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Proinsulin-transferrin fusion protein exhibits a prolonged and selective effect on the control of hepatic glucose production in an experimental model of type 1 diabetes Juntang Shao, Jennica L. Zaro, and Wei-Chiang Shen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00168 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 9, 2016

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Molecular Pharmaceutics

Proinsulin-transferrin fusion protein exhibits a prolonged and selective effect on the control of hepatic glucose production in an experimental model of type 1 diabetes Juntang Shao,a Jennica L. Zaro,a and Wei-Chiang Shena,* a Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, 1985 Zonal Ave, Los Angeles, CA 90033 * Address correspondence to: Wei-Chiang Shen, Ph.D. 1985 Zonal Avenue, PSC 404B, Los Angeles, CA 90033. Phone: (323) 442-1902; Fax: (323) 442-1390; Email: [email protected] Abstract graphic

Abstract An ideal basal insulin replacement therapy requires the distribution or action of exogenous INS to more closely mimic physiological INS in terms of its preferential hepatic action. In this paper, we introduce a novel strategy to exert liver-specific INS action by hepatic activation of INS’s precursor, proinsulin (ProINS). We demonstrated the conversion of human ProINS-transferrin (Tf) fusion protein, ProINS-Tf, into an active and immuno-reactive form of INS-Tf in the liver via the slow Tf receptor (TfR) mediated recycling pathway. ProINS-Tf displayed prolonged basal blood glucose lowering effects for up to 40 h in streptozotocin (STZ)-induced type 1 diabetic mice following a single subcutaneous injection. The effect of ProINS-Tf on blood glucose levels was observed predominantly under fasting conditions, with little effect under freefeeding conditions. In addition, both the pyruvate tolerance assay in normal mice and the Aktphosphorylation assay in H-4-II-E hepatoma cells indicated that the hepatic-activated ProINS-Tf possessed a much longer effect on the control of hepatic glucose production than INS. These results indicated ProINS-Tf may serve as an effective and safe hepatoselective INS analog to reduce the frequency of INS injections, as well as avert severe hypoglycemia episodes and other side effects frequently encountered with long-acting INS therapeutics due to their peripheral action. Keywords: insulin delivery, fasting hyperglycemia, hepatoselective INS analog, INS prodrug, transferrin fusion protein, diabetes

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Introduction Under physiological conditions, the liver is exposed to a 2-4 fold higher endogenous insulin concentration than peripheral tissues.1 However, during the treatment of diabetic patients, only 1% of subcutaneously (s.c.) injected INS reaches the liver,2 resulting in large fluctuations of blood glucose levels.3,4 Under these conditions, peripheral glucose disposal (PGD) continues as INS circulates throughout the body and is exposed to INS receptors (IR) 5 on peripheral muscle and adipose tissues, increasing the risk for severe hypoglycemia and weight gain.6 Because the liver is under-insulinized, excessive hepatic glucose output (HGO) in diabetes is not sufficiently regulated.7 Basal INS secretion plays a key role in regulating HGO and requires unique features on both temporal (a sustained INS pharmacologic effect) and spatial (a controlled INS concentration gradient between liver and peripheral tissues) dimensions.1 Current long-acting basal INS analogs such as INS glargine (Lantus®) and INS detemir (Levemir®) work well in sustaining the pharmacological effect of INS, but lack the liver: peripheral tissue gradient. Therefore, basal INS therapeutics that fulfill both of these characteristics have the potential to overcome the limitations of current INS therapeutics.8 Recently, the significance of the liver’s role in INS delivery has received increasing attention. Several INS analogs or formulations have been reported to exhibit preferential inhibition on HGO. Examples include PEGylated INS lispro (LY2605541) recently in phase 3 clinical trials, covalent INS dimers, thyroxin INS analogs and lipidized INS detemir.9,10,11,12,13 However, the hepatoselectivity of these analogs is largely dependent on passive targeting to the liver due to their larger molecular weight (LY2605541 and INS dimers) or increased molecular size due to protein binding following injection (thyroxin INS and INS detemir).14 The increased size of the INS analogs restricts their access to the peripheral tissues due to the tightly packed endothelium, but are small enough to pass through the larger pores of vascular sinusoids in the liver.15 However, these analogs retain their glycemic activity, leaving a large amount of active circulating drug. The resultant risk for severe hypoglycemia at high doses of these hepatoselective INS analogs, as well as the potential hepatotoxicity noted for LY2605541,16 make chronic administration to diabetic patients a concern. In this study, the hepatic activation and hepatopreferential INS action of a novel fusion protein, proinsulin-transferrin (ProINS-Tf),17,18 was investigated. ProINS-Tf consists of the Cterminal of the human INS prohormone, ProINS, fused to the N-terminal of human serum Tf using recombinant DNA technology. Previous studies have shown that the elimination half-life was extended from 0.5 hours for ProINS to 7.29 hours for ProINS-Tf. ProINS-Tf was converted from the inactive hormone to an immuno-reactive form of INS-Tf (irINS-Tf) in a rat hepatoma cell line via the TfR-mediated endocytosis pathway in an acidic intracellular compartment, and acquired increased INS activity following conversion.17 The exact form of irINS-Tf has yet to be identified, but is recognizable by INS specific radioimmunoassay (RIA) with ProINS cross activity of less than 0.2 %. Furthermore, studies in an in vivo mouse model demonstrated that ProINS-Tf preferentially activated hepatic IR, rather than IR in the muscle.18 In this report, pharmacodynamic (PD) features of ProINS-Tf were compared with the long-lasting INS analog, INS glargine, in a streptozotocin (STZ) induced type 1 diabetic mouse model. Experimental section Protein preparation Protein expression of ProINS-Tf from HEK-293 cells (American Type Culture Collection, ATCC) and purification were performed as previously described.17 Purified product


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was recognized by both anti-ProINS antibody (ab8304, Abcam) and anti-Tf antibody (T2027, Sigma) in Western blots. ProINS-Tf was quantified according to human serum Tf standards in anti-Tf antibody Western blot based on band density using Image Lab™ Software (Bio-Rad). Animals and Diabetic mice model Male C57BL/6J mice (6 weeks old, Jackson Laboratory) were housed at 12:12 h light/dark cycle under standard conditions at 22 ± 3 °C with access to regular rodent chow (Labdiet) and water. Animal use protocols were in accordance with NIH guidance and approved by the Institutional Animal Care and Use Committee (IACUC) at USC. For studies on diabetic mice, mice after a 6 h morning fast were administrated with freshly prepared STZ (150 mg/kg) in sodium citrate buffer (100 mM, pH 4.5) via intraperitoneal (i.p.) injection. After injection, mice were maintained under standard care for 4-5 days to develop diabetic symptoms. Mice with blood glucose (BG) over 300 mg/dL after 2 h fasting were used for later investigations. Dose response study ProINS-Tf (67.5 nmol/kg, 135 nmol/kg and 202.5 nmol/kg), INS glargine (Lantus®, Sanofi-Aventis) (135 nmol/kg) or saline (control) were injected s.c. into diabetic mice in respective groups. INS glargine was injected with the original formulation of Lantus®. BG levels were checked through tail veil blood samples by OneTouch® glucose meter (BG assay range of 20 – 600 mg/dL, Lifescan Technologies) at indicated time points. BG levels lower than 30 mg/dL were considered as severe hypoglycemia.19 Hypoglycemic efficacy study under fast/feed cycles ProINS-Tf (135 nmol/kg), INS glargine (135 nmol/kg) or saline solution was s.c. injected to diabetic mice in each group. Mice after injection were subjected to 8 h fasting/ 8 h freefeeding cycles. BG levels were monitored at the end of each fasting or feeding phase using OneTouch® glucose meter. Pyruvate tolerance test (PTT) PTT was performed on normal 6-7 weeks old male C57BL/6J mice. After 6 h fasting, mice were randomly grouped to receive s.c. injection of respective treatment (saline, 22.5 nmol/kg INS or 22.5 nmol/kg ProINS-Tf). Mice were then kept under an overnight fast (12 h) for hepatic glycogen depletion. Afterwards, the main precursor of hepatic gluconeogenesis, sodium pyruvate, (Sigma) was injected i.p. into mice (2g/kg). BG levels were recorded at indicated time points following pyruvate bolus using the same method described above. ProINS-Tf conversion assay on precision-cut liver slices (PCLS) Liver samples collected from wild type male C57BL/6J mice (6–7 weeks old) were precisely sliced (250 µm) using a McIlwainTM Tissue Chopper (The Mickle Laboratory Engineering Co. Ltd). Freshly prepared liver slices were cultured in William’s E medium (Life Technologies) supplemented with 25 mM D-glucose and 0.1 % BSA. For conversion assay, liver slices were treated with oxygenated (95 % air/5 % CO2) dosing medium containing ProINS-Tf (10 nM) with or without holo-Tf blockage (10 µM) and placed on an orbital shaker (50 RPM) at 37 °C. At indicated time points, the irINS-Tf conversion product was detected from aliquots of incubation medium using using a human INS specific radioimmunoassay (RIA) kit (Millipore) with ProINS cross activity of less than 0.2 %. The conversion assay was also performed using 10 nM human recombinant ProINS (R&D Systems). To ensure the viability of liver slices, the conversion assays were performed within 12 h of collection of liver samples Time courses of Akt dephosphorylation on H-4-II-E H-4-II-E cells after overnight serum starvation were treated with equimolar doses (1 nM) of ProINS-Tf, ProINS (R&D System), or INS (Sigma) in serum free DMEM for 4 h at 37 °C.



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After treatment and extensive washing, dosing medium was replaced with fresh DMEM. At the indicated time points, cells were solubilized in cell extraction buffer (Life Technologies) containing additional phosphatase inhibitors and protease inhibitor cocktail (Sigma). Cell lysates were extracted by centrifugation (13,000 g, 10 min) and subjected to SDS-PAGE. The residual phosphorylated Akt was detected by anti-Phospho Ser473-Akt antibody (Cell Signaling Technology). Band densities were quantified by Image Lab™ Software (Bio-Rad). Akt phosphorylation was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detected by anti-GAPDH (D16H11) XP® antibody (Cell Signaling Technology) based on band density. Data analysis BG levels were expressed as the group mean value (n ≥ 3) with standard deviation (SD). Data were analyzed by Student’s t-test, where P< 0.05 was considered as statistically significant. Results Dose response study of ProINS-Tf In the dose response study (Fig.1), BG levels gradually decreased with similar lowering rates among three dosages of ProINS-Tf in the first fasting phase. Even the highest dose of ProINS-Tf (202.5 nmol/kg) did not cause severe hypoglycemia in mice. In contrast, INS glargine (135 nmol/kg) induced an instant BG reduction. All mice (n=5) in the INS glargine group exhibited severe hypoglycemia from 2 to 8 h, which was recovered after 2 h feeding. Also, INS glargine exerted a robust inhibition of hyperglycemia during both fasting and feeding conditions. BG was below 100 mg/dL up to the end of the initial 4 h feeding periods. Once its hypoglycemic effect diminished after 12 h post-injection, the BG bounced back to hyperglycemia and showed no significant difference from the control group even under fasting condition. Contrarily, the BG lowering effect of ProINS-Tf was more prominent during the fasting phase. At 4 h post injection, three dosages of ProINS-Tf exerted similar efficacy and maintained fasting BG around euglycemic levels at the subsequent fasting time points. During the feeding phase, only the highest dosage of ProINS-Tf was able to restrain to a 40 % BG reduction compared to saline group. Duration of efficacy study under fast/feed cycles To explore the duration of the BG lowering effect of ProINS-Tf and its glycemic control between prandial and basal conditions, BG levels were monitored during the fast/feed cycles on diabetic mice after s.c. injection of INS analogs. As shown in Fig.2, INS glargine at 135 nmol/kg induced severe hypoglycemia at 2 h post injection, and lost efficacy after the first fast/feed cycle at 18 h post injection. ProINS-Tf exhibited a 20-40 % BG lowering activity during feeding phases. However, an effective and sustained blunting effect of fasting hyperglycemia was observed at 40 h post injection. In comparison with saline control, BG levels in ProINS-Tf group at the end of three subsequent fasting phases, i.e., 8, 24 and 40 h, were around euglycemia level in mice (100-120 mg/dL), while BG in the control group were all above 300 mg/dL. PTT Our previous study showed that ProINS-Tf displayed a longer BG lowering effect than 18 INS. Two contributing processes of HGO include glycogen breakdown (glycogenolysis), and gluconeogenesis, i.e., de novo glucose synthesis from pyruvate, lactate, and amino acids. During prolonged fasting after hepatic glycogen is depleted, gluconeogenesis becomes the main contributor of glucose released by the liver. Under these conditions, the level of HGO can be measured by determining BG levels after pyruvate i.p-injection using PTT.20 To evaluate the


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effect of ProINS-Tf and INS on inhibition of HGO, a PTT was conducted 12 h after protein injection. In saline and INS groups, pyruvate induced peak BG elevation at around 120 mg/dL (Fig.3). INS which shows short-term activity, lost restrictions of HGO at the 12-h post-injection time point. In contrast, ProINS-Tf reduced BG increment by 50% compared to control, therefore showing a prolonged effect on the inhibition of HGO. ProINS-Tf conversion studies on PCLS Previous studies showed that the ProINS-Tf conversion in H-4-II-E hepatoma cells was through the TfR mediated endocytosis and the recycling pathway.17 In this paper, conversion assay was performed on PCLS ex vivo liver models. PCLS show preserved liver tissue architecture and cell heterogeneity,21 closely mimicking in vivo hepatic processing, and are therefore a widely applied model to study liver glucose metabolism and INS action.22 The defined thickness (250 µm) is optimum to maintain liver slice function and viability in PCLS culture at least up to 24 h incubation.23,24,25 As shown in Fig.4A, liver slices were able to convert ProINS-Tf and release irINS-Tf into the medium after 8 h incubation. Complete blockage of conversion by holo-Tf indicated that hepatic conversion was mediated by TfR. TfR-mediated conversion was also confirmed by the lack of conversion when incubating PCLS with ProINS alone. (Fig.4B). Prolonged time course of Akt dephosphorylation After the 4 h stimulation (the initial time point 0 h in Fig.5), both ProINS-Tf and INS treatment induced significant phosphorylation of Akt. When the dosing solution was removed and cells were starved in serum-free medium, the residual Akt phosphorylation in the INS group rapidly decreased and returned to basal levels within 1 h. On the other hand, the phosphorylated Akt in the ProINS-Tf group was maintained at a similar level and lingered for at least 4 h after removing the fusion protein (Fig.5). ProINS did not lead to notable phosphorylation of Akt at the 1 nM dose due to its low potency to activate IR. Discussion Tf has long been used to improve the serum stability of fused protein drugs.26,27 In the case of ProINS-Tf, the fusion to Tf not only increased circulation time in plasma,18 but also enabled ProINS to irINS conversion, presumably in the slow TfR recycling pathway.17 Our previous studies have pinpointed the intracellular venue for conversion specifically to an acidic compartment along the TfR-mediated slow recycling pathway on H-4-II-E cells, and ProINS-Tf's ability to inhibit glucose production in vitro was diminished when the conversion was blocked.13 In addition, irINS-Tf generation required the presence of trypsin-like enzymes with restricted basic amino acids cutting sites, rather than non-specific protease degradation.18 Conversion from ProINS-Tf to irINS-Tf is not only necessary for the INS prodrug to become the active form, but also contributes to ProINS-Tf's hepatopreferential action and other glycodynamic features. In this report, conversion assays on PCLS further supported that the liver was able to activate ProINS-Tf to irINS-Tf (Fig.4). In dose response studies, the delayed onset of BG lowering efficacy of ProINS-Tf has been attributed to both the protracted absorption and the rate/activitycontrolled conversion.18 Saturation of conversion has been observed with increased doses of ProINS-Tf above 10 nM after prolonged incubation with H-4-II-E cells (Fig.S1). The rate/activity-limited conversion can explain the relative dose-independent BG reduction immediately after ProINS-Tf treatment (Fig.1), and demonstrated a major difference in tailoring BG lowering efficacy between INS glargine and ProINS-Tf. ProINS-Tf exhibited less reliance on dosage possibly due to a stringently regulated conversion in vivo. This type of dose-response



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regiment allows for the prevention of overdose-induced hypoglycemia, since the conversion of ProINS-Tf is conceivably regulated in vivo by the competition with the constant excess endogenous Tf in Tf/TfR pathway. ProINS-Tf exhibited a prolonged BG lowering efficacy, which was still active at 40 h (Fig.2) post-injection compared to 12 h for INS glargine (Fig.1). In addition to the prolonged half-life of ProINS-Tf,18 the sustained hypoglycemic effect might also be contributed by an increased retention in liver. As shown in Fig.5, either ProINS-Tf or irINS-Tf exploited TfR recycling pathways as intracellular reservoirs, and led to time-lapse INS signaling. Another conceivable reason contributing to the prolonged effect of ProINS-Tf was the intrinsic feature of its action site. Deactivation of INS suppression on HGO took two times longer than the disappearance of INS-induced PGD after ceasing INS infusion on healthy human subjects.28 Therefore, the prolonged efficacy of ProINS-Tf may be related to its preferential action on HGO in the liver. The bioactivity of ProINS-Tf in the liver was supported by the PTT results, where ProINS-Tf showed an inhibition of gluconeogenesis (Fig.3). It was also supported by the effect of ProINS-Tf on BG where a higher efficacy in maintaining euglycemia under fasting, rather than feeding conditions, was observed (Fig.2). Hepatic contribution in glycemic homeostasis varies between basal and prandial conditions.29 After meals, the liver takes up one third of absorbed dietary glucose in an INS independent manner. The main portion is disposed to the brain and the peripheral tissues via INS sensitive glucose transporters.30 During fasting, the liver plays the dominant role as the main supply of glucose in systematic circulation. Moreover, an excessive HGO is the cause of fasting hyperglycemia in diabetes.31,32 Hepatoselective INS analogs better mimic normal physiology, and lead to lower risks of hypoglycemia and metabolic side-effects including weight gain. ProINS-Tf has the same advantages with regard to increased molecular size which may lead to passive liver targeting, but also has the advantage of being administered as an inactive prodrug. In order to obtain sitespecific delivery, it has been previously suggested that the following three criteria should be optimized in prodrug design: (1) efficient absorption, (2) selective activation, and (3) retention at the target site.33,34 Due to its molecular weight and to the high number of TfR present in the liver, ProINS-Tf should have high absorption in the liver. Additionally, ProINS-Tf conversion requires additional factors other than TfR and specifically occurs in hepatocytes (Fig.S2). Finally, as mentioned above, the prolonged activity of ProINS-Tf (40 h) which is much longer than its halflife (7.29 h),18 and its sustained phosphorylation of Akt (Fig.5), both indicate that it is retained in the liver. Therefore, ProINS-Tf may meet all three of the important criteria for an effective prodrug. In conclusion, ProINS-Tf exhibits a liver-targeted hypoglycemic activity due to a combination of TfR-mediated endocytosis, intracellular activation, and extended IR binding of this fusion protein in hepatocytes. ProINS-Tf possesses a prolonged plasma half-life and, unlike other long-acting INS analogs, can avoid severe hypoglycemia even at high doses in a doseescalation study. Due to the long-lasting liver-targeted effect, ProINS-Tf can maintain a prolonged control on BG levels in fasted, but not in free-fed, Type 1 diabetic mice. Therefore, ProINS-Tf appears to be an ideal INS prodrug for the long-term control of basal BG levels in Type 1 diabetes. Acknowledgements These studies were supported by NIH grant GM063647 and USC Bensussen Innovative Challenge Grant.


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Figures

Figure 1. Dose response study. STZ induced diabetic mice after s.c. injection of respective treatment were kept under 8 h fasting (white zone)/feeding (grey zone) phase intervals. Data represented average value with SD. The dashed line indicated BG threshold (30 mg/dL) of severe hypoglycemia in mice.


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Figure 2. Hypoglycemic efficacy under fast/feed cycles. Equal dose of ProINS-Tf and INS glargine were s.c. injected to STZ induced diabetic mice in the respective group. After injection, mice were kept under fast/feed cycles and BG levels were monitored at the end of each phase (8 h). White zones represented fasting phases and grey zones indicated feeding phases. The dashed line indicated the severe hypoglycemia threshold (BG=30 mg/dL). BG curves reflected average values and error bars indicated SD (n=3).

Figure 3. BG increment after pyruvate bolus. After respective treatment (Saline, 22.5 nmol/kg INS or 22.5 nmol/kg ProINS-Tf), mice were restrained from food for 12 h. A single dose of sodium pyruvate (2g/kg) was then i.p. injected and BG levels were recorded at indicated time points. Data were presented as the average value (n=4) of BG changes in each group. (BG increment = BG at X time point -initial BG level before pyruvate injection). A

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Figure 4. Characterization of ProINS-Tf conversion with freshly-prepared liver slices in PCLS incubation. (A) 10 nM of ProINS-Tf in absence or presence of 1000-fold holo-Tf blockage (ProINS-Tf and ProINS-Tf+Holo-Tf respectively). In the medium only group, liver slices were incubated in supplemented WE medium without fusion protein. At indicated time points, aliquots of the incubation medium were applied to human INS specific RIA kit to detect irINS-Tf (pmol/ g tissue protein). (B) 10 nM human recombinant ProINS with or without 10 uM holo-Tf. Data were expressed as average value and error bars indicated SD (n=3).

Figure 5. Time course of Akt dephosphorylation. H-4-II-E cells were initially exposed for 4 h with control (DMEM), 1 nM ProINS-Tf, 1 nM INS or 1 nM ProINS. P-Akt was determined by Western blot at 0, 1 and 4 h after the removal of the treatment medium. Phosphorylated-Akt bands were normalized with GAPDH band density. Data were expressed as the average values and error bars indicated SD (n=3).


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