Diabetes Metab J.  2024 Jan;48(1):59-71. 10.4093/dmj.2022.0292.

A New Concept in Antidiabetic Therapeutics: A Concerted Removal of Labile Iron and Intracellular Deposition of Zinc

• Vinokur V
• Berenshtein E
• Chevion M
• Chevion D

• KMID: 2551263
• DOI: http://doi.org/10.4093/dmj.2022.0292

Abstract

Background
The inflammatory process is known to be an integral part of the pathophysiology of type 2 diabetes mellitus (T2DM). The “labile,” redox-active iron, serving as a catalyst in Fenton reaction, producing the deleterious reactive oxygen species, triggering and maintaining inflammation, is hypothesized to play a causative role in this process. Concenter Biopharma continued the development of a new platform of iron chelators (Zygosids), first initiated at the Hebrew University of Jerusalem, Israel (HUJI), acting via the novel mechanism, based on a sequestration of the labile redox-active iron and its substitution by zinc or gallium. The mode of action of Zygosids is based on the higher affinity of the metal-binding moiety of the complex to Fe³⁺ in comparison to already bound ion, leading to rapid release of the ion of another metal and chelation of Fe³⁺. Concomitantly, zinc ion, released by the complex, is known for its antidiabetic and anti-inflammatory role.
Methods
The therapeutic effect of zinc-desferrioxamine (Zygosid-50) and gallium-desferrioxamine, was tested on fat sand rat (Psammomys obesus) model of diet-induced T2DM and on Lepr^db transgenic diabetic mice.
Results
Zygosids demonstrated an ability to noticeably reduce blood glucose and insulin levels and improve the lipid profile. Moreover, an ability to mitigate insulin resistance by >90% was shown on the sand rat model. In addition, a potent anti-inflammatory effect, expressed as a diminishment of the proinflammatory cytokines in tissue levels, was demonstrated.
Conclusion
Zygosids demonstrated robust therapeutic efficacy in treatment of T2DM. Importantly, no adverse effects were detected, in all the experiments, indicating high safety profile.

Keyword

Diabetes mellitus, type 2; Inflammation; Insulin resistance; Iron chelating agents; Zinc

Figure

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  Fig. 1. Effect of the preventive administration of Zygosid-50 and gallium-desferrioxamine (Ga-DFO) on sand rats’ (A) blood glucose level (BGL), (B) body weight, (C) serum insulin, (D) total cholesterol, (E) high-density lipoprotein (HDL), (F) HDL as a percentage of the total cholesterol, (G) low-density lipoprotein (LDL), and (H) triglycerides. Ten 12 weeks old male sand rats (Psammomys obesus) from the diabetes-prone sub-strain were transferred onto the high-energy diet (HED) on day 1. The animals were divided into six groups. Group 1: low energy (LE) diet, untreated controls (n=12); Group 2: HED, sham-treated (n=10); Group 3: HED, treated with Zygosid-50 2 mg/kg (n=8); Group 4: HED, treated with Zygosid-50 6 mg/kg (n=9); Group 5: HED, treated with Ga-DFO 2 mg/kg (n=8); Group 6: HED, treated with Ga-DFO 6 mg/kg (n=8). Treatment was started from day 1 of the study. The drugs were given by intraperitoneal injection 3×/week. Throughout the experiment the animals’ blood glucose was monitored three times a week (A) body weight twice a week (B). On day 52 the animals were euthanized, and blood was taken for assessment of serum insulin (C), and lipid profile (total serum cholesterol, HDL, LDL, triglycerides) (D, E, F, G, H). (A) Starting from day 16, the values observed in all groups were significantly lower (P<0.05) than those in HED sham-treated one. Treatment with either Zygosid had no statistically significant effect on the body weight (B). The values are shown as average±standard error. aP≤ 0.05 vs. control group, bP≤0.05 vs. diabetes sham-treated group.

• 

  Fig. 2. Effect of treatment of the diabetic sand rats with Zygosid-50 and gallium-desferrioxamine (Ga-DFO) on (A) blood glucose level (BGL), (B) body weight, (C) serum insulin, (D) total cholesterol, (E) high-density lipoprotein (HDL), (F) HDL as a percentage of the total cholesterol, (G) low-density lipoprotein (LDL), (H) triglycerides, and (I) glucose tolerance, shown as results of intraperitoneal (i.p.)-glucose tolerance test (GTT). Ten 12 weeks old male sand rats (Psammomys obesus) from the diabetes-prone sub-strain were transferred onto the high-energy diet (HED) on day 1. The animals were divided into five groups. Group 1: low energy (LE) diet, untreated controls (n=8); Group 2: HED, sham-treated (n=14); Group 3: HED, treated with Zygosid-50 2 mg/kg (n=10); Group 4: HED, treated with Zygosid-50 6 mg/kg (n=12); Group 5: HED, treated Ga-DFO 6 mg/kg (n=10). Treatment was started from days 27/28 of the experiment. The drug was given by i.p. injection 3×/week. Throughout the experiment blood glucose was measured thrice a week (A), and body weight twice a week (B). On day 57 the animals were euthanized, and blood was taken for assessment of serum insulin (C) and lipid profile (D, E, F, G, H). (A) Starting from day 30, the values observed in all groups were significantly lower (P<0.05) than those in HED shamtreated one. (I) The i.p.-GTT and homeostasis model assessment of insulin resistance (Fig. 3) calculations were conducted at the end of the study. In i.p.-GTT results the values of HED sham-treated group from 30 minutes. were significantly higher than all other groups. The P values of all treatment groups versus the control increased to >0.05 after 60 minutes. The values are shown as average±standard error. aP≤0.05 vs. control group, bP≤0.05 vs. diabetes sham-treated group.

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  Fig. 3. Effect of treatment of the diabetic sand rats with Zygosid-50 and gallium-desferrioxamine (Ga-DFO) on insulin resistance, expressed as homeostasis model assessment of insulin resistance (HOMA-IR). The values are shown as average±standard error. aP≤0.05 vs. control group, bP≤0.05 vs. diabetes sham-treated group.

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  Fig. 4. Effect of treatment of the diabetic sand rats with Zygosid-50 and gallium-desferrioxamine (Ga-DFO) on indicators of diabetic complications, e.g., (A) alanine transaminase (ALT), (B) non-alcoholic steatohepatitis (NASH) severity score, and (C) cataract. (D) These representative images of type 2 diabetes mellitus-induced cataracts in sand rat. Immediately after euthanasia, ALT, the blood marker of NASH was measured (A). Samples from the liver underwent H&E staining and analyzed for NASH severity score as described by Brunt et al. [27], measuring the percent of intra-hepatocyte macro-vesicular fat, as following: 0%, score 0; <30%, score 1; 31%–50%, score 2; >51%, score 3 (B). On the last day of the experiment cataract formation was visually assessed, according to the following score: 3: cataract covering more than 75% of the visible area on at least one eye; 2–3: cataract covering more than 50% of the visible area on at least one eye; 1–2: cataract covering <50% of the visible area on at least one eye; 0: no signs of cataract detected (C). The values are shown as average±standard error. aP≤0.05 vs. control group, bP≤0.05 vs. diabetes shamtreated group.

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  Fig. 5. Effect of treatment of diabetic leptin receptor (Lepr)db mice with Zygosid-50 on (A) blood glucose level (BGL) and (B, C) intraperitoneal (i.p.)-glucose tolerance test (GTT). Eight male BKS.Cg-Dock7m+/+ Leprdb/J mice 8 to 9 weeks old were divided into two groups of n=4. Group 1 was treated with Zygosid-50 6 mg/kg body weight thrice a week for 60 days, while the second one served as a sham-treated control. i.p.-GTT was performed in the beginning of the experiment and 2 days before the euthanasia. (A, C) The P value between the treated and untreated groups was <0.05 in all time points. The values are shown as average±standard error.

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  Fig. 6. The therapeutic effect of Zygosid-50 delivered topically against croton oil-induced inflammation, expressed as (A) ear swelling, (B) interleukin 1α (IL-1α), (C) IL-6, (D) tumor necrosis factor-α (TNF-α), and (E) IL-17. Three groups of 12-week-old female Balb/c mice (n=6 per group) were used. Irritant contact dermatitis was induced by epicutaneous application of croton oil solution on ear. Ear thickness was monitored with an engineer’s micrometer, along the experiment: immediately before the exposure to the croton oil, and at 3, 6, 9 hours following the exposure. The pre-exposure thickness value was subtracted from the post-exposure values at each time point, quantifying the severity of inflammation. Group 1 remained untreated. A fingertip of Vaseline (Group 2) or Zygosid-50 0.5% ointment (Group 3) was applied once, 3 hours after the first exposure to the irritant. Six hours after the exposure the mice were euthanized, the ears were immediately harvested. Cytokine concentrations (relative to protein levels) were determined by enzyme-linked immunosorbent assay (ELISA) methods, employing commercially available kits, according to the Manufacturers’ protocols. The cytokines measured included: IL-1α (ELM-IL1a-1, RayBiotech), IL-6 (ELM-IL6-CL-1, RayBiotech), TNF-α (ELM-TNFa-CL-1, RayBiotech), and IL-17 (EKA51897, Biomatik). The values are shown as average±standard error. aP≤0.05 vs. control.

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