into liver and a 5 hour circulatory half-life (Table E3). The clearance from mouse blood , is given in Figure El.

(iv) Subcellular Fractionation of Mouse Liver after 125 Administration of____I PVP Liposomes 125 A2G mice were administered with I PVP in reverse phase liposomes composed of egg phosphatidylcholine-cholesterol- 125 phosphatidic acid (10:8:1). When PVP is entrapped in this composition of liposomes, maximum uptake into the liver is found between 20 minutes and 1 hour (Figure El). Sub- 155

Table El. Distribution of 1 2 5 I PVP (free) in A2G Mice

Jo Injected Dose Time (mins) Liver Spleen Blood

3 0.7 1.9 80 60 3.5 3.2 39 120 1.8 0.95 46 180 7.1 0.70 31 2^0 3.8 0.80 25 300 4.0 0.50 20

125 Table E2. Distribution of ^1 PVP entrapped in Egg Phosphatidylcholine-cholesterol-phosphatidic acid (10:8:1) Reverse Phase Liposomes

1 Injected Dose

Time (mins) Liver Spleen Blood Kidneys Lungs 00

5 • 2.1 42.9 2.5 2.9 15 54.7 6.5 35.3 1.7 1.6 20 71.5 5.1 22.0 1.3 1.0 60 61.0 8.0 6.4 1.3 0.5 120 59.0 4.3 6.9 0.5 0.5 180 38.2 4.9 2.7 1.0 NS - 156 -

Figure El

125 Clearance from blood of I PVP either free or entrapped in egg phosphatidylcholine-cholesterol-phosphatidic acid (10:8:1) reverse phase liposomes or in sphingomyelin-cholesterol (3:2) reverse phase liposomes. A2G mice were injected with 0.20inl liposomes containing 0.05uCi 12 I5 PVP. 157

l 25 Table E3« Distribution of J1 PVP entrapped in Sphingo myelin-cholesterol (3:2) Reverse Phase Liposomes

% Injected Dose

Time (mins) Liver S pleen Blood Kidneys Lungs

60 8.8 2.5 81.9 2.5 1.1 120 13.0 4.5 63 • 6 2.9 1.5 180 22.6 12.9 56.5 3.3 1.1 o H 1 — • 240 27.5 1 41.0 3.6 0.9 o H • 300 29.0 v_n 51.0 3.0 1 .0

Table E4. Sub-cellular Fractionation of A2G Mouse Liver 125 1 hour after administration of I PVP entrapped in Reverse Phase Liposomes (Egg Phosphatidylcholine- cholesterol-phosphatidic acid 10:8:1)

Fraction % Total Dose Found in Liver

Cytoplasm 21.6 Microsomal Fraction 3.5 Lysosomes ) 33 Mitochondria ) 9.2 Nuclear/Cell Debris 31 158

cellular fractionation of pooled mouse livers after one hour was performed in order to detect to which subcellular 125 organelle liposomes were being delivered. I PVP remains in tissues without being metabolised.

Enzyme assays of subcellular fractions included lactate dehydrogenase for cytoplasm, glucose 6-phosphatase for microsomes, succinate dehydrogenase for mitochondria and 3-glucosidase for lysosomes. Lysosomes and mitochondria were not distinctly separated, but this was considered non- essential for the purposes of this investigation. Separation of lysosomes and mitochondria can be obtained by sucrose density centrifugation. 125 The distribution of I PVP-containing liposomes in the liver is shown in Table E4-. Although liver was thoroughly homogenised, nearly 31$ remained with the cell debris. The percentage associated with the final centrifugation, the cytoplasmic fraction, and that associated with the lysosomal fraction comprised over 60$ of the dose found associated with liver.

2. Liposome-Entrapped___125I Histidase in vivo

When iodinated histidase was entrapped in negative reverse phase vesicles (phosphatidylcholine-cholesterol-phosphatidic acid 10:8:1) about 14-$ of the added radioactivity was entrapped. When iodinated histidase was entrapped in neutral reverse phase liposomes (phosphatidylcholine-cholesterol 10:8) 3-7$ of the added material was entrapped (Table E5). Considering only 5$ of added enzyme activity can be entrapped inside negative liposomes (Section D), the higher entrapment of radioactivity may reflect the loss of iodine-125 from the enzyme on sonication in formation of the liposomes, and its attachment to lipid. Any free iodine would be cleared to the thyroid immediately after administration.

■JO c (i) Distribution of Histidase Entrapped in Reverse Phase Liposomes in Mice

125 I histidase was entrapped in negative reverse phase liposomes (above composition),administered to normal A2G mice and its 159

Table E5« Entrapment of____125 I Histidase in Anionic and Neutral Reverse Phase Liposomes

^125i Histidase

Anionic Neutral Liposomes Liposome

Entrapped and Associated 19? 9.8? Associated 5% 6.1$ Entrapped in bilayer 1—1 3.7?

125 Table E6. Distribution after 1.hour of I Histidase in A2G Mice, either Free or Entrapped in Negative Reverse Phase Vesicles (PC;Ch:PA, 10:8:1)

% Injected Dose

Free 125 I Histidase Liposome entrapped ^ ^ 1 Histidase

Blood 4.6% 9? Liver 18% 53? Spleen 3% 16? Kidney 22% 9? Lungs 8% 12? 160 distribution after 1 hour monitored (Table E6). The fate of free 125 I histidase was also monitored.

(ii) Subcellular Fractionation of Mouse Liver after 125 Administration of I Histidase in Liposomes 125 The distribution of I histidase in negative reverse phase liposomes in liver, one hour after administration to normal A2G mice was examined (Table E7) . The greatest part of the radioactivity not associated with cell debris was found in the lysosomal/mitochondrial fraction, followed closely by the cytoplasmic fraction.

3. Administration of Histidase in Reverse Phase Liposomes to Histidinaemic Mice

(i) Subcellular Fractionation after One Hour

Histidase (30,000 units/ml; - lOmg protein) was added to negative reverse phase vesicles (PC:Ch:SA 10:8:1) to give a final entrapment of 882 units per ml liposomes. Liposomes were labelled with 5yCi cholesterol oleate.

Histidinaemic mice were administered with 0.25ml liposomes (5mg lipid; 220 units histidase) via a lateral tail vein.

Subcellular fractionation of 4- histidinaemic mouse livers one hour after administration of liposomes containing histidase showed no demonstrable histidase activity in any fraction (Table E8). An aliquot of each fraction was digested and counted for cholesterol oleate activity.

A normal A2G mouse liver assayed for comparison contained 285 units histidase activity in the whole liver.

(ii) Effect on Blood Histidine Levels over 24- hours

Histidase (15,000 units) was added to negative reverse phase vesicles to give a final entrapment of 375 units histidase/ml liposomes. Histidinaemic mice were administered with 0.2ml liposomes containing 4-mg lipid and 75 units histidase. Blood was taken from the tail at various intervals (Table E9)• Histidinaeinic mice were treated with empty liposomes mixed with free histidase. Lysine levels are given to show that any effect was specific (Figure E2). 161

Table E7. Subcellular Fractionation of A2G Mouse Liver 1 hour after Administration of 12 5 I Histidase Entrapped in Reverse Phase Liposomes (Phosphatidyl- choline-cholesterol-phosphatidic acid 10:8:1)

Fraction % Total Dose Found in Liver

Cytoplasm 23.0 Microsomes 13.5 Lysosomes ) 25.7 Mitochondria ) 3.4- Nuclear/Cell Debris 29.0

Table E8.

Fraction % Dose Found in Liver

^ C Cholesterol Oleate Histidase Activity

Cytoplasm 14.7 ND Microsomes 5.7 ND Lysosomes ) 46.0 ND Mitochondria ) 18.0 ND Nuclear/Cell Debris 15.2 ND

ND = Not detectable 162

Table E9« Effect of free and liposomally entrapped histidase on blood histidine levels in histidinaemic mice. Egg phosphatidylcholine-cholesterol-phosphatidic acid reverse phase liposomes were used to entrap histidase (final entrapment 375 units histidase/ml liposomes). Histidinaemic mice were administered with liposomes (0.2ml containing 4mg lipid and 75 units histidase) . Blood was analysed at intervals for histidine and lysine. Values expressed are in umoles per ml blood.

PREPARATION Time after Plasma Amino Acids injection (hours) pmoles/ml ± SE HISTIDINE LYSINE m C '(n*= 5) 1! o o H +1 . Liposomally 0 2.51 ± 0.166 • entrapped histidase 1 1.95 ± 0.25 0.41 ± 0.05 2 2.39 ± 0.11 0.39 ± 0.08 5 2.63 ± 0.05 0.32 ± 0.001 18 2.19 ± 0.23 0.325 ± 0.03 24 3.29 ± 0.36 0.53 ± 0.06

HISTIDINE LYSINE (n = 2) (n = 2) Histidase with 0 1.249 o . 646 empty liposomes 1 0.769 0.298 2 0.539 0.490 24 0.888 0.351

+ A more extensive experiment with free histidase administration to histidinaemic mice has been carried out in Section G. Amino Acid Level (% Initial Concentration) ciiy bcue yie ees fell as well. levels lysine becauseactivity, diitainddnt per o e specifically be todue tohistidase appearnotdid administration h fl i bod itdn lvl afterfree histidase levels histidine in blood fallThe Effect of free and liposomally entrapped histidaseentrapped on blood liposomally and offree Effect itdn ad yie ees nhsiiamc ie Reverse mice.histidinaemic in levels lysine andhistidine bleeding each mouse at time intervals. Amino acid Amino arelevels intervals. at time mouse each bleeding acid aminolevelswere analysed Blood 4.mg by histidase, lipid). 0.20ml (75liposomes units administered were mice Histidinaemic hs iooe (CC:A1::) wereto used entrap (PC:Ch:PA10:8:1) histidase. phaseliposomes xrse s ecnae fte nta ee (see initiallevel oftheTable E9)• percentageasexpressed Figure E2 □ Lysine levels afterLysineadministration □ of yie ees after levelsLysineadministrationH of .163 itds inreversehistidase phase liposomes - 164

SECTION F

ENTRAPMENT AND ACTIVITY OF HISTIDASE AND PHENYLALANINE AMMONIA LYASE IN ERYTHROCYTE GHOSTS IN VITRO

Studies in vitro were necessary to quantitate the degradcttive capacity of the above enzyme-loaded erythrocyte ghosts on their substrates.

1. Entrapment of Histidase in Erythrocyte Ghosts

Histidase was entrapped in erythrocyte ghosts by the hypotonic dialysis procedure at 70$ haematocrit. Dialysis buffers were supplemented with 4mM adenosine, 3mM glutathione, 4mM magnesium ions and 5mM glucose.

Resealed erythrocyte ghosts collected on centrifugation were normally about 50$ of the initial volume of erythrocytes used. Ghosts were centrifuged at low speed <500g to avoid damage to the membrane.

Entrapment of histidase in erythrocyte ghosts varied from 4.5$ to 7$ of the material added before haemolysis. Entrapment depended on the protein concentration added and the age of the enzyme. There was a linear relationship between the enzyme added and the enzyme entrapped.

Association of the enzyme to the outside of erythrocyte ghosts was not detectable.

2. Entry of Histidine into Erythrocyte Ghosts

The influx of histidine into erythrocyte ghosts was measured to determine if they could act as metabolic compartments for the metabolism of histidine in the circulation. The ability of circulating histidase entrapped in erythrocyte ghosts to degrade plasma histidine depends not only on the enzyme activity, but also on the rate of substrate entry and product diffusion.

The plasma histidine levels in histidinaemic mice are approximately 3mM. The influx and efflux of histidine through the erythrocyte ghost membrane when suspended at 50$ - 165

Figure FI Influx and efflux of histidine from murine erythrocyte ghosts. The incubation medium contained 3mM histidine with O.lpCi histidine. For efflux, the ghosts were preincubated with histidine for 3 hours. Ghosts were at 50% haematocrit.

Table FI The concentration of histidine inside erythrocyte ghosts was determined before and 30 minutes after incubation of ghosts in 3mM, 25mM and 50mM histidine. Intracellular histidine levels were determined by automated Pauly analysis.

Qiistidine] mM

Time (mins) 3 25 50

0 0.7 0.4-2 3.0

30 1.0 1.80 9-8

Increase in Intra cellular ^Histidine] 0.3 1.38 6.8 166

haematocrit in isotonic. 3mM histidine is shown in Figure FI. The distribution ratio is the ratio of the radioactivity in the intracellular medium to the radioactivity in the incubation medium. It can be seen that the ratio increases up to 2 hours, after which an equilibrium is reached with a distribution ratio of nearly 0.3.

In order to determine what concentration of histidine was actually present inside erythrocyte ghosts after incubation with the various concentrations of this amino acid, intra cellular histidine levels were determined by automated Pauly analysis. Erythrocyte ghosts were washed once in phosphate buffered saline and lysed with distilled water. Protein was precipitated with 10$ 5-sulphosalicyclic acid and the super natant analysed. Internal histidine concentrations were determined at zero time and after thirty minutes (Table Fl).

The actual concentration of histidine inside the erythrocyte ghosts when injected into histidinaemic mice would be about one-tenth (0.3mM) of the plasma concentration. This value is below the K for histidase. Therefore, it would seem m that if histidase entrapped in erythrocyte ghosts is administered to histidinaemic mice, the rate limiting factor in the degra dation of histidine would be its transport into the ghosts.

3. Activity of Histidase Entrapped in Erythrocyte Ghosts

Histidase (50 units) entrapped in erythrocyte ghosts was incubated with various concentrations of isotonic histidine pH 7.4- (3>10,25 and 50mM) at 37°C. The incubation medium and the erythrocytes were separated by centrifugation. Urocanate production in both the intracellular medium and the supernatant was followed by increase in optical density at 277nm (Figure F2) . The reaction was linear from 0 to 15 minutes after addition of the substrate. The rate of production of urocanate tails off after fifteen minutes in both the incubation medium (supernatant) and the erythrocyte ghosts (intracellular medium).

The results indicate that histidase is active when it is entrapped in erythrocyte ghosts. Incubation with histidine causes product formation, both intra- and extracellularly. After 15 to 30 minutes, levels of product have been built up ■JU-) Incubation Medium Figure P2

^Histidine] ★ 50mM ■ 25mM

□ lOmM 3raM

Production of urocanate by intact erythrocyte ghosts containing histidase incubated with various concentrations of histidine. At time intervals, erythrocyte ghosts were separated from the incubation medium. The optical density of the intracellular medium was determined after lysis of the cells. The optical density of the incubation medium at the same time points were determined. Urocanate concentrations corresponding to these optical densities were determined by Pauly analysis. 168

such that enzyme activity declines. The fact that urocanate is present in the incubation medium indicates that it can diffuse through the erythrocyte membrane. Even so, the closed system means that product inhibition occurs.

The measurement of total urocanate concentration in this system was not possible by spectrophotometric methods. Therefore automated Pauly analysis was performed on both the incubation and intracellular medium. The urocanate produced in the first fifteen minutes was 30-4-OpM for each of the four histidine concentrations used In the assay. From the kinetic data with free histidase (Section C), the for urocanate was found to be 0.26mM. Within the erythrocytes, urocanate concentrations below this may substantially inhibit enzyme activity.

By assuming the erythrocyte ghosts to be spheres, it was calculated that the volume of medium between packed erythro cyte ghosts after centrifugation was one-half of the total packed cell volume. The ratio of supernatant volume to packed cell volume in Figure F2 was 8:1. This demonstrated that there was a higher urocanate level found inside the cells than outside. This may mean that although urocanate can diffuse through the erythrocyte ghost membrane, equilibration had not occurred after fifteen minutes.

Although product inhibition was demonstrated in vitro, this may not be a problem in vivo, if the urocanate produced is removed from the plasma and diffusion from within the erythrocyte ghosts occurs at a fast enough rate.

When the incubation medium containing histidine was removed from the erythrocyte ghost containing entrapped histidase, and the cells resuspended in distilled water and lysed by sonication, histidase activity could be demonstrated. This fact means that irreversible inhibition or proteolytic inactivation had not occurred.

4.. Entrapment of Phenylalanine Ammonia Lyase in Erythrocyte Ghosts

The phenomenon of product inhibition of histidase entrapped in erythrocyte ghosts was further investigated using the - 169 -

structurally and functionally analogous enzyme phenylalanine ammonia lyase. Hubbard et al (1980b)showed that phenylalanine ammonia lyase from Rhodotinila glutinis could be entrapped in erythrocyte ghosts. Passage of phenylalanine through the erythrocyte membrane was not a limiting factor and it was suggested that this could be used for substrate depletion in phenylketonuria. The activity of entrapped phenylalanine ammonia lyase was not demonstrated.

Phenylalanine ammonia lyase has similar properties to histidase. It has a pH optimum of 8.7, depends on sulphydryl groups for activity, and has a similar molecular weight (275,000 for R. glutinis). It has the same ammonia elimination, catalytic sequence and has a dehydroalanine residue at its active site. It is found only in fungi and higher plants. It has been found to have complex kinetics. It shows negative cooperativity with its substrate (Le Vitzki and Koshland, 1969). The apparent K increases with increasing substrate concentration. The enzyme is also product inhibitable (Subba Rao et al, 1967).

The entrapment of phenylalanine ammonia lyase in hypotonic dialysis ghosts was performed as for histidase. Lysis at the

resealed erythrocyte ghosts demonstrated a 1% entrapment of the enzyme initially added (0.87 units). There was an approx imate 60% recovery of the initial volume of erythrocytes used in the preparation of ghosts.

There was no absorption of the enzyme to the outer surface of erythrocyte ghosts.

3 5. Passage of H Phenylalanine into and out of Erythrocyte Ghosts 3 The efflux and influx of H phenylalanine into erythrocyte ghosts was performed as for histidine (Section F(2)). Phenylalanine rapidly passed through erythrocyte ghost membranes, with a distribution ratio of 0.25 after 10 minutes (FigureF3 ).

6. Activity of Phenylalanine Ammonia Lyase Entrapped in Erythrocyte Ghosts

Erythrocyte ghosts containing 0.01 units phenylalanine ammonia lyase per ml packed cells were incubated with 3mM phenylalanine t Distribution Ratio ih .ui hnllnn. o effluxstudies, For the phenylalanine. O.luCiwith nlxad flxo phenylalaninethrougherythrocyte contained medium 3mM The incubation phenylalanine ghosts. of effluxand Influx hsswr a 50$ haematocrit. atwereGhosts phenylalaninewith for preincubated 1 hour.ghostswere 0.10 0.20 0.30 iue F3 Figure ie (mins)Time 170 Ifu o C phenyl C of Influx D flx of BEfflux alanine from erythrocyte ghosts erythrocyte from alanine ghosts alanine from erythrocyte erythrocyte from alanine 1/fC ,. phenyl 171 in isotonic phosphate buffered saline pH 7.$., at 50$ haematocr.it. The production of trans-cinnamic acid was measured at various time intervals in the supernatant (incubation medium). . Cinnamic acid production was linear for the initial three hours before activity ceased. Intact erythrocyte ghosts expressed nearly 33$ of their entrapped activity, as measured by the product extracellularly Intracellular cinnamic acid levels were not measured. Full activity could be recovered after lysis of the erythrocyte ghosts .

These results demonstrate more clearly than for histidase that enzymes remain active on entrapment in erythrocyte ghosts Phenylalanine ammonia lyase showed product inhibition after 3 hours, whereas histidase showed inhibition after 15 minutes. 172

SECTION G

ADMINISTRATION OF ERYTHROCYTE GHOSTS TO MICE

125 1 Distribution of____I PVP entrapped in Erythrocyte Ghosts in vivo

Characterisation of erythrocyte ghosts in vivo was performed using the inert, non-biodegradable molecule polyvinyl pyrrolidone (MW 4-0,000), iodinated at a specific activity of 20-60uCi/mg. Although this is not - an ideal marker to mimic the distribution of erythrocyte ghosts containing the higher molecular weight protein, histidase (MW 210,000), it gave a good indication of the circulatory half-life of intact erythrocyte ghosts. The time course of clearance of the erythrocyte ghosts from blood of normal A2G mice and the appearance in liver and spleen is shown in Figure G1 and Table Gl.

The erythrocyte ghosts show a biphasic removal from the blood. After 3 minutes 10$ of the label appeared in the liver and this did not increase significantly for over 22 hours. In the early hours after injection, the spleen only removed small amounts of the injected dose, but this increased to nearly 20$ after 22 hours. Blood radioactivity declined to 34-$ of the injected dose after 94- hours. The distribution of the radioactivity between plasma and erythrocytes is shown in Table G2. 125 The distribution of free I PVP is given in Figure Gl for comparison (see Section E).

The fact that only 25$ of the total injected dose of erythro- 125 cyte entrapped I PVP was lost after 94- hours demonstrates that there is very little lysis of erythrocytes in the circulation and that the majority of the circulatory loss is by uptake into liver and spleen.

125 2. Distribution of Erythrocyte Ghosts containing I Histidase in vivo 125 The distribution of I histidase entrapped in erythrocyte ghosts after administration to normal A2G mice was investigated Table G1 125 Distribution of I after Administration of Erythrocyte 1?5 Ghosts containing PVP in A2G Mice.

% Injected Dose

Time (hours) Blood Liver Spleen Total

3 mins 88 7 1.3 96.3 1 82.5 8.7 3.1 94.3 .2 75.6 9.6 12.8 98.0 3 69.8 10.8 15.0 95.6 5 72.0 11.5 10.5 94.0 22 57.6 12.3 18.0 87.9 O H

34- • 19.3 17.3 77.6 94 34.0 25.3 13.5 72.8

Table G2 125 Distribution of I PVP between Plasma and Erythrocytes after Administration of Erythrocyte Ghosts containing

1 2 5 I PVP.

% Injected Dose

Time (hours) 3 mins 1 2 3 5 22 34 94 00 ro Plasma 8.8 • 3.5 2.8 10.8 1.53 0.65 0.3

Erythrocytes 79.2 73.4 72.0 67.0 70.9 55.9 40.6 33.7 laac o rtrct hs etapd I orPVP entrappedghost oferythrocyte Clearance free ^ ^ 1 PVP from the blood ofmice.A2G the blood from PVP 1 ^ ^ free Blood Radioactivity {% Injected Dose) G1Figure —» i »1— 3 2 1 125-j-□ free pvp ^^^1 entrapped inPVP H erythrocytes Time (days) 125 ------1 ------k \ 17 5

(Figure G2 and Table G3) • The radioactivity associated with erythrocytes after 3 minutes had fallen to 27%, with plasma levels already at 28% of the injected dose at this time. The liver contained 11% of the injected dose after 3 minutes. After the initial drop in erythrocyte radioactivity, the levels remain nearly constant for up to 18 hours and have declined to 13% after 48 hours. Plasma radioactivity falls to 1% after 4-8 hours, while liver levels do not increase above 10% and have fallen to under 1% by 48 hours. Spleen radioactivity was never higher than liver radioactivity, the majority appearing 1 hour after administration of ghosts.

3. Administration of Histidase Entrapped in Erythrocyte Ghosts to Histidinaemic Mice

Histidinaemic mice with initial plasma histidine levels of 2.55 ± 0.22 hmoles/ml were administered with 20 units histidase either entrapped in erythrocyte ghosts (50% haematocrit) or on its own (free) . Histidinaemic mice were also administered empty erythrocyte ghosts at 50% haematocrit (Figure G3 and Table G4)• Blood histidine levels show a rapid fall to undetectable levels one hour after the administration of free histidase. This rises to 50% from one to six hours after injection and histidine levels have returned to the normal histidinaemic level after 24 hours (about 20 times that of normal mice).

Blood histidine levels fell to 50% of their initial value after administration of histidase containing erythrocyte ghosts. Histidine levels remain at 50% of the initial histidinaemic level (about ten times normal mouse level) for over five hours after administration of enzyme containing ghosts. Histidine levels rise to over the initial level after 24 hours.

Figure G3 shows that erythrocyte ghosts: alone have little effect on plasma histidine levels. Therefore the decrease in plasma histidine when erythrocyte ghosts containing histidase are administered is specifically due to entrapped histidase.

Table G5 shows the effect of erythrocyte ghosts containing histidase and free histidase on histidinaemic mice plasma lysine levels. There was no change in blood lysine levels 176

Table G3

Distribution of 125 I following administration of erythrocyte 125 ghosts containing I labelled histidase to A2G mice (n = 2) .

% Injected Dose

Time (hours) Plasma Erythrocyt es Liver Spleen

5 mins 28.0 27.0 10.8 1.7 (37,19) (27,27) (10,11.5) (2.5,0.9)

1 15 30.5 11.3 10.35 (15,15) (30,31) (9.0,13.5) (10.9,9.8)

3 16.9 22.0 11.0 6.0 (11.2,22.6) (21,23) (6.5,15.5) (4.2,7.8)

5 11.5 20 13 8.9

18 10.2 25.3 10.6 5.35 (14.8,5.5) (32,18.6) (14.2,6.9) (5.9,4.8)

24 2.2 15.5 0.5 0.67 (1.8,2.6) (11.4,19.5) (0.25,0.74)(1.H,0.22)

48 1.1 i—1 0.34 0.29 (1.76,0.48) (17.5,9.2) (0.45,0.23)(0.33,0.25) - 177 -

Table G4

Effect of erythrocyte ghosts containing histidase (20 units), histidase alone (20 units) or erythrocyte ghosts alone on blood histidine levels in histidinaemic mice (±S.E.).

iJ moles histidine/ml blood Histidase Alone Erythrocyte Erythrocyte - Ghosts Alone Ghost Time (hours) Containing Histidase n = 3 n = 1 n = 3

0 2.59 ± 0.15 1.850 2.548 ± 0.22 1 0.084 ± 0.030 1.990 1.238 ± 0.16 2 0.83 ± 0.14 3 2.30 1.28 ± 0.12 3.5 1.00 ± 0.12 5 2.02 1.106 ± 0.08 5.5 1.00 ± 0.24 24 3.10 ± 0.05 1.98 2.58 ± 0.03

Table G5

Effect of histidase entrapped in erythrocyte ghosts and free histidase on lysine levels in histidinaemic mice (±S.E.). Jl moles lysine/ml blood ~atment Histidase Erythrocyte Ghost Alone Containing Time Histidase n = 3 n = 3

a 0.319 0.425 ±O(04 ± 0.04 1 0.327 0.475 ±0.02 ±0.04 2 0.417 ±0.03 3 0.550 ±0.02 3.5 0.390 ±O.O3 5 o . /j ()1 ~().O3 5 . 5 o. !~OO ~O.OL(J

() . y;l) O.4!~j~ ±n_n?n ±n n~ Histidase (% Injected Dose) 100A Distribution of radiolabelled histidase between erythro of radiolabelled Distribution ye adpam fe amnsrto oferythrocyte administrationafter and cytesplasma nrpe nye oAG mice. toenzymeentrappedA2G Figure G 2 ie fe ijcin (hours) injection afterTime 178 A Erythrocytes A Plasma 179

with either treatment. The result demonstrates that the fall in blood histidine by erythrocyte ghosts containing histidase or free histidase is specific for histidine and has no effect on lysine, which is taken as representative of all other amino acids that are present in histidinaemic mouse blood. The effect on the imidazole derivates excreted in urine was not investigated.

These results indicate that free and erythrocyte entrapped histidase can deplete plasma histidine in histidinaemic mice for up to and probably over 6 hours. A time course from 6 to 24. hours showed that histidine levels rise slowly from 50% to 100% (results not shown). However, further data are required between 6 and 24- hours to compare erythrocyte- entrapped histidase treatment and free histidase treatment in histidinaemic mice. The rate of recovery of the plasma histidine levels may be different for the two conditions. A more extensive investigation may determine whether the response after erythrocyte-entrapped histidase is more prolonged than that for free histidase treatment.

When the clearance of erythrocyte entrapped 125 I PVP is compared to the clearance of erythrocyte entrapped 125 I histidase, the rate of removal of radioactivity associated with erythrocytes from the circulation is similar for both. This can be seen when the data from Table G2 for erythrocyte entrapped 125 I PVP and from Table G3 for erythrocyte entrapped 125I histidase is recalculated using the initial time point as 100% values (Figure G4-) • This indicates that, although enzyme-containing erythrocyte ghosts may undergo initial rapid lysis after injection into mice, surviving enzyme ghosts have a corresponding circulatory life close to PVP-containing erythrocyte ghosts. ±«u

Figure 0 3

Time after administration (hours)

Effect of erythrocyte ghosts containing histidase (20 units), histidase alone (20 units), or erythrocyte ghosts alone on blood histidine levels in histidinaemic mice. % Radioactivity found at Initial Time point laac o rtrct got nrpe I andPVP entrapped ghost erythrocyte ofClearance ie on a 10 vle. Bothprotein and polymer 100% value). astime point otiig hs hs iia circulatory survival similar times.containing has ghost (from histidasere G2and G3using initial the Tables in datacalculation of entrapped erythrocyte ghost iue G4Figure .18.1 12 5 H histidase n K 125i PVP 24 12 5 182

SECTION H

DISCUSSION

Rat liver histidase was examined as a potential supply of the mammalian enzyme for replacement therapy in histidinaemic mice. It was purified to a final specific activity of 309 units per mg protein. Published values for the homogeneous rat liver enzyme are 1910 (Brand and Harper, 1976) 3500-6800 from female rat liver (Cornell and Villee, 1968; Okamura et al, 1974) and 138 for guinea pig liver (Kato et al, 19 55)* The purified protein gave a major band at R^ = 0.44 on analytic gel electrophoresis. Previous studies have shown the in situ detection of histidase after electrophoresis on similar 7.5$ analytic gels by incubating the gels in medium containing histidine (Hassall et al, 1970). These gave histidase as a uv absorbing band at R^. = 0.4-7.

Although the starting material was 150g of liver, there was insufficient yield of histidase to be effective for replacement therapy attempts. Normal hepatic histidase levels in mice are between 200 and 250 units per gram tissue. The relation ship of flux through the histidine to glutamate pathway has been discussed in Section A. Since heterozygous histidinaenic mice (his/+), with 50% of the normal mouse hepatic histidase level, have an unaltered flux through the pathway and homozygous histidinaemic mice (his/his), with <5% normal histidase levels, have 4-0% of normal flux, replacement of only a small fraction of total wild type activity is necessary (Figure Al). Even so, the amount of enzyme that can be administered is limited by the quantity that can be encapsulated in liposomes and erythrocyte ghosts. It was demonstrated that approximately 5% histidase added to the preparation could be encapsulated in either carrier (Section D). Consequently, activities much greater than can be purified from rat liver were required.

Induction of histidase from a bacterial source, a Pseudomonas species, resulted in a high starting specific activity (2140units/mg protein). The regulation of histidine catabolism in Pseudomonas is known to involve induction and derepression 183

(Lessie and Nudhart, 1967). The production of 4.0,000 units from nearly 20g wet weight of cells enabled purification of sufficient quantities of histidase for entrapment and in vivo studies to be conducted. A simple purification procedure was developed and after four steps, a specific activity of 2104- units per mg protein was obtained. Homogeneous Pseudomonas histidase has been shown to have a specific activity between 11,000 and 12,000 units per mg protein (Klee, 1970c). The enzyme used in these experiments was not homogeneous but over 70$ of the protein present was histidase, as demonstrated on analytical polyacrylamide gel electrophoresis, with two major peaks (polymeric forms) at R^ 0.4-4- and 0.66 (Figure CIO) . Further purification could have been performed with hydroxyapatite and another ion-exchange column, but as initial studies required high yields of activity, the partial purification established here was judged adequate for this work.

Both the bacterial and mouse liver enzymes were found to be product inhibitable. Inhibition of mouse liver histidase by urocanate was found to follow normal competitive inhibition characteristics (Figure C6) . Although some studies on the rat liver enzyme have reported product inhibition (Edlebacher, 194-3) , other studies on the kinetics and inhibitors of the rat liver enzyme have not mentioned this (Brand and Harper, 1975; Brand and Harper, 1976). A study by Burns and Kacser (personal communication) on mouse liver histidase has found a for urocanate of 0.05mM. The significance of this on the control of histidase activity in the liver in vivo may be to limit the catabolism of the essential amino acid histidine. Histidase present in epidermis, being the same gene product as the liver enzyme (Kacser et al, 1973), probably has similar properties. In the epidermis, the enzymes comprising the urocanate to glutamate pathway are absent. Therefore urocanate may play an essential role in the control of histidase activity in skin.

The liver enzyme was found to be slightly less active at high histidine concentrations. The assay was performed on a high speed supernatant (cytoplasmic fraction) of a crude mouse liver homogenate. It is possible that urocanase, the enzyme that converts urocanate to imidazole prop ionate, was present 18/, with histidase in this fraction. In this case, the apparent substrate inhibition may be explained by urocanase activity at high histidine concentrations. Kinetics on the purified enzyme, after heat treatment to destroy urocanase activity, would have been advisable. Substrate inhibition has not been detected by other workers.

Pseudomonas histidase was found to be both substrate and product inhibitable. Product inhibition of Pseudomonas histidase at low histidine concentrations was observed to be competitive with a of 0.26mM (Figure C13). Previous studies on the Pseudomonas enzyme have shown product inhibition with a of 0.13mM (Hug et al, 1968). Substrate inhibition was detectable with histidine concentrations above lOmM. Extrapolation of the Lineweaver-Burk plot for low concentrations of histidine, gave a of 1.25mM at pH 7.4 (Figure Cll). All the kinetic studies were performed at physiological pH to reflect the activity found in vivo or when the enzyme was entrapped in liposomes or erythrocyte ghosts.

These observations may be explained by the partial reactions of histidase that were reported by Peterkofsky (1962). The formation of an amino-enzyme intermediate means that the reaction may either go irreversibly forward to form urocanate and free enzyme or reversibly backwards to form histidine. At high urocanate levels the reverse reaction may predominate, or urocanate may form a dead-end complex with histidase (Hanson and Havir, 1974) • The overall and partial reactions have been shown to have distinct bell-shaped dependence upon pH (Peterkofsky, 1962). When equimolar histidine and urocanate were treated with the enzyme, the maximal rate for the partial reaction was at pH 7.$ and for the forward reaction at pH 8. Since all experiments in this study were performed at pH 7.4> this may account for the product inhibition at low histidine concentrations obviously established 185 here, but not well documented by others. Although histidase appeared to obey Michaelis-Menten kinetics at lower substrate concentration, the velocity fell off at high histidine concentrations. As the velocity shown in Figure Cll is the initial rate (ie. before urocanate is at a level to inhibit activity), there may be some effect of the first molecule of histidine bound, making it more difficult for subsequent molecules to be bound. This phenomenon has been observed with phenylalanine ammonia lyase (Conway and Koshland, 1968; Levitzki and Koshland, 1969)- An alternative explanation is that urocanase activity manifests itself at high histidine concentration.

It is highly unlikely that urocanase was present with the purified Pseudomonas histidase, both because of gel electro phoretic evidence and because it is very unstable at 4-°C (Tabor and Mehler, 1955). At low histidine concentrations, enzyme inhibition was directly proportional to urocanate present in the assay. This points against any urocanase being present with histidase. A decrease in urocanate levels in the presence of histidase and in the absence of histidine was never seen.

Histidase activity decreased when ATP was added (Figure C14) , The effect of this nucleotide triphosphate was investigated because histidase was to be entrapped in erythrocyte ghosts with supplemented substrates for ATP synthesis. It was found that histidase was 60$ inhibited by ImM ATP, the concentration found in normal erythrocytes. A similar observation has been made by Nash and Phillips (1979) where the ATP concentration required to give 50$ inhibition of histidase was 1.5mM. The physiological significance of this effect has been suggested to be the regulation of one carbon units for purine formation, since formylglutamate, an intermediate in histidine degradation, can donate a formyl group to tetrahydrofolate.

Histidase from rat liver and Pseudomonas was labile at temperatures below 0°C, in both its crude and purified form. 186

This is a confirmation of the findings of Klee (1970b; personal communication) .

Entrapment of histidase in liposomes was found to be maximum in negative reverse phase liposomes constructed from egg phosphatidylcholine-cholesterol-phosphatidic acid (10:8:1) at 5% of the added material. Histidase could also be entrapped in liposomes of a neutral (phosphatidylcholine-cholesterol) composition but none could be detected in multilamellar vesicles, sonicated vesicles or reverse phase vesicles constructed from sphingomyelin or DSPC. The captured volume of reverse phase vesicles is much greater than other forms of vesicle (Szoka and Papahadjopoulo s, 1978). Small uni lamellar vesicles have a volume of 0.5ul/mg lipid, multilamellar vesicles of 4.0pl per mg lipid, whereas reverse phase liposomes have a captured volume of lOpl/mg lipid. Electron micrographs presented in Section B show reverse phase vesicles as a heterogeneous uni- or oligo-lamellar population from 0.2-ljjm in diameter. The fact that sphingomyelin reverse phase liposomes did not entrap histidase may result from the temperature of preparation (32°C) or the fact that these lipo somes are smaller with a tighter bilayer packing, sphingomyelin interacting with cholesterol even more strongly than phospha tidylcholine (Demel et al, 1977). This also means that sphingomyelin-cholesterol liposomes are more stable in the circulation as they are highly resistant to high density lipoprotein attack. Distearoylphosphatidylcholine reverse phase liposomes prepared by the Battelle method are also smaller than egg phosphatidylcholine liposomes (80-200nm) and more stable in plasma. But inability to entrap histidase limited their usefulness in this study.

The efflux of histidine from liposomes was shown to be extremely low. This is a confirmation of the work of Klein et al (1971), who found that histidine had the lowest rate of efflux of all amino acids. They suggested that the feature that distinguishes histidine is the positive charge that is carried on its imidazole ring. It may interact by hydrogen bonding or the anomalous behaviour compared to other amino acids may be because it can only pass through the hydrophobic 187 region of lipid in the non-polar rather than the highly polar zwitterionic form. The permeability of amino acids through liposomes has also been studied by entrapment of D-amino acid oxidase and determination of D-amino acid permeability by oxygen consumption (Naoi et al, 1977). They found histidine and proline had the lowest rates of oxidation. Both these studies observed that the rate of amino acid efflux depended on the composition of the lipo somes. Amino acids diffused more quickly from liposomes formed from highly unsaturated phosphatidylcholines. In this study, the diffusion of histidine was marginally faster through liposomes made from sphingomyelin than through those composed of distearoylphosphatidylcholine. However incubation of distearoylphosphatidylcholine liposomes with plasma caused an increase of histidine efflux whereas plasma had no effect on efflux from sphingomyelin liposomes. The efflux was very small in all cases, and would not be sufficient for in vivo depletion of circulating histidine in histidinaemic mice. This assumes that diffusion histidine into liposomes occurs at the same rate as the efflux shown in these experiments. The instability of distearoylphosphatidylcholine liposomes in the presence of plasma proteins is probably due to phospho lipid exchange. Sphingomyelin has increased stability in vivo (Dunnick et al, 1975, 1976).

The distribution of egg phosphatidylcholine reverse phase liposomes in mice showed a maximum uptake into the liver between 20 minutes and 1 hour after administration. Since 125 free aqueous phase marker, I PVP, was not taken up into tissues, it demonstrates liposomal localisation in liver. Although over 70$ of the injected dose was found in the liver after 20 minutes, only 38$ was left after 3 hours. Considering the fact that 125 I PVP is metabolised at a slow rate, the fall in hepatic radioactivity could be due to extracellular break down of liposomes associated with hepatic endothelial cells, with release of entrapped marker. There was no parallel rise in blood radioactivity, which implies that excretion of the free marker must have been rapid. Evidence that this is a general phenomenon has been produced by Souhami et al (1981), Tuzel (1983), Freise et al (1981). They suggest that initially liposomes are associated with the vascular endo thelium of the liver, where some may be internalised and others broken down. 188

Reverse phase liposomes composed of sphingomyelin-cholesterol (3:2) had a half-life in the circulation of over 5 hours compared to less than 5 minutes for egg phosphatidylcholine anionic reverse phase liposomes. Sphingomyelin has been observed when mixed with cholesterol to have very little interaction with cells in vivo (Gregoriadis & Senior, 1980; Hwang et al, 1980). This, together with the fact that liposomes composed of sphingomyelin retain their integrity in the circulation (Ellens et al, 1981), make them an ideal carrier for long term survival of enzyme in the circulation. Unfortunately, not only is the entrapment of histidase low in these vesicles but also, the diffusion of histidine through the bilayer is almost undetectable..It would seem that any type of liposome that is stable to interactions with high density lipoproteins would have a low permeability to histidine and would be of little use as a long circulating protective metabolic capsule for histidine depletion in the circulation of histidinaemic mice.

Liposomes are cleared from the circulation in relation to their size (Juliano and Stamp, 1975)> large vesicles being removed to the liver quicker than small. In all liposome preparations, cholesterol was present in 4-0 mole % to increase liposomal stability by reducing the loss of phosphatidylcholine to high density lipoproteins (Kirby et ai, 1980; Tall and Small, 1977). Incorporation of cholesterol increases liposomal size but does not increase liposomal uptake into liver (Tuzel, 1983) .

Subcellular localisation of ^"^I pyp anc} 125j hiStidase entrapped in egg phosphatidylcholine-cholesterol-phosphatidic acid (10:8:1) showed a 1+0% deposition in hepatic lysosomes. 125 The endocytosis of I PVP containing liposomes by the hepatic reticuloendothelial system has been well documented (Roerdink et al, 1981; Mayhew et al, 1980; Hwang and Pagano, 1975). Following liposomal breakdown in lysosomes, there should be a concomitant transfer of label to the cytoplasmic fraction (Gorden and Cohn, 1973). The difference between this and initial appearance of the liposomal aqueous phase label in the cytoplasm by fusion of the liposome with the plasmalemma is very difficult to distinguish and has been extensively examined (Chapter 1, Section C). In this study, the radioactive 189 markers were used to determine the time of maximum uptake of enzyme-containing liposomes into the subcellular fractions of liver. The uptake of exogenous histidase activity into the liver of histidinaemic mice, where there is little endogenous enzyme activity, provided a very good model for the assessment of cytoplasmic enzyme delivery. The optimum time for enzyme uptake from the radiolabelled histidase was 1 hour. Therefore a maximum possible amount of liposomally entrapped histidase was administered to histidinaemic mice to investigate whether active enzyme could be delivered to the cytoplasm or if lysosomal deliveiy of liposomes was prevalent. Only by fusion would active enzyme be delivered because, not only is histidase a cytoplasmic enzyme which cannot function at lysosomal pH, but also it has been shown that proteins delivered to lysosomes are only passed into the cytoplasm after extensive proteolysis (Dingle and Fell, 1969) .

Unfortunately, it is extremely unlikely that vesicles above lOOnm could penetrate the sinusoidal fenestrations for fusion with hepatocyte plasmalemma. The size of liposome required for histidase encapsulation was much larger than lOOnm. However, this sort of preliminary investigation was essential in a search for a carrier that would protect and deliver intact enzyme. As has been discussed, liposomes of a smaller size for hepatic enzyme delivery or prolonged enzyme circulation were inadequate in all parameters - permeability of histidine, entrapment of histidase, etc. The indetectability of histidase in hepatic subcellular fractions after administration of 220 units entrapped in reverse phase liposomes to histidinaemic mice and the concurrent delivery of 6 0 % of the lipid phase cholesterol oleate marker implied the internalisation and destruction of the majority of the protein in lysosomes. The liposome bilayer had probably afforded no protection against the proteases and cathepsins of the lysosomal apparatus.

Therefore, although rapid delivery of histidase in large vesicles to the liver would certainly minimise vesicle disruption in the circulation, it also signifies vesicle uptake into lysosomes with subsequent inactivation of histidase, 190 rather than fusion with hepatocyte plasmalemma and delivery to the cytosol.

Plasma histidine levels were found to be unaltered up to 24- hours after liposome administration. However, injection of free histidase did cause lowering of histidine levels. This will be discussed later, but it does confirm liposomal rapid delivery to the liver and their integrity. An advantage of quick removal of protein-containing liposomes from the circulation is the prevention of a humoral or cellular immune response. When proteins are exposed on the surface of liposomes, it has been observed that the liposomes act as adjuvants (Hudson et al, 1980; Heath et al, 1976). Since association of histidase with the outer liposomal bilayer has been demonstrated (Section D) , problems with immunogenicity of long-circulating liposomes might result.

The solution to these problems seemed to be, instead of tailoring a carrier for histidase delivery to the liver, using one which would be circulated for a prolonged time for depletion of plasma histidine. It has already been discussed above that liposomes stable in the plasma do not have the correct characteristics for this. The extensive work of Hubbard et al (1981) illustrated that erythrocyte ghosts might serve this purpose. Erythrocyte ghosts, supplemented with substrates for ATP synthesis, were used to entrap histidase. Cells with supplemented ATP have been found to have a much longer in vivo half-life (Hubbard et al, 1981).

The rate of histidine entry into erythrocyte ghosts was small with a distribution ratio of 0.3 after two hours. Published values for amino acid distributions across the erythrocyte give 0.3 for L-histidine after 60 minutes, 1.06 for L-phenylalanine, 1.25 for L-tyrosine, 0.95 for proline, 0.81 for alanine (Winter and Christensen, 1964-) • The rate of entry of neutral amino acids is directly related to the size of their hydrocarbon side chains. Three modes of uptake have been demonstrated into erythrocyte ghosts (Winter and Christensen, 1964-) , a mediated transport used by almost all neutral amino acids, prefering long chain amino acids and saturable; a low capacity uptake for alanine 1 9 - l and glycine; a non-saturable uptake limited for amino acids with large hydrocarbon side chains. Since histidine carries a positive charge, and substances cross membranes in the undissociated form (Aubert and Motais, 1975), the rate of diffusion of histidine is limited to its non-polar form. The initial influx over thirty minutes demonstrated a non saturable uptake for histidine. Other amino acids have been shown to have saturable uptake with values for phenyl alanine, leucine, valine and alanine of 4..3mM, 1.8mM, 7.0mM and 0.34-mM respectively.

When the intracellular concentrations of histidine were determined by amino acid analysis, about one-tenth of the extracellular concentration was present inside erythrocytes after 30 minutes. As discussed in Section F(2), the rate determining factor in decrease of histidinaemic plasma histidine levels may be the transport of the substrate into erythrocyte ghost-entrapped histidase. However, subsequent investigations into the activity of histidase entrapped in erythrocyte ghosts indicated that in vitro, the level of urocanate production after only fifteen minutes was sufficient to totally inhibit the enzyme. Measurement of the * concentrations of urocanate intracellularly gave approximately 30-4.0pM. Although equilibration between the intra- and extra-cellular medium had not occurred, urocanate could diffuse through the erythrocyte membrane more easily than histidine, probably because it has lost the positively charged a-amino group, making it less polar than histidine (see above). Although in the closed, in vitro situation, product inhibition occurred, this may not be a problem in the in vivo situation if urocanate is quickly removed from the extracellular surroundings, ie. the plasma. The ATP substrates added to the erythrocyte ghosts may also have inhibited enzyme activity.

When the mechanistically similar enzyme, phenylalanine ammonia lyase, was entrapped in erythrocyte ghosts, nearly 3 3 % of the entrapped activity could be demonstrated in the extracellular medium. This demonstrated that the lipid environment had little detrimental effect on the enzyme, in terms of its active site or thiol groups and that the difference shown between histidase and phenylalanine ammonia 192 lyase was probably solely due to substrate transport. Phenylalanine, on account of its long hydrocarbon side chain, was more permeable than histidine to lipid membranes, and equilibrium was reached after 20 minutes with 3mM phenylalanine. These results are compatible with those of Hubbard et al (1980), who showed an entrapment of between 5.8$ and 9.4-$ for phenylalanine ammonia lyase on lysis of erythrocyte ghosts and an equilibration of 4-mM phenylalanine into erythrocytes of 20 minutes. However, they did not demonstrate the activity of entrapped phenylalanine ammonia lyase. In the present work, the fact that entrapped phenyl alanine ammonia lyase activity fell off after 3 hours while histidase activity ceased after fifteen minutes shows that both are product inhibitable but the substrate and product of one are more permeable to erythrocyte membranes than those of the other.

The circulatory half-life of erythrocyte ghosts was demonstrated 125 with entrapped I PVP to be 28 hours. Restoration of erythrocyte ghost ATP concentrations has been found to increase survival of erythrocytes in vivo (Hubbard et al, 1981). Since reduced glutathione is also important for survival and for histidase activity, this was included in the dialysis buffers in the formation of ghosts. 125 Release of entrapped I PVP from erythrocyte ghosts into the plasma of A2G mice was observed to a small extent, with less than 9$ on injection, falling to less than 1$ after 22 hours. After 94- hours 27$ of the injected dose had been excreted (Table Gl), with 25$ taken into liver and 13$ in spleen. Since the polymer is rapidly excreted when released in the plasma (Figure Gl), it can be concluded that there was a gradual lysis of erythrocyte ghosts in the circulation which was approximately equivalent to the uptake into liver. However, the distribution of erythrocyte-entrapped 125 I histidase after 125 injection was different to that of erythrocyte-entrapped I 125 PVP. Plasma levels of I histidase were high on administration, indicating a large initial lysis of ghosts (Table G3). After the initial lysis, the rate of clearance of both erythrocyte- entrapped histidase and erythrocyte-entrapped 5I PVP was similar (Figure G4-) • It appears that the remainder of the 193

ghosts were stable in the circulation. 125 I histidase uptake into liver does not significantly increase with decreasing erythrocyte-associated radioactivity (Table G3). Since only 125 7% of erythrocyte-associated histidase is lost between 5 hours and /f8 hours, it is difficult to determine if 125 I histidase was being eliminated by uptake into liver and spleen or by excretion via the kidneys.

Histidinaemic mice, administered histidase entrapped in erythrocyte ghosts, showed a 50% fall in the plasma histidine levels after one hour, which persisted for 5 hours. Histidin aemic mice administered free histidase showed a fall in plasma histidine levels to 3 % of the initial value after one hour (Figure G3). In vivo, the same amount of histidase (20 units) caused a 2-fold drop in histidine when entrapped in erythrocyte ghosts and a 30-fold drop when free. Since it has been demonstrated, using histidase, that 28% of the dose was released into plasma immediately on injection (Table G3) , there would be only a %,-fold difference in plasma-associated histidase activity between mice administered free histidase and mice administered erythrocyte entrapped histidase. It would therefore be expected that, disregarding the erythrocyte- associated activity, only a 4--fold difference in histidine depletion between the two conditions would be seen. There was a 15-fold difference between the free histidase depletion and the erythrocyte entrapped histidase depletion of plasma histidine levels. There may be many explanations for the low response to the erythrocyte entrapped histidase treatment.

One solution may be on account of the greater concentration of protein which was entrapped in ghosts for histidinaemic mouse treatment (20 units was O.Olmg protein) than for histidase distribution studies. This may have caused a distortion of the erythrocyte ghost membrane. When injected into histidin aemic mice, these ghosts may have been removed by the reticulo endothelial system of the liver and spleen to a far greater extent than I histidase-containing ghosts. The liver is responsible for removal of badly damaged erythrocytes, while the spleen can detect small changes in erythrocyte membrane shape (Chapter l). A further experiment is required where 125 I histidase is entrapped with non-labelled histidase to m give a high protein concentration and the subsequent distribution of erythrocyte ghosts determined.

Another solution may be the inactivity of erythrocyte- entrapped histidase in vivo. It has already been demonstrated that erythrocyte entrapped histidase is subject to product inhibition in vitro. Urocanate may build up within the erythro cyte ghosts and prevent histidase activity in vivo. It is known that urocanate is rapidly excreted from the plasma via the kidneys. Urocanate levels in plasma after histidase administration were measured by Pauly analysis and found to be undetectable. Product inhibition, therefore, would certainly not prevent histidase activity in the plasma. Erythrocyte entrapped histidase may be significantly inhibited if diffusion of urocanate through the erythrocyte membrane was limiting. Further studies on the equilibrium of urocanate between plasma and erythrocytes are required. A comparison of the data for 125 I histidase distribution between plasma and erythrocytes (Figure G2) and the histidine levels in histidinaemic mice (Figure G3)> demonstrates that histidase activity is still associated with intact erythrocytes at a time when histidinaemic plasma levels have returned to their initial levels. This may support the suggestion that product inhibition of histidase activity had occurred within the erythrocytes.

A combination of the above two solutions may explain why free histidase is more successful at plasma histidine depletion than erythrocyte entrapped histidase.- There may be a more rapid removal of the latter into the reticuloendothelial system immediately on injection, while the remainder may be subject to product inhibition. Further work in vivo is necessary to determine whether erythrocyte associated radioactivity after 6 hours represented histidase which had become inhibited inside ghosts or if it represented radiolabel attached to other erythrocyte proteins. Histidine levels in histidinaemic mice returned to normal within 24- hours due to large pools of this amino acid in liver and other tissues in equilibrium with that in the plasma.

Although erythrocytes show great promise for replacement therapy in vivo on account of their low immunogenicity and 195 long circulatory half-life, further studies are required to determine the reason for the initial lysis on administration to mice. Turbulence-induced lysis may have resulted in passage through the fine needle needed for injection.

Conclusions that can be drawn from this chapter are numerous. Amounts of enzyme that would be effective for histidinaemic therapy can only be entrapped in large reverse phase vesicles. The majority of these are cleared to the lysosomal apparatus of the liver where the enzyme is inactivated. The majority of large vesicles are always cleared to the reticuloendothelial system (RES) (Juliano, 1981) and the only parameters that can be changed (size, charge, lipid composition) will only alter the pharmokinetics of RES uptake. Entrapment in small vesicles was not possible, but the disadvantages of long circulating vesicles with exposed protein has already been discussed. It would appear from this work that the limiting factor in therapy using any carrier-mediated circulation of histidase is the inability of histidine to diffuse through hydrophobic membranes. The properties of substrate and product inhibition of Pseudomonas histidase are such that rapid diffusion of substrate and product is required. Erythrocyte ghosts, providing an ideal long circulating carrier, are also subject to this limitation. Other enzyme activities entrapped in erythrocyte ghosts have been found to have similar dependence on the rate of substrate diffusion, for example uricase (ihler e t al, 1975). Further experiments are required to reduce the initial lysis of histidase- containing ghosts on injection into mice. This might be done by improving the purification procedure in order to increase the specific activity of the enzyme, enabling a lower total protein content for a similar enzyme activity. Parameters such as erythrocyte ghost glucose, glutathione and ATP content were not measured as this has been performed in a previous study (Hubbard, 1981). Adenosine was found to be the best substrate for ATP restoration, which is deaminated to form inosine and provides adenine nucleotides by the action of adenosine kinase. The latter alternative only occurs in the presence of magnesium ions (Lerner and Rubinstein, 1970). Therefore it was found that adenosine and magnesium ions restored erythrocyte ghost levels of ATP better than inosine alone. - 196 -

In the present work, glutathione supplementation was also considered necessary. But there may be other factors, beside membrane distortion or glycolytic nucleotide deficiencies, that cause the rapid lysis of histidase containing erythrocyte ghosts in vivo. 197

CHAPTER 3

THE POSSIBLE USE OF LIPOSOMES AND ERYTHROCYTE

GHOSTS FOR ENZYME THERAPY IN MURINE HYPERPROLINAEMIA 198

SECTION A

INTRODUCTION

Two disorders of proline metabolism involving the degradation of proline to glutamate have been characterised in man and mouse. Type I hyperprolinaemia involves a disorder in the oxidation of A1-pyrroline-5-carboxylate. It is caused by the deficiency of proline oxidase (Efron, 1966). Type II hyperprolinaemia involves a disorder of the oxidation of A 1-pyrroline-5-carboxylate to glutamate semialdehyde. It is caused by a deficiency in A1-pyrroline-5-carboxylate dehydro genase (Figure Al).

The condition in humans is often detected as it is accompanied by renal disease (Shafter et al, 1962). The condition itself is asymptomatic, but the high level of plasma proline "over flows" through the kidneys, and renal dysfunction may be secondary to the hyperprolinaemia. There is no association with mental retardation (Mollica and Pavone, 1976). Decreasing dietary proline to decrease plasma proline levels would be difficult because nearly all food proteins contain proline.

1. Hyperprolinaemic Mice

One of the very few murine animal models of a human inborn error of metabolism is the PRO/Re mouse. It has elevated proline in plasma and urine and a deficiency of proline oxidase (Blake, 1972). The specific activity of liver proline oxidase in the mitochondrial preparations of PRO/Re mice was 0.4. enzyme units/mg protein, whereas normal mice have an activity of approximately 10 units/mg protein. Proline oxidase in kidney and brain are also 1-2% of the control values. In spite of this, proline oxidation in the kidneys is still 20% of normal.

Studies have shown that Type I hyperprolinaemia in the mouse is caused by an abnormal allele at a single locus, pro-1. The normal and Type I hyperprolinaemic alleles at this locus £L b are designated pro-1 and pro-1 , respectively. Two proline oxidase components have been detected (Blake et al, 1976). Figure A1 METABOLISM OF PR0LIK5

:-:2o N

to. l 7 U A I

Glutamate

a keto glutamate 200

In PRO/Re mice, component 1 is missing, and residual activity is due to component 2. The K of the enzyme in PRO/Re mice is /OOmM, while that in C57B1 mice is 2.9mM. Component 2 is more stable at /6°C, shows a slower electrophoretic mobility and is less reactive with menadione (Vitamin which can be reduced by proline oxidase).

The proline excretion in PRO/Re mice is far more than expected from plasma proline levels. Intracellular kidney levels of proline are /-fold elevated. Transepithelial reclamation is impaired because the high intracellular levels of proline means there is unidirectional backflux at the luminal membrane. This causes a 50-times normal excretion of proline. Although the membrane transport and intracellular metabolism of substrate are independent functions, the metabolism of proline by the kidney has a profound effect on its transcellular transport (Scriver et al, 1975)• This does not occur in histidinaemia because the role of the kidney in oxidation of histidine is negligible so that the intracellular concentration of this amino acid is not modulated by its renal metabolism.

The PRO/Re mice are also characterised by an altered polyamine metabolism. Putrescine and spermidine are all excreted at 1-3 times the level found normally (Manen et al» 1976). The metabolism of taurine is also altered in PRO/Re mice. This primary amine or the polyamines are responsible for the bright yellow coloration of urine, which causes pine wood shavings to be stained yellow.

An acceleration in the synthesis of polyamines (Figure A2) may be a mechanism for decreasing the overload of proline. The higher concentrations of proline cause an increase in ornithine (Efron, 1965) . Polyamines are synthesised by the action of ornithine decarboxylase. PRO/Re mice for some reason have 200% ornithine decarboxylase activity of normal mice. Elevated proline levels may derepress the synthesis of this enzyme.

The PRO/Re mouse in the heterozygous state does not have an abnormality of proline metabolism. Proline levels in plasma of hyperprolinaemic mice are about 2mM.

A comparison of hyperprolinaemia in mouse and man is given in Table Al. 20.1

Figure A2

C02H I (CH2]2 CHO I HC—NH2 H2C- -CH2 H2C------CHj [C H2]2 , 1 C02 H H2( Xn^CH— c o2h h c^ n^ ch—C02H HC—NH2 I Glutamic acid H C02H Proliuc A1-Pyrrol ine-5- Glutamicy- carboxylatc semialdehydc NH2 NHa I [CH212 [C H 21i HC—NH2 NH| NH2| co2h lCHa]« 5’-Adenosyt- [CHJ, •J-Adenosyt- 1 m ethionine m ethionine Ornithine NH NH n h2 | decarboxylase 1 decarboxylase fCH2]2| [CHJ, [c h2]4 n h2 n h2 n h2 Spermine Spermidine Putrescine

Schematic involvement ofproline in the metabolism of ornithine and the biosynthesis of the polyamincs (Manen et al, 1976) 2 0 2

Table A1 Comparison of Type I Hyperprolinaemia in Man and PRO/Re Mouse. (From R. L. Blake, 1972)

Characteristics Man Animal Model

Genetics 1. Mode of transmission autosomal autosomal recessive recessive 2. Locus pro-1 3. Experimental control congenic. (pro-1t a/ pro-la) mice

B Physiology

1. Aminoaciduria prolinuria prolinuria imunoglycinuria 2 . Blood proline increased several increased several concentration fold fold 3. Staining reaction of unknown positive to pine urine shavings

C Biochemistry 1. Metabolic disorder ”overflow" type ”overflow” type 2. Enzyme defect low mitochondrial deficiency of proline oxidase proline dehydro genase component 1 3. Polyamine metabolism unknown alteration in enzyme activity of kidneys 2 . Proline Oxidase

(i) Mammalian Proline Oxidase

Proline oxidase is a membrane-bound enzyme found associated with the inner mitochondrial membrane. The proline oxidase system comprises a proline dehydrogenase linked to the electron transport chain, in an analogous manner to succinate dehydro genase. It is a flavoprotein with a tightly bound prosthetic group, FAD. There are two components, showing low and high proline oxidase activity, detectable on gel electrophoresis. Mammalian proline oxidase has not been extensively purified, studies mainly having been performed on mitochondrial suspensions from rat liver and kidney (Johnson and Streker, 1962; Lang and Lang, 1958) and human liver, brain and kidney (Taggart and Kraukaur, 194-9). Proline oxidation, as measured by oxygen utilization, has not been demonstrated in rat heart, muscle or brain nor in pig liver or kidney. The primary electron acceptor from proline oxidase is not known. Electron transport chain inhibitors, such as cyanide and antimycin A, also inhibit proline oxidasp. Electrons are probably fed to ubiquinone. The succinate dehydrogenase complex, at Site II in the electron transport chain, has been found to consist of FAD, three iron- sulphur centres and cytochrome b. Proline oxidase may have a similar mechanism of electron transport to ubiquinone. The particulate preparation of proline oxidase can be made dependent on cytochrome c by depletion of endogenous cytochrome c. There may be a coupling site for ATP synthesis. Proline oxidase can be reoxidised by artificial electron acceptors such as ferricyanide, phenazine methosulphate, INT (iodophenyl nitro- phenyltetrazolium) and dichlorophenolindophenol.

Proline oxidase is inhibited by lactate (Kowaloff et al, 1977 a) . Since proline can serve as a gluconeogenic precursor, regulation of proline degradation by lactate may be a mechanism for allocation of metabolic fuel sources. In lactic acidosis, hyperlactacidaemia blocks proline degradation and produces hyperprolinaemia. In two patients with lactate levels 10-fold normal, plasma proline levels were also 3-fold elevated (Haworth et al, 1976). Glucocorticoids also stimulate proline oxidase (Kowaloff, 1977 b) increasing the synthesis of the 20/f enzyme. Steroids increase proline release from muscle and absorption from the gut.

An intercellular proline cycle has been demonstrated between hepatocytes and erythrocytes (Phang et al, 1981). In hepatocytes, proline is converted to A 1-pyrroline-5-carboxylate by proline oxidase. In erythrocytes, A 1-pyrroline-5-carboxylate is converted to proline by A 1-pyrroline-5-carboxylate reductase (Figure A3).

This metabolic link stimulates the hexose monophosphate pathway by oxidation of NADPH. This is important in erythro cytes for the production of phosphoribosylpyrophosphate (PRPP) for nucleotide synthesis. In hepatocytes oxidation of proline can be a glucose-independent mechanism of ATP generation via the electron transport chain, without the requirement for NADH oxidation.

(ii) Bacterial Proline Oxidase

Only two highly purified preparations of proline oxidase have been reported, from E .coli (Scarpulla and Soffer, 1978) and from Salmonella typhimurium (Menzel and Roth, 1980). The native molecular weight for the E .coli enzyme was estimated to be 200,000 to 260,000 and the denatured reduced molecular weight of 124»000, suggesting that it is a dimer. The S.typhimurium enzyme was found to be a dimer of identical 132,000 dalton subunits. Lactate and pyruvate were competitive inhibitors. In bacteria, the enzyme is associated with a particulate fraction consisting of a membrane bound electron transport chain.

The put A gene may code for both proline oxidase and A 1-pyrroline-5-carboxylic acid dehydrogenase. The enzyme purified from E .coli (Scarpulla and Soffer, 1978) appeared to catalyse only the proline oxidase reaction, whereas the S .typhimurium enzyme demonstrated both activities (Menzel and Roth, 1978). These conflicting reports appear to be due to the different procedures employed for purification of each enzyme.

Proline degradation in bacteria is inducible and subject to catabolite repression (Frank, 1963; Ratzkin, 1978). A mutation of the E . coli proline oxidase resulted in an elevated iueA Te neclua proline cycle (Phang intercellular The al et FigureA3 HEPATOCYTE ERYTHROCYTE

P5C = A 1-pyrroline-5-carboxylate 6PG = 6-phosphogluconate fp = flavoprotein RuP = ribulose 5-phosphate G6P = glucose 6-phosphate HMP = hexose monophosphate pathway 19817 ^Ub synthesis of this enzyme due to a loss of a leucyl, phenyl- alanyl-t RNA protein transferase (Deutch and Soffer, 1975). This enzyme is a regulator of proline catabolism. The mutant could be useful in the production of proline oxidase for the extensive quantities needed for therapy for hyperprolinaemia.

3. Objectives of this Study

The purification of proline oxidase from a mammalian and bacterial source was investigated for a replacement attempt in hyperprolinaemia. The enzyme was encapsulated in liposomes and erythrocyte ghosts and the resultant activity determined. The enzyme was administered, after encapsulation, to PRO/Re mice and the effect on blood proline levels monitored. The carriers were not used to direct the enzyme to the subcellular sites of pathology, because it is extremely unlikely that liposomes or erythrocyte ghosts could deliver proline oxidase to the mitochondrial membrane. Carriers were used to prolong and protect the life of the enzyme in the circulation.

This approach was of only limited success in histidinaemia due to the rapid lysis of erythrocyte ghosts containing histidase in the plasma and the inability of liposomes to deliver active enzyme to the liver. This may not be the case with hyperprolinaemia. Proline oxidase is a membrane-bound enzyme and encapsulation in liposomes and erythrocyte ghosts may enhance its activity. The characteristics of proline diffusion through membranes may be more favourable than those of histidine. If proline could diffuse quickly through liposomal and erythrocyte membranes, these carriers may serve as metabolic compartments for the degradation of the elevated plasma proline levels found in hyperprolinaemia.

The problem of reoxidation of the enzyme after entrapment in liposomes and erythrocyte ghosts ivas investigated as these carriers possess no electron transport chain. 207

SECTION B

MATERIALS AND METHODS

MATERIALS

1. Animals

PRO/Re mice and C3H mice were provided by Dr. Kacser, Depart ment of Genetics, University of Edinburgh. These had been received as a gift from Dr. Blake, The Jackson Laboratory, Bar Harbor, Maine, USA.

A2G mice were from the Animal House, Charing Cross Hospital Medical School.

2. Bacteria

Proline oxidase was induced from a strain of Escherichia coli K12 by growth on proline as the sole carbon and nitrogen source. Bacteria had been previously isolated from a urinary infection.

3. Reagents

The following chemicals were obtained from Sigma:-

p-iodophenyl tetrazolium violet (INT; 2-(4--iodophenyl)-3- ( 4--nitrophenyl)-5-phenyl tetrazolium chloride)

cytochrome c (horse heart) o-aminobenzaldehyde L-proline Tween 20 Dowex 50W (hydrogen form, strongly acidic cationic-exchange resin, 8% cross linked, 100-200 mesh)

L(U-^C) proline was obtained from Amersham International.

k . Apparatus

Apparatus used was as in Chapter 2. 208

METHODS

1. Proline Oxidase Assays

(i) o-aminobenzaldehyde

The membrane-bound enzyme was assayed using o-aminobenzaldehyde (Strecker, 1971). The product of the reaction, A1 -pyrroline- 5-carboxylate combines with the o-aminobenzaldehyde to form a dihydroquinazolium derivative. The extinction coefficient is 2710 M"^cm’^ at 443nm. The reagents used were: - A: Potassium phosphate buffer 0.5M, pH 7.5 B: L-proline C: 0.5$ o-aminobenzaldehyde in 5$ trichloroacetic acid

The incubation mixture containing 0.3ml A and 0.1ml B was placed at 37°C in a shaking water bath. Mitochondria (0.3- 6.0mg) or the membrane bound bacterial enzyme were added to the flask and the reaction incubated with shaking for aerobic conditions for 30 minutes. The reaction was stopped by addition of 2ml C. The flasks were left at room temperature for twenty minutes and the solutions were centrifuged at 5000xg for ten minutes. The white pellet was discarded and the yellow supernatant was read at 443nm. oxidase Units of proline activity are the ymoles product formed per hour at 37°C.

(ii) iodonitrophenvl tetrazolium violet (INT)

After isolation from membranes, proline oxidase requires the presence of an electron acceptor. To measure purified proline oxidase activity, the reduction of the electron accepting dye iodonitrophenyl tetrazolium was followed at 520nm. The molar extinction coefficient of the reduced dye is 11500 M~^cm_^ at 520nm at 25°C. Units of enzyme activity are in umoles A 1-pyrroline-5-carboxylate per hour. The solutions used were:- 0.1ml 0.2M L-proline 0.16ml 99$ ethylene glycol 0.1ml 4-$ Tween 20 0.54ml 0.3M Tris-HCl pH 8.9 0.1ml 5mM INT 209

When the purified enzyme was entrapped in liposomes and erythrocyte ghosts, the buffer used was 5mM potassium phosphate containing 5mM INT pH 7.4-. Entrapped enzyme was assayed by addition of L-proline, final concentration 20mM, in 5mM potassium phosphate pH 7.4«

(iii) A radioisotopic assay for proline oxidase

In order to detect small quantities of proline oxidase, an assay using proline was developed. The radioisotopic assay used by Phang et al (1975) gave non-reproducible results. A modification of this assay involved reaction of the enzyme with proline and separation of the radioactive product from the substrate by Dowex ion-exchange chromatography.

It was found essential to purify the proline. This was performed by ion-exchange chromatography on Dowex 50 (acidic form). The column was equilibrated with 20mM citrate buffer pH 2.8. 1^'C proline was acidified with 33$ 5-sulphosalicyclic acid (SSA) and added to the column. The A 1-pyrroline-5-carboxylate was eluted 10ml 20mM citrate pH 2.8. Fractions of 1ml were collected (Figure Bl) . *^C proline was eluted 20ml phosphate buffered saline. 1ml fractions were collected. The purified proline was used for all radioisotopic assays of proline oxidase.

Proline oxidase was assayed as in (ii) with INT as the electron acceptor. 0.25uCi proline were added to the assay. After various time intervals the reaction was stopped by the addition of 150ql 33$ SSA. The samples were passed through a Dowex 50 column (2x0.5cm) equilibrated with 20mM citrate pH 2.8, and A 1-pyrroline-5-carboxylate eluted with 10ml of the same buffer. Fractions of 1ml were collected, the first ten representing the total production of A 1-pyrroline-5-carboxylate. The activity of proline oxidase was calculated in the following manner: n n counts in first peak . qmoles P5C/min =100 x ------e--- x prolme m assay total counts added (in praoles) to assay

The production of A 1- pyrroline-5-carboxylate was linear with time. i— 1 -vi- o CPM x l O ; /fraction a eue ih1m o h sm bfe (first buffer samepeak). ofthe10ml eluted with was o oe 5 + o-xhne columnequilibrated 50 ion-exchange 20mMwithH+ ato Dowex h rdostpcasyo rln oxidaseinvolvedofprolinethe purificassay The radioisotopic irt p .. Contaminating Theradioactivity applied pH2.8.was citrate use. before proline ofation rln ws ltdwt 2m popae buffered saline phosphate 20ml eluted 7.4. pHwithproline was (second peak). 4 ------0M citrate20mM H 2.8 pH ------» ------H 20 - - 210 hsht buffered salinephosphate A 1-pyrroline-5-carboxylate H 7./ pH 211

2. Entrapment of Proline Oxidase in Liposomes

Proline oxidase was entrapped in ten minute sonicated anionic liposomes comprised of phosphatidylcholine-cholesterol- phosphatidic acid (molar ratio 10:8:1) as in Chapter 2. Concentrated proline oxidase was dialysed against 5mM potassium phosphate buffer pH 7.4-- After dialysis, FAD was added to give a final concentration of ImM. INT was added to give a final concentration of 5mM. 1ml enzyme solution was added to 3ml diethyl ether containing the above molar ratio of lipids. Free enzyme was separated from entrapped by gel filtration through Sepharose 6B equilibrated with 5mM phosphate pH 7 ./+• Proline oxidase entrapped in liposomes was determined by addition of proline at a final concentration of 20mM, and reduction of the entrapped INT measured by increase in optical density at 520nm. Latent enzyme activity was determined by addition of 0.2$ Triton X100 to disrupt the liposomes, followed by the above assay. Absorption of proline oxidase to the outside of liposomes was determined by addition of the enzyme to pre-formed liposomes.

3. Entrapment of Proline Oxidase in Erythrocyte Ghosts

Proline oxidase was entrapped in murine erythrocyte ghosts by the method of Hubbard & Chalmers (1981) with supplemented adenosine (4-mM) glutathione (3mM), magnesium chloride (4.mM) and glucose (5mM). In initial experiments 5mM INT was added to the lysing and resealing buffers. Since there are several endogenous oxidising agents in erythrocytes, with the potential to reoxidise proline oxidase, it was subsequently omitted from further experiments. It was found that INT caused haemoglobin to change from a bright red to a dark red colour. The haemoglobin may have become oxidised from the low spin Fe 2+ to the high spin Fe 3+ methaemoglobin state.

Entrapment of proline oxidase in erythrocyte ghosts was measured by the radioisotopic method. Proline oxidase was always supplemented with FAD before entrapment. Intact or lysed erythrocytes were incubated with various concentrations of proline with O.luCi ^ C proline in phosphate buffered saline, pH 7.k . The reaction was stopped by addition of 33^ 212

SSA. The reaction mixture was passed through Dowex 50 equilibrated 20mM citrate buffer, pH 2.8. The product, A 1- pyrroline-5-carboxylate, was eluted in the first peak and counted.

4-. Detection of Hyperprolinaemic Mice

Hyperprolinaemic mice were identified in two ways. Their urine was tested for its ability to stain pine wood shavings yellow. Hyperprolinaemic mice were also tested for by paper chromatography of the urine. Since urine proline levels of homozygotes should be 50 times the level found in normal or heterozygous mice (about lmg/ml), this could be identified by staining with isatin. 50ql urine from each mouse was spotted onto Whatman 3MM chroma tography paper. It was sprayed with 0.2$ isatin in n butanol, 4$ with respect to acetic acid. The stain was dissolved by heating to 105°C before use. Proline gives a blue spot. The limit of detectability is about ljjg proline.

5. Passage of Proline through Erythrocyte Ghost Membranes

Influx of proline into erythrocytes was performed as for histidine in Chapter 2. Erythrocyte ghosts were formed from mouse blood as described previously. The resealed cells were incubated at a 50$ haematocrit with 3mM L-proline containing O.lyCi 1^C proline in isotonic phosphate buffered saline pH 7.4-. The cells were incubated at 37°C with shaking. Aliquots (0.2ml) were removed at various time intervals. After immediate addition of 1ml ice cold phosphate buffered saline, they were centrifuged at 15000xg for 2 minutes. The supernatants were removed and placed in scintillation vials for scintillation counting. The pellets were lysed by addition of 1ml distilled water and brief sonication. The protein was precipitated with 100ql 10$ TCA and the precipitate removed by centrifugation. The clear supernatants (intracellular media) were placed in vials for scintillation counting.

Efflux of proline from erythrocyte ghosts was investigated by preincubation of ghosts with proline (O.ljjCi) in 3mM isotonic proline at pH 7.4- at 37°C for 2 hours. The cells were pelleted and resuspended in isotonic phosphate buffered saline pH 7.4* Aliquots (0.2ml) were taken at various time intervals and added to 1ml ice cold PBS. After centrifugation, the pellets and the supernatants were treated as above. 213

SECTION C

PROLINE OXIDASE

1. Growth of Bacteria

A strain of E. coli K12 was grown on proline as its sole carbon and nitrogen source. The medium used was 0.2$ L-proline, 0.15$ dipotassium hydrogen phosphate, 0.05$ potassium dihydrogen phosphate, 0.1$ yeast extract, 0.02$ magnesium sulphate. The production of proline oxidase in this medium was followed by growing the bacteria in shake flasks over 12 hrs.At various time intervals the cells were centrifuged at 20000xg for 10 minutes and the supernatant was decanted. The cells were resuspended in 1ml sodium cacodylate buffer pH 6.8 with lOpl of toluene. The cells were shaken for 10 minutes at 37°C. Then an equal volume of 200mM proline was added. After 30 minutes, the reaction was stopped by addition of 0.5$ o-aminobenzaldehyde in 5$ trichloroacetic acid. The solutions were incubated for 20 minutes at room temperature, centrifuged at 5000xg for 10 minutes and the optical density of the supernatants at 4-43nm estimated. The time at which proline oxidase production was maximum was determined to be when bacterial growth had just entered stationary phase. Bacteria were grown in batches in a 10 litre fermentor in the above medium at 37°C for eight hours with a 10$ inoculum. They were pelleted by centrifugation at 2000xg for 2 hours in a 6 x 1 litre centrifuge. They were stored at 4°C overnight before purification.

2. Purification of Proline Oxidase

All procedures were carried out at 0-4°C.

Step I Crude Extract

Proline oxidase was purified essentially by the method of Menzel and Roth (1981). Bacteria (25g wet weight) were suspended in 500mM cacodylic acid, pH 6.8. They were broken open by two passes through an ice cold French Pressure cell at 11,000 psi. The disrupted cells were adjusted to 25$ solution with 500mM cacodylic acid and centrifuged at 5000xg 214

for 10 minutes. The supernatant was decanted and the pellet was washed in the above buffer. The cell debris was separated by centrifugation and the second supernatant combined with the first.

Step II Washed Membranes

The supernatant from Step I was diluted to 200ml with 500mM cacodylic acid pH 6.8 and centrifuged for 8 minutes at 110,000xg in a MSE superspeed 60. The supernatant was decanted very carefully and the pellet was retained.

Step III Detergent Extract

The pellet from Step II was resuspended in 100ml 0.1M Tris HC1 pH 8.2 with 0.1$ Tween 20. After careful stirring until the solution was homogeneous, it was centrifuged at 110,000xg for 10 minutes. The supernatant was retained. The pellet was resuspended in the above buffer and centrifuged again. The supernatant was added to the first supernatant. The enzyme required the presence of INT in the assay for activity after this stage because it was no longer in association with the membrane bound electron transport chain.

Step IV Ammonium Sulphate Precipitation

The supernatant from Step III was brought to 50$ ammonium sulphate saturation by dropwise addition of a 90$ ammonium sulphate solution pH 7.0 (475g/l). The solution was stirred for 30 minutes, before centrifugation at 20,000xg for 10 minutes. The supernatant was discarded and the pellet was resuspended in 70mM Tris HC1, pH 8.2 with 0.5$ Tween 20 and 30$ glycerol. The enzyme was desalted by passage through a Sephadex G50 column (2.5x30cm) equilibrated with the above buffer. The enzyme eluted in the void volume and was of a yellow colour.

Step V DEAE-Column Chromatography

A DEAE Sephadex A50 column (5x10cm) was prepared and equilibrated with 70mM Tris HC1, pH 8.2 with 0.5$ Tween 20 and 30$ glycerol. The enzyme eluent from Step IV was applied to the column and washed through with 100ml 70mM Tris HC1 pH 8.2 with 0.5$ Tween 20 and 30$ glycerol and then with 100ml 215 of the same buffer containing 60mM KC1. A linear gradient was applied between 60mM KC1 and l60mM KC1 in the same buffer.

Fractions were assayed for proline oxidase activity. The enzyme was pooled and dialysed overnight against 1 litre

70mM Tris HC1 pH 8.2 containing 0.5% Tween 20 and 3 0 % glycerol.

Step VI Concentration and Storage

The dialysed enzyme was concentrated in an Amicon pressure cell with a PM 30 filter. The enzyme was stored in 30% glycerol at -20°C. It rapidly lost activity if glycerol was removed. Glycerol was always removed by dialysis immediately prior to use.

A typical purification procedure is shown in Table Cl.

3. Characteristics

The of proline oxidase was determined by the Lineweaver- Burk method to be 8.0mM for proline (Figure Cl). It was neither substrate nor product inhibited. It depended on the presence of an artificial electron acceptor for activity (INT). The oxidase assay only was measured. It has been suggested that proline oxidase is a bifunctional enzyme, not only carrying out the proline to pyrroline-5-carboxylate reaction but also capable of converting pyrroline-5-carboxylate to glutamate (Menzel and Roth, 1981).

1. Purification of Mouse Liver Proline Oxidase

Mitochondria were isolated by sub-cellular fractionation of a 30% mouse liver homogenate in 0.25M sucrose and ImM EDTA. The homogenate was centrifuged at 1020xg for 10 minutes. The pellet was discarded and the supernatant was centrifuged at 3300xg for 10 minutes. The pellet contained the heavy mito chondrial fraction. The supernatant was centrifuged at !9,000xg for 10 minutes. This pellet contained the light mitochondrial fraction and the lysosomal fraction. The supernatant was discarded. Both fractions were assayed for proline oxidase activity using both the o-aminobenzaldehyde (0AB) method and the INT method. 216

Table Cl

Purification of E, coli proline oxidase. A typical purific ation started with 30g wet weight bacteria. Units are pmoles A 1-pyrroline-5-carboxylate per hour. The INT assay at pH 7.5 was used.

r' PURIFICATION PROTEIN UNITS SPECIFIC PURIFIC- P STEP (mg) (ymole/ ACTIVITY ATION YIELD hr) (units/mg FOLD protein)

Crude Extract 960 960 1 1 100.0

Washed Membrane 80 2 U 3.05 3.05 25.0

Detergent Extract 5.0 4-6 9.20 9.20 5.0

50$ Ammonium Sulphate/G50 1.8 32 17.7 17.7 3.3

DEAE Sephadex 0.6 25 4-1.6 4-1.6 2.6 217

Figure Cl

Double reciprocal plot of proline oxidase activity against proline concentration. The is 8.0mM for proline. 218

PRO/Re and C3H mouse liver proline oxidase were assayed by preparing mitochondria as above (Table C2) . Fresh mitochondria of PRO/Re mice gave 28$ of the activity of C3H mice, using the OAB assay method. The INT assay method on fresh PRO/Re mitochondria gave 13.7$ of the proline oxidase activity found in C3H mice (Table C2).

Frozen mitochondria assayed by the INT method showed 12.8$ of the proline oxidase activity of normal mice. This is the same value as obtained by the OAB assay for fresh and frozen mitochondria. 219

Table C2

Proline Oxidase Activity in Mitochondria of C3H and PRO/Re Mouse Liver

INT Assay Proline Oxidase % Difference unioles/hr/mg protein

Fresh mitochondria PRO 0.077 28% C3H 0.275 ^(20%)

Frozen mitochondria PRO 0.034 12.8/, C3H 0.275 +(10$) o-Aminobenzaldehyde Assay

Fresh mitochondria PRO 0.007 12.7$ C3H 0.057 + (6.4$)

Frozen mitochondria PRO 0.007 12.7$ C3H 0.057 + (2$)

Percentages given in brackets indicate the values obtained by Blake et al (1976) for differences between PRO/Re mouse liver and C57 mouse liver. 220

SECTION D

ENTRAPMENT OF PROLINE OXIDASE IN LIPOSOMES AND ERYTHROCYTE GHOSTS

1. Entrapment of Proline Oxidase in Liposomes

Small unilamellar vesicles, with a long circulatory half-life and which were stable in the circulation, were used to entrap proline oxidase. The aim was to decrease plasma proline levels in hyperprolinaemic mice and not to direct the enzyme to liver or other organs. Small unilamellar vesicles have a longer half-life than multilamellar vesicles in the circulation, before being cleared to the liver (juliano and Stamp, 1975). A high cholesterol content was used to make them stable (Kirby et al, 1979).

Phosphatidylcholine-cholesterol-phosphatidic acid (molar ratio 10:8:1) with a total lipid weight of 22mg were used to construct liposomes. 1ml proline oxidase containing 76.8 units in 5mM potassium phosphate buffer pH 7 . J+ with 5mM INT was added as the aqueous phase. Liposomes were sonicated for a total of 10 minutes with intermittent cooling.

The profile after gel filtration is shown in Figure Dl. 87$ of the added activity is expressed by intact liposomes, when INT is entrapped together with the enzyme. When Triton X100 was added about 66$ of the added activity was expressed by the liposomes fraction. The decrease in activity after disruption may have been due to the release of INT from the aqueous phase.

Absorption of proline oxidase to liposomal membranes was 5% of the added activity.

Liposomes containing proline oxidase and INT demonstrated the ability to degrade external proline. It was not determined whether proline oxidase was present in the aqueous phase or in the lipid bilayer but the fact that activity did not increase on disruption of the liposomes by detergents suggests the latter. rti rLpd otcldniy 280nm) density (optical Lipid or Protein 0.2 0.1 0.8 1.2 1.0 . . •0.6 - Fraction Number 0.06 0.12 0.21 0.18 0.36 0.30 0.12 1 / h P X O 0 P 0 > CD O P P P, P P O 0 P 0 CO O — P •H i •H •H O •H -P ■P •H -p •H -P _ . j p H P O H- CD H c+ CD P' o a P CD a H* c+ 3 c+ o pr p CO 4 C/D >-1 H* ON ttf o w 0 0 CO Hj O P c*- H* *—t5 CD Q O P KJ *■0 crq liposomes containing proline oxidase and preformed liposomes with added proline oxidase. 87% of the added proline oxidase was entrapped in sonicated liposomes (PC:Ch:PA, 10:8:1). Only 5% of the added activity was associated with preformed liposomes 222

2. Entrapment of Proline Oxidase in Erythrocyte Ghosts

Erythrocytes do not possess proline oxidase. They carry out aerobic glycolysis to form lactate, but they have no mitochondria, no tricarboxylic acid cycle enzymes and they do not carry out oxidative phosphorylation. However, erythrocytes do possess a redox system in their cell membranes (Zamudio and Cenessa, 1966). This comprises a flavoprotein NADH dehydrogenase, a NADH cytochrome c reductase located in the inner half of the bilayer (Miller and Cusanovitch, 1975)> cytochrome b (Bruder et al, 1980) and other undefined species capable of carrying electrons. Erythrocyte ghost-entrapped proline oxidase could be reoxidised without the presence of the artificial electron acceptor, INT.

When proline oxidase was entrapped in ghosts, nearly 60% of the added activity could be demonstrated on lysis of the cells. This was assayed by the radioisotopic method.

When proline was incubated with intact erythrocyte ghosts, the entrapped activity at high proline concentrations was about 60$ of the initial material added.

The free and entrapped enzymes were incubated at various proline concentrations and the production of A 1-pyrroline-5-carboxylate determined by the radioisotopic assay (Figure D2). The free enzyme showed typical Michaelis Menton kinetics with a V at 50mM proline and a Km of 3mM proline. This was slightly lower than that found using the INT spectrophotometric method (8mM) . The entrapped enzyme gave a sigmoidal increase in activity with increasing proline concentration. It was approaching Vmax at 200mM proline and had a Km of approximately 50mM. Increase in the proline concentration from 0 to 4-OmM had. only a very little effect on erythrocyte entrapped proline oxidase. When proline levels were increased above 4-OmM, there was a sudden increase in proline oxidase activity. Since this effect was not seen with free enzyme, it must be attributed to its entrapment in erythrocyte ghosts. It could be a transport phenomenon of proline through the erythrocyte membrane. Measurement of proline transport (see later) showed that this was not the case. Erythrocytes contain endogenous A 1-pyrroline-5-carboxylate reductase which converts the product of the Proline Oxidase Activity (units) rdcin f 1-pyrroline-5-carboxylate byfree A proline ofProduction ih nraig ocnrtos f rln. re proline Free proline. of increasing concentrationswith xds soe tpclMcalsMne kineticsMichaelis-Menten showedoxidase erythro typicalwhile ye nrpe poie xds gvs sigmoidal oxidasea gives curve. entrappedcyte proline xds ad rtrct entrappedproline oxidase erythrocyteoxidase and on incubation iue D2Figure 223 22/f

proline oxidase reaction back to proline. Therefore an explanation of the behaviour of erythrocyte entrapped prolinc oxidase, given in Section F, is based on an intracellular proline cycle which might be occurring.

3. Passage of Proline Through the Erythrocyte Membrane

Influx of 3mM proline into erythrocyte ghosts was maximum after 15 minutes. Efflux of proline showed a similar passage outwards through the erythrocyte ghost membrane (Figure D3).

Influx of proline into erythrocyte ghosts occurred at similar rates for increasing concentrations of proline (Table Dl). The uptake of proline may be saturated at proline concentrations over 0.5mM. Distribution Ratio (intracellular medium) rln (M after 30minutes (mM)Proline Table D1Table ocnrto o CPM inErythrocyte Ghosts ofConcentration iueD Ifu n efu ofprolinethrough efflux andtheerythrocyte Influx Figure D3 nlxo poie t aiu concentrations various at proline into minutes. thirtyerythrocyte ghosts after Influxof ih3Mpoie ih .yi rln. Foreffluxstudies, proline. O.lyCi3mMproline withwith ebae Eyhoye hss t 50$ athaematocrit ghosts were incubated Erythrocyte membrane. erythrocyte ghosts were preincubated for 2forhours.preincubated erythrocyte ghostswere 10.0 3.0 1.0 0.5 14 225 1612 1748 1504 2042 226

SECTION E

ADMINISTRATION OF PROLINE OXIDASE TO PRO/Re MICE

1. Liposomally Entrapped Proline Oxidase Administered to PRO/Re Mice

PRO/Re mice (males) were administered proline oxidase entrapped in sonicated liposomes (phosphatidylcholine-cholesterol- phosphatidic acid 10:8:1). Liposomes (2mg lipid containing 10 units proline oxidase in a volume of 0.2ml) were injected via the lateral tail vein. Blood samples (60pl) were taken from the end of the tail at time intervals. Proline levels were determined by amino acid analysis.

Individual mouse proline levels after administration of liposomally-entrapped proline oxidase, free proline oxidase or saline were determined (Table El; Figure El).

The blood proline levels before treatment are between 0.10 and 0.15mM. Hyperprolinaemic PRO/Re mice have been found to have a blood proline level of between 2mM and 3mM (Blake., 1972). The low blood proline levels found in the PRO/Re mice used in this study raised the question of whether they were truly Mhyperprolinaemic" mice. An isatin test for elevated proline in the urine of PRO/Re mice was performed (see Section E(3))»

The effect of free or liposomally-entrapped proline oxidase

was a 50l drop in plasma proline levels, 1.5 hours after administration. Proline levels declined to undetectable

levels after 3 hours and rose again to 75%> k hours after injection of liposomally-entrapped enzyme. Free proline oxidase caused a 50$ drop in plasma proline levels for over U hours. This may demonstrate that liposomally-entrapped activity is greater, but free activity is more prolonged. However, the proline levels were too low and the mice too few to make any sweeping conclusions.

Levels of proline in PRO/Re mice were too low to make precise statements about circulatory half-life of the enzyme or to determine the response due to enzyme action from that due to 227

Table El

Plasma proline and lysine levels in PRO/Re mice after administration of proline oxidase, either free or entrapped in sonicated liposomes (PC:Ch:PA, 10:8:1). Control mice were administered with saline. Amino acid levels are given as pmoles/ml plasma (mM) ± SE.

Time (hours)

Treatment Amino Acid (nmole s/ ml) 0 1.5 3 l

Liposomally Entrapped Proline 0.091 0.062 ND 0.100 Proline Oxidase (n = 2) 0.152 0.071 ND 0.08^

Lysine 0.185 0.216 0.150 0.41 4 (n = 2) 0.271 0.108 0.36A 0.486

Proline Oxidase (free) Proline 0.126 0.063 0.052 0.063 (n = 3) ±0.035 ±0.023 ±0.027 ±0.032

Lysine 0.32 0.32 0.26 0.45 (n = 3) ±0.009 ±0.010 ±0.006 ±0.03

Saline Proline 0.087 0.107 0.072 O.C63 (n = 1 )

Lysine 0.260 0.291 0.350 0.312 (n = 1 )

ND = not detectable % Initial Plasma Proline Level lsapoielvl i R/e ie after miceadministrationof inprolinelevelsPRO/Re Plasma diitrdwt saline.withadministered rln oiae 1 nt) ihr free or (10 entrapped either in oxidaseunits)proline oiae lpsms P:hP, 081. Control mice were (PC:Ch:PA, 10:8:1).sonicated liposomes Figure El 228 rln oiae (20 O proline oxidase ■ Saline nt enzyme;units 2m^ lipid) Liposome entrapped % Initial Plasma Lysine Level lsalsn ees nPOR mice afteradministration inlysinelevelsPRO/Re of Plasma oto mc ee diitrdwt saline.with administered miceControl were rln oiae 1 uis eitherfree or (10entrapped oxidase units)proline in liposomes. FigureE2 229 230 diurnal variation. Although saline alone caused a small decrease in proline, administration of free or liposomally- entrapped enzyme caused larger, more immediate responses, demonstrating that, indeed, there is an effect over and above that due to the injection.

Lysine levels were not affected by the administration of proline oxidase (Figure E2) . This indicates that the effect was specifically on proline in the circulation.

2. Erythrocyte Entrapped Proline Oxidase Administered to PRO/Re Mice

PRO/Re mice were injected with erythrocyte ghosts containing 10 units proline oxidase. Mice were bled at intervals via the tip of the tail. Blood samples were analysed for changes in proline level (Table E2; Figure E3).

Both free enzyme and erythrocyte entrapped enzyme caused a decrease in plasma proline levels. Because the levels of proline were low, the differences between erythrocyte entrapped proline oxidase and proline oxidase alone are difficult to determine. Lysine levels were unchanged (Table E2), indicating that there was an effect which was specific for proline.

3. Isatin Test on Urine of PRO/Re Mice

The isatin test for proline in urine of PRO/Re mice indicated that proline levels were not above that of normal mice. Hyperprolinaemic mice should possess a proline urine level of 9ijmole/ml, about 50 times that of normal mice. (Blake and Russell, 1972). This is well within the detection limit of the isatin test. Although urine of PRO/Re mice used in this study did not show elevated proline it did stain pine wood shavings yellow.

These mice had much lower blood and urine proline levels than expected and possessed more than the residual 2% proline oxidase activity normally associated with PRO/Re mouse liver (as measured by Blake et al, 1976). These facts indicated that the mice in this study were probably heterozygotes for hyperprolinaemia. The yellow staining of pine wood shavings may mean that the elevated polyamine excretion, characteristic of these PRO/Re mice, was unaltered. 231

Table E2

Plasma proline and lysine levels in PRO/Re mice after administration of proline oxidase, either free or entrapped in erythrocyte ghosts. Amino acid levels are given as umoles/ml plasma (mM) ± SE.

Time (hours)

Treatment Amino 0 1 2 5.5 24- Acid (pmoles/ ml)

Erythrocyte Proline 0.188 0.071 0.084 0.047 0.250 Entrapped Proline (n = 6) Oxidase ±0.014 +0.018 ±0.009 ±0.013 ±0.003

Lysine 0.284 0.410 0.272 0.269 0.470 (n = 3) ±0.016 ±0.012 ±0.020 ±0.011 ±0.008

Proline Oxidase Proline 0.126 0.060 0.072 0.120 0.046 (free) (n = 3) ±0.035 ±0.021 ±0.015 ±0.014 ±0.012

Lysine 0.32 0.214 0.271 0.250 0.317 (n = 3) ±0.021 ±0.025 ±0.020 ±0.015 ±0.013 •&Q. Initial Plasma Proline Level 120 lsapoie ees nPOR mice after ofadministration inproline levelsPRO/Re Plasma rln oiae ihr re orentrappedin freeerythrocyte either oxidase ghosts.proline - iue E 3 Figure —, 2

------"~i » 4-6 ie (hours)Time 232 ------

I 1 ------□ 2k r ■ entrapped proline■ repoie oxidaseD Freeproline Erythrocyte xds (10oxidaseunits) (10units) 233

SECTION F

DISCUSSION

Proline oxidase was induced from a strain of E. coli by growth on proline to give a higher starting specific activity than from rat or mouse liver. Purification was by a simple procedure, involving extraction from the membrane fraction with detergent. After removal from the membrane, the enzyme required an artificial electron acceptor, INT, for activity. It was found to have a Km of 3.0mM for proline when measured using the radioisotopic assay. Previous work on this enzyme has shown that it possesses both proline oxidase activity and A 1-pyrroline-5-carboxylate dehydrogenase activity (Menzel and Roth, 1981). The substrate for the second reaction A 1-pyrroline-5-carboxylate must be chemically synthesised (Williams and Frank, 1975) . This was not performed in this study. However, since the dehydrogenase activity is linked to the reduction of NAD, production of NADH could have been monitored at 34-Onm. The production of glutamate could have also been followed. It would have been interesting to pursue these reactions, if time had permitted.

The starting activity of proline oxidase in isolated mitochondria was nearly 5-fold less in mouse liver (0.275 units/mg protein) than in E. coli (1 unit/mg protein). A murine source for isolation of the enzyme would probably not be sufficient for enzyme therapy. The difference in prbline oxidase activity from fresh mitochondria when assayed using INT and o-aminobenzaldehyde may be because of the action of A1-pyrroline-5- carboxylate dehydrogenase. The INT dye is also reduced by this second dehydrogenation reaction. If pyrroline-5-carboxylate dehydrogenase is preferentially destroyed by freezing (Blake et al, 1976), this would explain the difference in the fresh and frozen mitochondrial assay using INT.

Entrapment of proline oxidase in liposomes was very high (87$). This reflects the lipophilic nature of the enzyme. Although the enzyme is still active when dissociated from membranes, provided it has an electron acceptor, the entrapment in liposomes may enhance its activity. This has been found for 23 K

other mitochondrial enzymes, such as D-3-hydroxybutyrate dehydrogenase, a mitochondrial enzyme, although this is entirely dependent on phosphatidylcholine for activity (Hexter and Goldman, 1973)* Proline oxidase entrapped in liposomes is entirely dependent on the INT entrapped with it. This may lead to less efficient electron transfer than when proline oxidase is in contact with the electron transport chain. Reconstitution of parts of the electron transport chain from both bacteria and mammals into liposomes has been established (Kagawa and Racker, 1971). The terminal oxidase of the E .coli K12 respiratory chain (cytochrome complex) has been reconstituted in liposomes (Kita et al, 1982). It was capable of forming a membrane potential. This protein, if it is capable of accepting electrons from proline oxidase, may be better than INT at reoxidising the enzyme. Cytochrome oxidase has been reconstituted into liposomes (Eytan and Brcza, 1978). The undefined complex which normally accepts electrons from proline oxidase may also be able to be reconstituted into liposomes, but itself would need reoxidising. Detergent, which disrupted the liposomes, caused a decrease in pr.oline oxidase activity. This may have been due to a loss of INT in the aqueous phase or demonstrate the fact that proline oxidase works better in lipid vesicles.

Erythrocytes normally catalyse the reverse reaction of proline oxidase, that is the reduction of A 1-pyrroline-5-carboxylato to proline (Phang et al, 1981). It has been found that erythrocytes incubated with A 1-pyrroline-5-carboxylate release proline into the medium. With a A 1-pyrroline-5-carboxylate concentration of 0.3mM, proline accumulated at a rate of 1 . 1 4 ymole/hour per ml of cells. The sigmoidal kinetics (Figure D2) of proline oxidase entrapped in erythrocytes may be explained by the fact that it is in' the presence of endogenous erythro cyte pyrroline-5-carboxylate reductase. At low proline concentrations, the reverse reaction may remove any product formed, converting it back to proline. This intracellular proline cycle normally occurs intercellularly between erythro cytes and hepatocytes (Phang et al, 1981). At higher prolire concentrations, the reductase activity may be negligible compared to the proline oxidase activity. 235

In this study, A 1-pyrroline-5-carboxylate reductase activity was not measured because its substrate has to be chemically synthesised. The fact that proline oxidase activity in erythrocyte ghosts is low at 3mM proline may mean that the erythrocyte ghost entrapped enzyme would not be useful at lowering plasma proline levels. The reverse reaction would certainly significantly recycle any A1-pyrroline- 5-carboxylate that is produced in erythrocyte ghosts.

Proline transport through the erythrocyte membrane had reached a maximum after 15 minutes. Since increase in proline concentrations did not increase proline uptake by erythrocytes, the transport process was saturated at 0.5mM proline. This excludes the possibility of proline transport into erythrocyte ghosts being the factor which increases proline oxidase activity above 4-OmM proline. However, proline entry into erythrocyte ghosts could be a limiting factor for maximum proline oxidase activity if the actual concentration inside the erythrocyte ghosts is below the of the entrapped enzyme. In vivo proline passes out of the erythrocytes into hepatocytes and so internal erythrocyte proline levels are low. The equilibrium between hepatocytes and erythrocytes may be disturbed in hyperprolinaemic mice. Although hepatocytes have less than 3$ of the normal level of proline oxidase, this sustains 20% of the flux from proline to glutamate, and proline levels are only four-fold elevated.

Administration of free and liposomally-entrapped proline oxidase caused a fall in plasma proline levels due specifically to the enzyme action. Since free proline oxidase required an electron acceptor for activity, oxidising activity must have been provided by a plasma electron acceptor, maybe an erythrocyte membrane protein. This implies that the proline oxidase administered in liposomes need not necessarily have remained within the lipid vesicles. However, liposomally- entrapped proline oxidase did cause a slightly greater drop in plasma proline levels, which may indicate that activity was greater in liposomes. The proline levels were too low to justify repeating these experiments. It would be interesting to repeat them on a different colony of PRO/Re mice, that were hyperprolinaemic. 236 -

Erythrocyte ghost entrapped proline oxidase decreased plasma proline levels. This possibly could mean that proline oxidase had been released into the circulation. If the reaction had occurred in ghosts, it would have been reversed, by the pyrroline-5-carboxylate reductase reaction, and there may have been no change in proline concentrations intracellularlj

However, proline levels in the PRO/Re mice were too low to make accurate statements about the effects of proline oxidase, either free or entrapped in liposomes or erythrocyte ghosts.

In hyperprolinaemia, liposomes may provide the best carrier for proline oxidase because they do not possess pyrroline-5- carboxylate reductase. Liposomes have been shown to act as adjuvants for entrapped protein when administered subcutaneously (Heath et al, 1976). Prolonged circulation after intravenous injection, which is a requirement for proline oxidase therapy, may provoke an immune response, especially if proline oxidase is exposed on the liposomal membrane. Erythrocyte ghosts have the advantage of possessing endogenous electron acceptors, such as NADH cytochrome c reductase and cytochrome b (see Section D(2)) although it is not known if any of the species are at the correct potential for reoxidation of proline oxidase. As in Chapter 2, it seems that erythrocyte ghosts containing exogenous protein may not be stable in the circulation. The fate of the enzyme alone or ghosts could be followed by radio labelling both. It could then be determined if the enzyme remained associated with ghosts and its half-life in vivo.

It appeared that this stock of PRO/Re mice had become hetero zygous for hyperprolinaemia. It was quite interesting that urine still stained pine shavings yellow, indicating that the elevated polyamine metabolism found in the original PRO/Re mouse was unaltered. If this is caused by an increased synthesis of ornithine decarboxylase (Manen et al, 1976), either derepression of this enzyme is still occurring or it is a separate genetic trait from hyperprolinaemia.

For the assessment of the effects of enzyme replacement in hyperprolinaemia with liposomes or erythrocyte ghosts, it is essential that hyperprolinaemic mice are used with elevated 237

plasma proline levels and deficient liver and kidney proline oxidase. The results shown in this Chapter are only very small changes in proline peak heights after amino acid analysis. More accurate data with hyperprolinaemic mice are needed before any firm conclusions as to the usefulness of therapy in this disease are drawn. 238

CHAPTER k

THE POSSIBLE USE OF LIPOSOMES FOR CHELATION THERAPY IN A RAT MODEL OF IRON OVERLOAD 239

SECTION A

INTRODUCTION

Idiopathic or primary haemochromatosis is a result of an inherited metabolic defect in which excessive quantities of iron are absorbed from the diet. It results in hepatic deposits of insoluble haemosiderin. Secondary iron overload can also occur in anaemias, such as thalassemia major, where iron is absorbed over and above body requirements. The multiple transfusions needed increase the iron overload. When there is excessive iron deposition with no evidence of tissue damage, the condition is referred to as haemosiderosis. The extent of haemosiderin deposition and fibrosis varies from organ to organ. In the liver, iron is found in highest concentration in hepatocytes at the periphery of nodules and, to a lesser extent, in the Kupffer cells. The accumulation of iron in reticuloendothelial cells is relatively innocuous, whereas parenchymal siderosis is responsible for most of the clinical manifestations of haemochromatosis.

Iron in biological systems is tightly bound to proteins because free iron may oxidise or hydrolyse, forming insoluble ferritin hydroxide and hydroxide polymers. Iron in the body occurs in three main pools: iron involved in catalytic function and in oxygen transport; storage iron in cytosolic ferritin and lysosomal haemosiderin; transport iron, bound to transferrin in plasma for iron exchange between the different body compart ments. Iron is stored outside lysosomes in the form of ferritin which consists of a protein shell surrounding an iron core. Haemosiderin is a degraded form of ferritin in which the iron cores are no longer associated with intact protein shells. Lysosomes are involved in the breakdown of ferritin. In iron overload, lysosomal damage occurs so that lysosomal hydrolyases are released into the cytoplasm. Lysosomal fragility increases in proportion to the haemosiderin content. Fibrosis occurs in most tissues containing excessive amounts of storage iron. Iron may stimulate collagen synthesis. The classical clinical tetrad of haemochromatosis comprises hepatomegaly, skin pigmentation, diabetes mellitus and hypogonadism, but over half the homozygotes f or idiopathic haemochromatosis 240

are asymptomatic. The HLA system is a linked marker for the iron-loading gene.

Treatment of iron overload is by venesection or with chelating agents. The most effective chelating agent in clinical use today is desferrioxamine (Barry et al, 1974-)* Desferrioxamine is a natural sideramine of Streptomyces pilosus (Figure Al). It has a maximum theoretical iron-binding capacity of 85mg per gram. It has a higher affinity for iron than any other trace metal. It is administered parenterally and it is rapidly cleared from the plasma with a half-life of 5 to 10 minutes. It has been shown to remove iron from reticulo endothelial cells of the liver and spleen and from hepatic parenchymal cells (Cook et al, 1972). Desferrioxamine is less toxic than other chelating agents. Exchange of iron from protein to desferrioxamine can be greatly enhanced by ATP and citrate (Morgan et al, 1978) . The principal pool of iron tapped by chelates is the ferritin/haemosiderin pool.

Previous studies have shown that liposomes can enhance the delivery of chelating agents to the liver (Rahman, 1975). Liposomes can prolong the retention of the chelating agent in the tissues (Guilmette et al, 197 8). Liposomally-entrapped EDTA and DTPA were shown to be able to remove both intra- and extracellular plutonium from plutonium-loaded mice, whereas the free chelating agent could only remove extracellular plutonium (Rahman et al, 1973). Liposomes enabled the chelating agents to cross the cell membrane, to which they were otherwise impermeable.

In order to investigate from which cellular compartment liposomally-entrapped chelating agent was removing iron, hepatic parenchymal cells were labelled with '^9 Fe ferritin while Kupffer cells were labelled with 59 Fe damaged red blood cells (Lau et al, 1981). It was found that small unilamellar vesicles containing desferrioxamine were more effective at removing 59 Fe ferritin, while multilamellar vesicles were more effective at removing 59 Fe damaged red blood cells. Liposomes containing desferrioxamine have been targetted to hepatocytes by inclusion of galactocerebroside in the lipid bilayer (Lau et al, 1981) . These removed more 59 Fe ferritin Figure Al. Desferrioxamine

(CH0). fCH0)5 (cri2)2 CH ( /h2}5 3 VIZ / I

NH II 0 0 2/, 2 than non-targetted liposomes.

When desferrioxamine, entrapped in liposomes, was administered orally or intraperitoneally to iron-overloaded Fe labelled mice, entrapment did not enhance the effect of the chelator. However, liposomally-entrapped desferrioxamine given intra venously, did increase iron excretion over a longer period than the free drug (Young et al, 1979). DTPA and EDTA are unable to cross cell membranes. When DTPA was entrapped in liposomes in the presence of ionophores, the ionophores were able to transport Fe 2+ across the lipid membrane after the iron had been chelated in vivo.

Erythrocyte ghosts have been used for chelator delivery in iron overload (Green, 1981a). Since damaged erythrocytes are phagocytosed into the reticuloendothelial system, entrapped desferrioxamine would be delivered to cells in which surplus iron was present. In both animal and human experiments, the effect of desferrioxamine on urine iron excretion was enhanced when the drug was given in erythrocyte ghosts. Reticulo endothelial but not parenchymal iron excretion was increased (Green, 1981b) . Erythrocyte ghosts were prepared by the hypotonic dilution method in order to remove most of the endogenous iron bound to haemoglobin. 60-80$ of the red cell haemoglobin was removed !in the preparation of ghosts.

The objective of this study was to continue the previous work performed in this laboratory, using the same animal model. Seymour (1980) found that neither liposomally-entrapped DTPA and desferrioxamine nor the free drugs increased iron excretion in the urine or faeces of iron-overloaded rats. Liver lysosomes and crude liver homogenates showed no decrease in iron levels after treatment. The results conflict with those of Young et al (1979) and Rahman et al (1982), who both found that liposomally-entrapped desferrioxamine increase iron excretion in urine over the free drug.

In the present study, entrapment of desferrioxamine in lipo somes was increased by using reverse phase liposomes, which have a larger entrapped volume than the multilamellar liposomes used in the previous study (Seymour, 1980). The excretion of radiolabelled iron from overloaded rats was followed more closely by keeping the animals in metabolic cages. Liposome were administered when radiolabelled iron excretion was well above background so that changes could be detected. The excretion of iron was followed over a longer time period and the cumulative effects of chelating agent-induced iron removal monitored. - 2 U ~

SECTION B

MATERIALS AND METHODS

MATERIALS

1. Animals

CFY rats were provided by the Animal House, Charing Cross Hospital Medical School.

2. Reagents

Jectofer (iron Sorbitol Injection) containing 50mg iron per ml was obtained from the Pharmacy, Charing Cross Hospital. 59Fe citrate was obtained from Amersham International. o-Bathophenanthroline, thioglycollic acid, Triton X100 were from Sigma. Isoamyl alcohol - May and Baker. Desferrioxamine - Ciba Laboratories (Horsham). All glassware for iron determination was washed in aqua regia and rinsed thoroughly.

METHODS

1. Animal Model for Haemochromatosis

An animal model for haemochromatosis was induced by intraperitoneal injection of Jectofer (iron sorbitol). CFY rats were used, as this strain have been found to survive iron loading with no ill effect (Seymour, 1980). 0.1ml Jectofer (50mg/ml) was injected intraperitoneally every other day over a 4- week period. Animals received a total of 60mg iron. The final injection consisted of 5mg Jectofer with 5-10uCi radioactive iron (>7Fe citrate). Rats were placed in metabolism cages and the urine and faeces collected every day for one week.

2. Assay for Iron

Iron was assayed using o-bathophenonthroline (Doeg and Zeigler, 1962). Samples containing 2-30nmoles of iron (final volume of 0.1ml) were pipetted into iron-free tubes. Each tube received 0.1ml 5% mercaptoacetic acid (thio glycollic acid) and 0.2ml glacial acetic acid. The tubes were covered and vortex mixed. The pH of the solution was raised by addition of 0.28ml of saturated sodium acetate solution and the volume was adjusted to 1ml. One ml of bathophenanthroline- isoamyl alcohol was added (83mg bathophenanthroline/lOOml isoamyl alcohol). The tubes were mixed vigorously. The ferrous iron was extracted into the organic phase where the pink ferrobathophanthroline complex was formed. The tubes were centrifuged to separate the aqueous and organic layers. The organic layer was removed and the optical density at 535nm was measured. A standard curve was constructed using ferric chloride.

3. Assay for Desferrioxaiaine

The assay followed was as modified by Seymour (1980) from a method by Young et al (1979)• 27Omg ferric chloride was dissolved in 100ml 0.1M HC1. Aliquots of solution containing desferrioxamine were made up to 1ml. The ferric chloride reagent (2ml) was added. The tubes were mixed and the optical density was read at 4-28nm.

When liposome entrapment of desferrioxamine was measured, Triton X100 (0.2%) was added before the ferric chloride reagent. In construction of a standard curve for desferri oxamine, liposomes and Triton X100 were included in the assay, to compensate for the absorbance due to these compounds.

4-. Entrapment of Desf errioxamine in Reverse Phase Vesicles

Anionic reverse phase liposomes (phosphatidylcholine- cholesterol-phosphatidic acid, h i l l ) were used to entrap desferrioxamine. Reverse phase liposomes were made as previously described (Chapter 2). Lipid (25mg) was dried in a found-bottomed flask and resuspended in 3ml diethyl ether. 200mg desferrioxamine (lml) was added. Inverse micelles were formed by sonication on ice (10 x 30 second bursts). After removal of diethyl ether by rotary evaporation, the inverse micelles collapsed into a bilayer state. A viscous gel was formed. 0.5ml 5mM potassium phosphate pH 7.4- was added. The liposomes were shaken until a homogeneous suspension was formed.

Free desferrioxamine was separated from entrapped by passage through a Sepharose 2B column (1.5 x 15cm) equilibrated in 5mM potassium phosphate pH 7.4* 5. Subcellular Fractionation of Rat Liver

Rat liver was homogenised in ice cold 0.25M sucrose with ImM EDTA. Subcellular fractionation was carried out as in the scheme in Chapter 2. Cell debris and nuclei were removed by centrifugation at 1020xg for 10 minutes. The heavy mito chondrial sediment was removed by centrifugation at 3300xg for 10 minutes. Lysosomal containing fraction was isolated by centrifugation of the supernatant at ll,700xg for 30 minutes. S-Glucosidase was the enzyme used for a lysosomal marker.

6. Radioactive iron excretion

Rats were housed in stainless steel metabolic cages, and urine and faeces were collected for 7 days following administration of 59 Fe citrate. Urine and faeces were measured for 59 Fe content. Urine was placed directly in a gamma counting tube and immediately counted. Faeces were digested in 33% potassium hydroxide and counted. All radioactive calculations have been corrected for decay, the half-life 59 of Fe being 4-4- days. - 247 -

SECTION C

RESULTS

1. Entrapment of Desferrioxamine in Reverse Phase Vesicles

When desferrioxamine was added to anionic reverse phase liposomes, approximately 2% of the added material could be entrapped. Free desferrioxamine was separated from that entrapped in liposomes by passage through Sepharose 2B. The reverse phase vesicles containing entrapped desferrioxamine were eluted in the void volume.

The entrapment of desferrioxamine in neutral reverse phase vesicles was also about 2%,

2. Animal Model for Haemochromatosis

The model used for iron overload was identical to that used by Seymour (1980), where a histological picture showed a deposition of iron mainly in the macrophages of the portal tracts. Randomly situated and less heavily stained Kupffer cells were also observed with a faint dusting of iron in the hepatocytes. This model had been used to observe the effect of liposomally-entrapped DTPA on the removal of iron from the liver. In this present study, the excretion of iron from iron-overloaded rats administered with desferrioxamine entrapped in liposomes was followed.

In order to follow the rate of 59 Fe excretion from iron- overloaded rats, urine was collected from rats, which had been iron-overloaded for 4- weeks with the last injection containing 5yCi 59 Fe citrate. Radioactivity in urine was counted. (These rats were not treated with liposomes).

Radioactive iron was rapidly excreted from 0 to 24- hours after injection, in total about 6.5% of the injected radioactivity. From 1 day to 7 days after injection of 59 Fe citrate, there was a gradual decrease in radioactivity excreted, falling from 0.6% of the injected dose 4-8 hours after administration to 0.2% of the injected dose 14-4- hours (6 days) after ^Fe citrate administration (Figure Cl). The remainder of the administered radioactivity was either excreted in faeces (results not shown) or retained in the body. ON F

An initial experiment in which liposomally-entrapped desferrioxamine and free desferrioxamine were administered 5 days after 59 Fe citrate showed no increase in iron excretion, in agreement with the results of Seymour (1980). However, it was shown above (Figure Cl) that ^Fe excretion was only 0.02$ of the injected dose by this time. Any small changes would not be detectable. It was therefore decided to administer liposomes 24- hours after 59 Fe citrate, when radio active levels in urine were much higher.

Reverse phase liposomes, composed of 14-Omg phosphatidylcholine, 10mg cholesterol and 17mg of phosphatidic acid (molar ratio 7:7:1) were used to entrap desferrioxamine. Desferrioxamine (4-500mg) was suspended in 16ml 5mM potassium phosphate buffer pH 7.5. Free desferrioxamine was removed by passage through Sepharose 2B. Liposomes, eluted in the void volume were concentrated using carbowax. Iron overloaded mice were administered with 2.0ml liposomes (l0.5mg lipid) containing 5*5mg entrapped desferrioxamine. Liposomes were administered intravenously twenty-four hours after the administration 59 of 77Fe citrate.

Iron-overloaded rats were divided into five groups. Rats weighed from 250 to 300g. 59 Fe citrate was administered on Day 0. The groups of rats received the following treatments: -

I Untreated II Liposomally-entrapped desf errioxamine administered 24- hour (Day l) after ^^Fe citrate. III Desferrioxamine (5«5mg) and empty liposomes (2.0ml) , \ 59 administered 24- hours (Day 1) after Fe citrate. IV Liposomally-entrapped desferrioxamine administered 24- hours (Day 1) and 72 hours (Day 3) after ^Fe citrate. V Desferrioxamine (5»5mg) and empty liposomes (2.0ml) administered 24- hours (Day l) and 72 hours (Day 3) after 59Fe citrate.

Urine and faeces were collected every 24- hours for seven dajs after 59 Fe citrate injection, for each of the above treatments. 250

Tables Cl and C2 show excreted radioactivity on Day 1-2, Day 2-3, Day 3-4-, Day 4.-5 and Day 5-7 after ^Fe citrate injection.

Although each rat received the same quantity of radioactivity intraperitoneally, there was a wide spread of radioactivity excreted by individual rats. However, the increases in excretion of iron for rats in the same group were similar. Rats were excreting differing amounts of the initial injected material. In order to compensate for this, for individual rats, cumulative totals on subsequent days are divided by the radioactivity excreted on Day 1-2. The cumulative fold increases for rats in each group are then averaged.

The cumulative total of radioactivity found in faeces showed no increase in treatment with liposomally-entrapped desferrioxamine over the untreated level (Table Cl). Free desferrioxamine showed a slight increase over the untreated total after seven days. Iron level in faeces was not measured in rats given a second dose of liposomally-entrapped or free desferrioxamine.

In urine, the cumulative totals are shown in Table C2. Liposomally-entrapped desferrioxamine increased radioactive iron excretion over 7 days compared to free desferrioxamine. When liposomes were administered on Day 1, there was a greater excretion than when two doses of liposomes were administered on Days 1 and 3. It appeared that the first of the two doses was unsuccessful.

Free desferrioxamine increased iron excretion above the untreated level after two doses (llmg) had been given (Treatment V).

4-. Iron Content in Liver of Iron-Overloaded Rats after Administration of Liposomally-Entrapped Desferrioxamine

Animals in groups I to V were killed on Day 7. Livers were homogenised and iron levels measured in the crude homogenate and in lysosomes after sub-cellular fractionation. Iron was measured using the o-bathophenantholine assay (Table C3) • It appeared that liposomally-entrapped desferrioxamine decreased iron in liver homogenate to a greater extent than free desferri- oxamme . 251

Table Cl

Cumulative total of 59 Fe in faeces of iron-overloaded rats. 5 9 7Fe citrate (5uCi/rat) was injected intraperitoneally on Day 0. Desferrioxamine (5.5mg) was administered on Day 1, either in liposomes (10.5mg lipid) or free. Untreated mice received only 59 Fe citrate on Day 0. Results are shown as cumulative fold increases over 7 days (n = 4-) .

Cumulative Increases m 59 Fe in Faeces (fold increase over first 24- hours)

Day 1-2 2-3 3-4 4.-5 5-7 Treatment

I Untreated 1 2.15 2.83 3.25 3.50 ±0.04- ±0.12 ±0.15 ±0.17

II Liposomally- Entrapped Desferri- 1 1.91 2.68 3.14 3.37 oxamine (5.5mg) on ±0.06 ±0.18 ±0.22 Day 1 ±0.09

III Free Desferrioxamine 1 2.86 3.81 (5-5mg) with Empty 1.85 3.31 Liposomes on Day 1 ±0.13 ±0.21 ±0.27 ±0.14 252

Table C2

Cumulative total of 59 Fe in the urine of iron-overloaded rats. 597Fe citrate (5pCi/rat) was injected intraperitoneally on Day 0. Desferrioxamine (5*5mg) was administered, either in liposomes (I0.5mg lipid) or free. Rats received either one dose on Day 1 or two separate doses on Days 1 and 3. Untreated mice received only 59 Fe citrate on Day 0. Results are shown as cumulative fold increases over 7 days (n = 4) .

Cumulative Increases in Urine Radioactivity(fold increase over first 24- hours)

\ D a y 1 -2 2-3 3-4- 4-5 5-7 Treatment

I Untreated 1 1.22 1.37 1.49 1.59 + 1 0 0 • ±0.03 ±0.05 ±0.04

II 1 Dose Liposomally- 1 1.78 Entrapped Desferri- 1.69 1.91 2.45 oxamine (5*5mg) ±0.18 ±0.21 ±0.23 ±0.22

III 1 Dose Free Desferri- 1 1.38 oxamine (5-5mg) with 1.31 1.43 1.56 Empty Liposomes ±0.02 ±0.02 ±0.03 ±0.07

IV 2 Doses Liposomally- 1 1.32 1.52 Entrapped Desferri- 1.91 2.12 oxamine (5.5mg) ±0.04- ±0.04- ±0.06 ±0.06

V 2 Doses Free Desferri- 1 1.61 oxamine (5.5mg) with 1.33 1.4-5 1.93 Empty Liposomes ±0.11 ±0.1^ ±0.13 ±0.21 253

In the lysosomal fraction, untreated iron levels were 0.02pg/mg protein. Treatment with 5*5mg desf errioxamine, either entrapped in liposomes or free, did not significantly decrease iron levels. However, lysosomal iron levels were decreased to 0.013ug/mg protein with llmg free desf errioxamine and to O.Ol^pg/mg protein with llmg liposomally-entrapped desferrioxamine.

It therefore appears that desferrioxamine can decrease liver iron concentration in both its free form and its liposomally- entrapped form.

Unexpectedly, free desferrioxamine decreased iron in the lysosomal fraction slightly more than liposomally-entrapped desferrioxamine. Since the liposomally-entrapped drug is cleared more quickly to the hepatic lysosomal fraction (Guimette et al, 1978), it would be expected that encapsulated drug would be more effective than free drug. Tabic C3

Iron concentration in crude liver homogenate and in liver lysosome fraction of iron-overloaded rats after administration of desferrioxamine, either free or entrapped in reverse phase liposomes (PC:Ch:PA 7:7:1). Iron levels are in yg per mg protein. Values are the average of 4 animals ± standard error.

Ug iron/mg protein

Homogenate Lysosome

I Untreated 0.086 0.019 ±0.030 ±0.013

II 1 Dose Liposomally-Entrapped 0.027 0.0163 Desferrioxamine (5.5mg) ±0.009 ±0.a09

III 1 Dose Free Desferrioxamine 0.051 0.0182 (5.5mg) with Empty Liposomes ± 0.011 ± 0.012

IV 2 Doses Liposomally-Entrapped 0.019 0.0142 Desferrioxamine (5.5mg) ±0.007 ± 0.002

V 2 Doses Free Desferrioxamine 0.046 0.0127 (5.5mg) with Empty Liposomes ±0.024 ±0.004 255

SECTION D

DISCUSSION

A number of investigations for a suitable animal model of haemochromatosis have been carried out. In one study an experimental model has been generated by injection of the soluble, low molecular weight, inorganic complex, ferric acetohydroxamate (Corden and Hansen, 1981). Secondary haemochromatosis, which can occur after the frequent trans fusions required in diseases such as 3-thalassemia, can be reproduced in rats by hypertransfusion. Selective labelling of hepatocytes with 59 Fe ferritin and Kupffer cells with 59 Fe damaged red blood cells has been performed to demonstrate iron removal from specific cellular compartments after therapy. A well defined animal model is essential in any attempt at chelation therapy.

Although the model of haemochromatosis used in this study does not represent a chronic picture of iron overload, with little disruption of liver lysosomes, iron levels in Kupffer cells and hepatocytes of liver were elevated (Seymour, 1980). The efficacy of liposome-entrapped chelating agents for the removal of iron could be estimated. It has been shown (Drysdale and Ramsay, 1965) that iron in this model is almost entirely present as haemosiderin within lysosomes, whereas the soluble fraction contained high levels of ferritin. Haemosiderin is only seen within lysosomes. The transformation of lysosomal ferritin into haemosiderin is a consequence of digestion of the apoferritin coat (Fischbach et al, 1971) .

Ideal chelating agents should have a high affinity for iron but a low affinity for other metals such as magnesium, calcium, and copper. The drug must be able to bind to the iron stored in the excess pools of haemosiderin and not to the iron necessary for enzyme catalysis or haem iron. The chelating agent must be excreted after it has bound to iron. This may be in the faeces after biliary excretion from hepatocytes or in the urine after excretion from Kupffer cells. Chelation of excess iron in haemochromatosis is necessary from heart as well as from liver. Desferrioxamine has been shown to be the best - 256 -

iron chelator for clinical use. It has a very high affinity for iron and it is cleared very quickly from the plasma (ti 10 minutes). It is effective at producing a readily measurable net reduction of iron burden in older, heavily loaded patients and at preventing the build-up of iron in young patients. But because it is expensive and has to be infused daily, a method for enhancing its effect is required.

Previous work from this laboratory (Seymour, 1980) has shown that DTPA and desferrioxamine entrapped in multilamellar vesicles (MLV) could not reduce iron levels from crude liver homogenate or from the lysosomal fraction of iron- overloaded rats.

However, only 2.5mg DTPA and ling desf errioxamine were administered in MLV because of the low encapsulation efficiency (1% of the added material). In the present work, reverse

phase liposomes, with a higher encapsulation of 2% of the added material, were used to administer 5.5mg to iron-overloaded rats.

In the previous work, liposomes were administered 5 days after the injection of the last dose of iron-sorbitol containing 5yCi 59 Fe citrate. It has been shown that 59 Fe in urine was down to background level after 7 days and so collection of urine six days after 59 Fe injection would not reflect any increased iron excretion that may occur after chelation therapy. In this study, liposomes were administered 24- hours after 59 Fe citrate injection. In this way changes in urine radioactivity were measurable. However, this still may not reflect the true picture of iron excretion, if the radioactive iron is not mobilised at the same rate as the unlabelled iron. Unlabelled iron was injected every other day for four weeks. Radioactive iron was injected on the last of iron administration. Uniformly distributed radiolabelled iron may be attained by injection of the radioactive iron with the unlabelled iron throughout the four weeks.

Using the same animal model as in the former study in this laboratory, the present work shows that liposomally-entrapped desferrioxamine increased excretion of iron compared to free desferrioxamine. Lysosomal iron levels were decreased more by the free drug than the liposomally-entrapped drug. This 257

was unexpected because reverse phase liposomes are engulfed into endocytic vacuoles of the reticuloendothelial system. Since most of the stored iron is present as haemosiderin in the lysosomal apparatus, liposomal delivery of the chelating agent should have enhanced the excretion of lysosomal iron. This was not the case. Vacuoles into which the desferrioxamine- containing liposomes had been engulfed may not have fused with those containing haemosiderin. Alternatively, if haemosiderin was present mainly in hepatocyte lysosomes, liposome-entrapped chelating agent, being delivered to the reticuloendothelial system, may only have had access to a minor proportion of stored iron.

Liposomally-entrapped drug removed more iron from the crude liver ho mogenate than free drug. This may reflect the protective effect of liposomes in the circulation, preventing degradation of desferrioxamine in the plasma and enhancing its uptake into liver. It is difficult to perform tissue distribution studies on radiolabelled 59 Fe desferrioxamine in liposomes because interpretation of radioiron labels is complicated by isotope exchange in vivo (Rahman, 1980). However, liver uptake of other liposomally-entrapped chelating agents has been shown to be greater than the free drug (Rahman et al, 1974-) •

Although only one experiment was performed in this study, it demonstrated the need for an animal model in which the removal of radioactive iron would reflect the movement of the total iron deposits. The isolation of ferritin or haemosiderin from liver would be interesting in order to determine from which storage form iron was mobilized by the free or liposomally— entrapped chelating agent.

Reverse phase vesicles proved much better than the multilamellar vesicles used in the former study (Seymour, 1980).

A carrier that has been shown to entrap a large amount of desferrioxamine is the erythrocyte ghost prepared by the hypotonic dilution method (Green et al, 1977). A total dose of 70mg of desferrioxamine was administered in 3 doses, which caused a 29% loss of 7Fe in faeces and urine compared to 6.8% 258

in control rats. Those damaged red blood cells are phagocytosed by the reticuloendothelial system, where the majority of the iron is stored. It would be interesting to encapsulate desferrioxamine in erythrocyte ghosts prepared by the hypotonic dialysis method. These can be manipulated to have a circulation time in the blood near that of normal erythrocytes. Although they would be less useful than hypotonic dilution ghosts at directing desferrioxamine to the liver, they might either decrease plasma ferritin or transferrin levels, or act as a slow release depot for the chelating agent after their slow lysis in the circulation.

Desferrioxamine is the most useful chelating agent used in therapy today, but it is not ideal. It is toxic in high doses, a disadvantage that liposomal entrapment may overcome. It is expensive. Liposome~entrapped desferrioxamine only slightly increases iron excretion over the same amount of free drug. It is unlikely that liposome entrapment will significantly decrease the cost of chelation therapy. The ideal chelating agent would be administered orally. Liposomes may be able to increase desferrioxamine uptake after oral administration by protecting it from degradation in the intestinal tract. 259

CHAPTER 5

CONCLUDING REMARKS 260

Animal models analogous to human inborn errors of metabolism are important tools in the search for strategies for enzyme replacement therapy.

The iron-overload animal model represents a treatment by 'environmental manipulation’, enhancing iron excretion with chelating agents. The two amino acidopathies represent models for treatment by 'enzyme engineering', infusion of the deficient enzyme. The aim in both groups is to deplete excess metabolites. When the metabolite is sequestered in cells, such as in iron overload or lysosomal storage diseases, the chelating agent or enzyme has to be targetted to the site of storage. If sequestration does not occur, as in disorders of amino acid metabolism, the levels of metabolite are generally elevated throughout the body. In this case, the enzyme does not have to be delivered to target cells, but may be required to be active in the circulation. The enzyme and chelating agent must be stable and interact with their substrate long enough to produce clinical benefits.

The use of liposomes as therapeutic carriers has shown that they are specifically directed towards the reticuloendothelial system (Gregoriadis et al, 1972). It is uncertain whether liposomes can be introduced into any other type of cell. Alteration of the size, charge and lipid composition changes liposomal pharmokineties into the reticuloendothelial system.

Rapid clearance of a lysosome-directed enzyme or drug is an advantage for diseases affecting the reticuloendothelial system. For iron overload, this approach has proved successful for the enhanced excretion of the stored iron. However, tie use of liposomes to retain therapeutic enzymes in the circulation has shown several complications. The requirement for protection of enzymes from inactivation in the plasma entails the use of stable liposomes. Apart from a high cholesterol content (Kirby et al, 1980), stable liposomes require lipids that are below their transition temperature in physiological fluids (Allen, 1981). This means that it is very difficult for substrates to diffuse through the liposomal membrane. Liposomes composed of sphingomyelin 261

and cholesterol (3:2) or distearoylphosphatidylcholine and cholesterol (2:1) proved impermeable to histidine. Liposomes containing histidase would therefore be of little use in degrading plasma histidine in histidinaemia.

As discussed previously, hypotonic dialysis erythrocyte ghosts can be made so that they resemble normal erythrocytes, with a half-life of 28 hours in the circulation. Their best application to human therapy is to act as a circulating compartment for metabolite degradation after enzyme entrapment The present work has shown that the ability of the entrapped enzyme to degrade its substrate depends on the properties of the enzyme itself. Pseudomonas histidase is readily inhibited by its product and so its activity depends on the rate of diffusion of the product out of the erythrocyte. It also depends on the rate of diffusion of the substrate into the erythrocyte. Proline oxidase is a lipophilic enzyme and therefore is found in the erythrocyte membrane after entrap ment. Proline degradation may therefore not depend on its transport through the membrane. The rate of degradation of proline, however, is limited by the reverse reaction catalysed by a different enzyme found in erythrocytes.

If erythrocyte ghosts containing protein are to be useful in therapy, the reason for their rapid initial lysis on injection must be determined. This is particularly important before human therapy can be attempted because of the immuno logical complications that might result. The present work has provided a basis for further studies on enzyme replacement for amino acidopathies especially those which have more serious consequences than histidinaemia or hyperprolinaemia, e.g. phenylketonuria. In this case, it is important for maternal blood levels of homozygous phenylketonurics to be decreased during pregnancy to prevent the teratogenic effects of phenylalanine on the foetus. The histidinaemic mouse is an excellent laboratory model for this because of the teratogenic effects of histidine on the foetal inner ear. This study has shown that one must consider the characteristic of each enzyme used for therapy before deciding on the carrier for immunological and proteolytic protection. 262

Further work is needed on the in vivo half-lives of carrier entrapped enzymes. The work with proline oxidase in vivo was unfortunately not performed on hyperprolinaemic mice. It would be interesting to repeat all the in vivo proline oxidase studies on mice in which proline levels were elevated, to acquire more accurate data.

The work with iron-overloaded rats has shown the distinct advantages of using liposome-encapsulated chelating agents in relieving iron deposition. This is a confirmation of the work of Lau et al (1981) and Young et al (1979). However, this approach may still not significantly decrease the cost of chelation therapy for human haemochromatosis sufferers.

The results with all three models are potentially very interesting and applicable in serious clinical conditions. Further work on the stability, half-lives and kinetic properties in vivo of histidase and proline oxidase and the pharmokinetics of erythrocyte ghosts and liposomes is required.

Enzyme therapy in genetic diseases has been a long-sought- after goal for many years. The advent of genetic engineering may enable larger quantities of enzyme more useful for therapy to be produced. Only then would trials on humans be possible, especially if human enzymes could be supplied in large amounts. This gene product therapy has a more sound near-future potential than actual gene therapy, in which the missing gene itself would have to be spliced into the human genome. 263 -

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