Potency and stability of C terminal truncated human epidermal growth factor.
BACKGROUND
Epidermal growth factor (EGF) is normally present as EGF(1-53). A variety of C terminal truncated forms have been used in preliminary trials for treating gastrointestinal injury but their relative potency and stability when used in a clinical setting are unclear. Therefore, we compared the biological activity of recombinant EGF(1-53), EGF(1-52), EGF(1-51), and the C terminal peptides EGF(44-53) and EGF(49-53).


METHODS
Purity of forms was confirmed by mass spectrometry. Bioactivity of the different EGF forms was determined using [methyl-(3)H] thymidine incorporation into primary rat hepatocytes and their ability to reduce indomethacin (20 mg/kg subcutaneously)/restraint induced gastric injury in rats. Stability of EGF peptides was determined by serial sampling from a syringe driver system containing EGF/4% albumin in saline.


RESULTS
Biological activity assays of EGF(1-53), EGF(1-52), and EGF(1-51) gave almost identical thymidine uptake dose-response curves (maximal responses increasing baseline uptake from 4400 (600) cpm (mean (SEM)) to about 22 000 (2000) cpm when EGF was added at 1. 6 nM). EGF(44-53) and EGF(49-53) did not stimulate (3)H thymidine uptake. Control rats had 47 (4) mm(2) damage/stomach, EGF(1-51), EGF(1-52), and EGF(1-53) at 0.16 and 0.80 nmol/kg/h each reduced gastric injury by about 50% and 80%, respectively (both doses p<0.01 compared with control but no significant difference between the different forms). EGF was stable at room temperature for seven days but biological activity decreased by 35% and 40% at two and three weeks, respectively (both p<0.01). Exposure to light did not affect bioactivity.


CONCLUSIONS
EGF(1-51) and EGF(1-52) are as biologically active as full length EGF(1-53) but the C terminal penta- and decapeptides are ineffective. Clinical trials of EGF can probably use infusion systems for at least 48 hours at room temperature and with exposure to light, without reducing biological efficacy.
Calnan
D P
DP
University Division of Gastroenterology, Leicester General Hospital, Leicester, UK.
Fagbemi
A
A
Berlanga-Acosta
J
J
Marchbank
T
T
Sizer
T
T
Lakhoo
K
K
Edwards
A D
AD
Playford
R J
RJ
D000704:Analysis of Variance; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000627:therapeutic use; D005753:Gastric Mucosa / Q000187:drug effects / Q000293:injuries; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry* / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry* / Q000627:therapeutic use; D013272:Stomach Diseases / Q000139:chemically induced / Q000188:drug therapy / Q000473:pathology; D013936:Thymidine / Q000493:pharmacokinetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; C102345:epidermal growth factor (1-53); D004815:Epidermal Growth Factor; D013936:Thymidine