Spray-dried insulin particles retain biological activity in rapid in-vitro assay.
The purpose of this study was to rapidly determine, without the use of extensive animal studies, whether biological activity is retained after spray drying insulin with two excipients, lactose and xanthan gum. This was achieved by the detection of protein kinase B (PKB), which is activated by phosphorylation in response to insulin binding to cellular receptors. A myeloid cell line was cultured and stimulated with the reconstituted insulin powders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then utilised to allow in-vitro detection of phosphorylated PKB using an anti-phospho-PKB antibody. A single band specific to phosphorylated PKB was found on the Western blots, indicating that the active conformation of insulin was retained when spray dried in combination with lactose and with xanthan gum over the spray-drying inlet temperature range of 110-170 degrees C. Evidence of inactivation/denaturation was observed when insulin was spray dried at an inlet temperature of 200 degrees C. The assay may be of use as a more rapid and economic means to screen insulin formulations for inhalation and other purposes as opposed to conventional monitoring of blood glucose levels in animals.
Patel
N
N
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, UK.
Craddock
B L
BL
Staniforth
J N
JN
Tobyn
M J
MJ
Welham
M J
MJ
D003890:Desiccation; D004591:Electrophoresis, Polyacrylamide Gel; D005079:Excipients; D007004:Hypoglycemic Agents / Q000008:administration & dosage* / Q000494:pharmacology*; D007328:Insulin / Q000008:administration & dosage* / Q000494:pharmacology*; D007785:Lactose; D008855:Microscopy, Electron, Scanning; D010766:Phosphorylation; D011135:Polysaccharides, Bacterial; D013696:Temperature
D005079:Excipients; D007004:Hypoglycemic Agents; D007328:Insulin; D011135:Polysaccharides, Bacterial; D007785:Lactose; C002563:xanthan gum