Biodistribution, activation, and retention of proinsulin-transferrin fusion protein in the liver: Mechanism of liver-targeting as an insulin prodrug

Liu, Yuqian; Wang, Hsuan-Yao; Zhou, Li; Su, Yang; Shen, Wei‐Chiang

doi:10.1016/j.jconrel.2018.02.030

Cited by 10 publications

(11 citation statements)

References 34 publications

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“…7C, solid circles). The lag-time for activation, liver-preferential activity, and long-lasting BG-lowering effect of ProINS-Tf in INS-resistant NOD mice were similar to that observed in non-INS-resistant diabetes mouse models as described in our previous publications 7,8,28 . This finding indicates that a normal glycemic regulation efficacy of ProINS-Tf can be achieved in INS-resistant diabetic NOD mice.…”

Section: Discussionsupporting

confidence: 84%

“…1A), suggesting its enhanced ability on activating IR. ProINS-Tf by itself had little activity on Akt phosphorylation and became much more active after the H4IIE cells-mediated conversion 8 . The increased p-Akt levels induced by irINS-Tf could be due to the increased stability of the fusion protein in the dosing medium, and/or the enhanced and sustained binding of irINS-Tf with the IR.…”

Section: Discussionmentioning

confidence: 96%

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Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance

Liu

Wang

Shao

et al. 2020

Sci Rep

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Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease. Insulin (INS) resistance is the major cause of the development of type 2 diabetes (T2D), and it is often referred as a state in which a higher than normal level of INS is required to achieve the normal response 1-3. INS resistance may result from alterations at different cellular levels, including insulin deficient signaling, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction 3. Type 2 diabetic patients often require intensive insulin treatment to maintain glycemic control, which leads to increased risk of hypoglycemia, weight gain, and further deterioration of INS resistance state 1,4. Previously, INS X10 with higher IR binding affinity have been studied as a rapid-acting INS analogue to treat type 2 diabetes 5. INS X10 demonstrated sustained effect in inducing IR-mediated signaling; however, the development of INS X10 was discontinued due to disproportionate increase in mitogenic activity and higher breast cancer incidence in the long-term rat studies 6. It is still a great challenge to develop novel INS analogues with enhanced binding affinity to the IR to overcome INS resistance safely and effectively. A proinsulin-transferrin (ProINS-Tf) fusion protein was previously developed as a novel long-acting and liver-targeted INS prodrug for treating type 1 diabetic (T1D) mice 7,8. Proinsulin (ProINS), as a precursor of insulin (INS), has a much lower binding affinity to INS receptor (IR) and the resultant biologic potency is only 1% or less relative to INS 9. Therefore, ProINS-Tf is initially inactive and requires a lag time to be activated before exhibiting activity in stimulating Akt phosphorylation in H4IIE ce...

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 96%

Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance

Liu

Wang

Shao

et al. 2020

Sci Rep

Self Cite

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“…In addition, colchicine inhibits the macropinocytosis and cationic polylysine is an inhibitor to uptake the cationic NPs (Wu et al, 2014;Voltan et al, 2017). The caveolae-mediated process and clathrindependent endocytosis was blocked by flipin and PhAsO, respectively (Kim et al, 2007;Liu et al, 2018b). The results showed that the cell uptake of CRD-PEG-T7/YOYO1-pPMEPA1 was proportional to energy and inversely to the presence of T7, which reflected that T7 competitively uptake with CRD-PEG-T7/pPMEPA1.…”

Section: Discussionmentioning

confidence: 99%

Study on the cellular internalization mechanisms and in vivo anti-bone metastasis prostate cancer efficiency of the peptide T7-modified polypeptide nanoparticles

Chen

Zhang

et al. 2020

Drug Delivery

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Bone-metastasis prostate cancer (BMPCa)-targeting gene therapy is gaining increasing concern in recent years. The peptide T7-modified polypeptide nanoparticles for delivery DNA (CRD-PEG-T7/ pPMEPA1) was prepared as our previous study. However, the feasibility of CRD-PEG-T7/pPMEPA1 for BMPCa treatment, the mechanisms underlying cellular uptake, anti-BMPCa effect, and administration safety requires further research. LNCaP cells treated with endocytosis inhibitors and excessive T7 under different culture condition were carried out to investigate the mechanisms of cellular uptake of the CRD-PEG-T7-pPMEPA1. A transwell assay was applied to evaluate the cell migration ability. Besides, the tumor volume and survival rates of the PCa xenograft mice model were recorded to estimate the antitumor effect. In addition, the weight profiles of the PCa tumor-bearing mice, the blood chemistry, and the HE analysis of visceral organs and tumor was conducted to investigate the administration safety of CRD-PEG-T7/pPMEPA1. The results showed that PCa cellular uptake was decreased after treating with excessive free T7, endocytosis inhibitors and lower incubation temperature. Besides, CRD-PEG-T7/ pPMEPA1 could inhibit the LNCaP cells chemotaxis and tumor growth. In addition, the survival duration of the PCa tumor-bearing mice treating with CRD-PEG-T7/pPMEPA1 was significantly prolonged with any systemic toxicity or damage to the organs. In conclusion, this research proposes a promising stratagem for treatment BMPCa by providing the biocompatible and effective carrier for delivery DNA therapeutic agents.

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“…In the case of insulin and its analogues, radioactive tracer techniques using 125 Iodine isotope have been widely applied to the investigation and evaluation of pharmacokinetics, tissue distribution and excretion of macromolecules. [29][30][31][32] Given the fact that free iodine or smaller protein fragments labeled with 125 I could be released from the influence of metabolic system in RA method, 33 TCA precipitation has been introduced to couple with radiolabeling (TCA-RA) for reliable quantification of protein drugs. Since peptide fragments are generally not precipitated by TCA, the measurement of the radioactivity of TCA-precipitated part could objectively reflect the content of prototype drug.…”

Section: Discussionmentioning

confidence: 99%

Efficacy, Pharmacokinetics, Biodistribution and Excretion of a Novel Acylated Long-Acting Insulin Analogue INS061 in Rats

Pan

Shi

et al. 2021

DDDT

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Purpose Long-acting insulin analogues are known to be a major player in the management of glucose levels in type I diabetic patients. However, highly frequent hypo- and hyperglycemic incidences of current long-acting insulins are the important factor to limit stable management of glucose level for clinical benefits. To further optimize the properties for steadily controlling glucose level, a novel long-acting insulin INS061 was designed and its efficacy, pharmacokinetics, biodistribution and excretion profiles were investigated in rats. Methods The glucose-lowering effects were evaluated in a streptozocin-induced diabetic rats compared to commercial insulins via subcutaneous administration. The pharmacokinetics, biodistribution, and excretion were examined by validated analytical methods including radioactivity assay and radioactivity assay after the precipitation with TCA and the separation by HPLC. Results INS061 exhibited favorable blood glucose lowering effects up to 24 h compared to Degludec. Pharmacokinetic study revealed that the concentration-time curves of INS061 between two administration routes were remarkably different. Following intravenous administration, INS061 was quickly distributed to various organs and tissues and slowly eliminated over time with urinary excretion being the major route for elimination, and the maximum plasma concentrations (Cmax) and systemic exposures (AUC) increased in a linear manner. Conclusion The present structural modifications of human insulin possessed a long-acting profile and glucose-lowering function along with favorable in vivo properties in rats, which establish a foundation for further preclinical and clinical evaluation.

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Biodistribution, activation, and retention of proinsulin-transferrin fusion protein in the liver: Mechanism of liver-targeting as an insulin prodrug

Cited by 10 publications

References 34 publications

Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance

Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance

Study on the cellular internalization mechanisms and in vivo anti-bone metastasis prostate cancer efficiency of the peptide T7-modified polypeptide nanoparticles

Efficacy, Pharmacokinetics, Biodistribution and Excretion of a Novel Acylated Long-Acting Insulin Analogue INS061 in Rats

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