Synthesis and evaluation of human serum albumin-modified exendin-4 conjugate via heterobifunctional polyethylene glycol linkage with protracted hypoglycemic efficacy.
Albumin conjugation is considered to be one of the most effective means of protracting the short in vivo lifespans of peptides and proteins. Here, we present a new long-acting antidiabetic exendin-4 conjugate linked with human serum albumin (HSA) via polyethylene glycol (PEG). As a first step toward synthesizing this conjugate, three artificial sulfhydryl groups were introduced in HSA using 2-iminothiolane at pH 8.0. This thiolated HSA was further reacted with the monomer fraction of exendin-4 (6 equiv) conjugated with maleimide-PEG(5k)-N- hydroxysuccinimide (MAL-PEG(5k)-NHS) for 3 h. Because of the presence of PEG molecules, the resulting conjugate (HSA-PEG-Ex4) was found to have a greater apparent molecular weight and a larger particle size (ca. 195 kDa and 9.48 +/- 0.74 nm) than those of HSA-exendin-4 without the PEG linker (HSA-Ex4, ca. 84.3 kDa and 7.77 +/- 0.98 nm). Although the receptor binding affinity of HSA-PEG-Ex4 on RIN-m5F cells was significantly lower than that of Ex4, its antihyperglycemic efficacy was slightly higher than that of Ex-4 and HSA-Ex4 in type 2 diabetic db/db mice. Furthermore, HSA-PEG-Ex4 had greater circulating t(1/2) and AUC(inf) values than HSA-Ex and native exendin-4 by 2.1- and 10.3-fold, respectively. Accordingly, its hypoglycemic duration was greatly increased to 31.0 h at a dose of 250 nmol/kg vs that of native Ex4 (7.0 h). Results show that the HSA-PEG-Ex4 conjugate produced has distinct advantages over HSA-Ex4 without PEG. We believe that this exendin-4 derivative, which has the merits of albumin conjugation and PEGylation, has considerable potential as a novel type 2 antidiabetic agent.
Kim
Insoo
I
Pusan National University, 30 Jangjun-dong, Geumjeong-gu, Busan, Korea. [corrected]
Kim
Tae Hyung
TH
Ma
Kyungwan
K
Lee
Eun Seong
ES
Kim
Dongin
D
Oh
Kyung Taek
KT
Lee
Don Haeng
DH
Lee
Kang Choon
KC
Youn
Yu Seok
YS
D000818:Animals; D045744:Cell Line, Tumor; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000188:drug therapy*; D000077270:Exenatide; D005951:Glucose Tolerance Test; D006801:Humans; D007003:Hypoglycemia / Q000097:blood / Q000188:drug therapy*; D007004:Hypoglycemic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000627:therapeutic use*; D008297:Male; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D015394:Molecular Structure; D008970:Molecular Weight; D010316:Particle Size; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000627:therapeutic use*; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D012709:Serum Albumin / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties; D014688:Venoms / Q000138:chemical synthesis / Q000737:chemistry / Q000627:therapeutic use*
D007004:Hypoglycemic Agents; D008301:Maleimides; D010455:Peptides; D012709:Serum Albumin; D013388:Succinimides; D013438:Sulfhydryl Compounds; D014688:Venoms; C043592:maleimide; D011092:Polyethylene Glycols; D000077270:Exenatide; C001426:N-hydroxysuccinimide