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. 2000 Nov;47(5):622–627. doi: 10.1136/gut.47.5.622

Potency and stability of C terminal truncated human epidermal growth factor

D Calnan 1, A Fagbemi 1, J Berlanga-Acosta 1, T Marchbank 1, T Sizer 1, K Lakhoo 1, A Edwards 1, R Playford 1
PMCID: PMC1728116  PMID: 11034576

Abstract

INTRODUCTION—Epidermal growth factor (EGF) is normally present as EGF1-53. 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 EGF1-53, EGF1-52, EGF1-51, and the C terminal peptides EGF44-53 and EGF49-53.
METHODS—Purity of forms was confirmed by mass spectrometry. Bioactivity of the different EGF forms was determined using [methyl-3H] 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 EGF1-53, EGF1-52, and EGF1-51 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). EGF44-53 and EGF49-53 did not stimulate 3H thymidine uptake. Control rats had 47 (4) mm2 damage/stomach, EGF1-51, EGF1-52, and EGF1-53 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.
CONCLUSION—EGF1-51 and EGF1-52 are as biologically active as full length EGF1-53 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.


Keywords: epidermal growth factor; intestinal injury; nutrition

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Figure 1  .

Figure 1  

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Effect of epidermal growth factors EGF1-53, EGF1-52, and EGF1-51 on [methyl-3H] thymidine uptake into primary rat hepatocytes. The dose-response curves of the various forms of EGF are virtually superimposable. Results are expressed as mean (SEM), n=4.

Figure 2  .

Figure 2  

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Comparison of the biological activity of various forms of epidermal growth factor (EGF) using attenuation of gastric damage as a bioassay. Rats were restrained and given a continuous subcutaneous infusion of saline (control), EGF1-53, EGF1-52, or EGF1-51. Thirty minutes later, all animals also received indomethacin (20 mg/kg subcutaneously). Three hours after administration of indomethacin, all animals were killed and the amount of gastric injury assessed. (A) All forms of EGF reduced the amount of total ulcerated area (mm2/stomach) (all p<0.01 v control) but there were no differences in potency between the forms. (B) Assessment using a microscopic damage score, where each stomach was given a score of 0-4 (0, no damage; 1, one small erosion (less than 0.5 mm); 2, two small or one large erosion (greater than 0.5 mm); 3, two or more large erosions; and 4, any area of ulceration extending to the muscularis mucosa). Results are expressed as mean (SEM), n=6 animals per datum point.

Figure 3  .

Figure 3  

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Stability of epidermal growth factor (EGF) when placed in a clinical setting. A 60:40 mixture of EGF1-51 and EGF1-52 is commercially available in an intravenous formulation. To determine its stability under likely clinical conditions, the EGF1-51/EGF1-52 mixture was diluted to 1 µg/ml in a 4.5% solution of human albumin and divided into two samples. The test solutions were pumped at room temperature (24-26°C) through an infusion system. One infusion system was covered in silver foil and the other was placed under a phototherapy lamp to mimic the conditions of phototherapy treatment commonly prescribed for the jaundiced neonate. Samples were subsequently analysed for biological activity using the rat hepatocyte assay. Results are expressed as mean (SEM), n=4.

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