1. EARLY DAYS OF GROWTH HORMONE THERAPY: IT PAYS TO TAKE CARE Phil Lowry Leeds University 1962-1969 Ciba/Geigy 1969-1973 St Bartholomew’s Hospital “Barts” 1973-86 Research funded by Medical Research Council DHS therapeutic hGH research 1974-1981 University of Reading 1986-2009 (MRC funded)

2. PITUITARY GLAND

3. REPROD- UCTION GROWTH STRESS THYROID

4. Human Growth Hormone: From Cadaver to Clinic Impure hGH Height (cm) hGH 5mg/gland Late 60s simple precipitation…no chromatography Pain after injection

5. The original extraction methods started with acetone dried human pituitary glands Raben method used hot glacial acetic acid Wilhelmi method used alkali pH Isolation was achieved by precipitation Neither method used any chromatography Both methods caused some denaturation Both preparations were associated with significant antibody generation which blocked hGH bioactivity 1984 first cases of CJD (Creutzfeldt-Jakob Disease) hGH (ex-cadaver) therapy discontinued

6. An example of therapeutic hGH isolated without using chromatography containng 60% “non-specific junk” Deceasing in size

8. At Barts we used frozen pituitary glands Extracted in mild alkali, then by isoelectric precipitation in mild acid/10% ethanol Filtered through 0.22 micron membrane Gel chromatographic step Ion exchange chromatographic step Freeze dried. 5mg/gland … 2.5 times purer At Wellcome Laboratories Redissolved and refiltered through 0.22 micron membrane Freeze dried in ampoules for clinical use

9. Therapeutic human growth hormone purification at Barts 1975-1981 Gel filtration chromatographic cloumn Ion exchange chromatographic column

14. Transmitted Spongioform Encephalopathies…TSEs Originally called “Slow Viruses” up to 14 years after infection “Scrapie” endemic in sheep whose brains were fed to cows Bovine Spongioform Encephalopathy “BSE” or mad cow disease Creutzfeldt Jacob Disease was very rare in humans before BSE infected meat in hamburgers lead to new variant CJD “Fore” tribe in New Guinea ate brains of their dead..KURU All these diseases are transmitted by an infectious protein called PRION

15. Prion protein found in membrane of neural cells but in its infectious state has folded into an extremely stable form which can withstand autoclaving and digestion by proteases. When ingested (BSE, new variant CJD) or injected (hGH therapy) finds its way into neural tissue where it very slowly catalyses the conversion of normal prion proteins into the infectious form leading to spongioform encephalophathy. Can also get infected from e.g. corneal transplant and blood transfusion.

16. An example of therapeutic hGH isolated without using chromatography containng 60% “non-specific junk” contained infectious particles Deceasing in size

17. The Lancet, 1985 Aug 3; 260-2. 1978 Alan Dickinson contacted MRC Head Office about possibility of contracting CJD from hGH extracted from human pituitary glands

18. Polyacrylamide gel electrophoresis, under non-dissociating conditions, of a sample of Barts therapeutic human Growth Hormone De-amidated forms of human growth hormone But in 1995 I ended up on the stand in the High Court ! Since 1986 hGH purified from genetically engineered bacteria load + Since production at Barts stopped in 1981 there have been no cases of CJD associated in patients treated exclusively with this preparation. Antibody titres dropped after switching over to Barts hGH.

19. rhGH 1986 Human Growth Hormone Cloned: purified recombinant hGH used in Clinic mRNA Pituitary gland Clone