Novel COVID-19 Therapeutic
DIBI, an Iron-Binding Polymer, Provides a Potential New Therapeutic for COVID-19 Infections
The challenge: “Currently there are no broad-spectrum antivirals or immunotherapies available for the fight against emerging pathogens, and no proven treatments approved for use against COVID-19.”
Chelation Partners proposes a broad-spectrum mobilization of host defense through the sequestration of iron required for growth and proliferation of pathogens including viruses, bacteria and parasites. Iron regulation and availability is a key but often overlooked factor in pathogenesis. Starving pathogens and/or infected cells of biologically available iron is a natural defense against infection. Many pathogens possess countermeasures such as siderophores, to scavenge iron. Chelation Partners developed a next generation iron sequestration agent, DIBI, powerful enough to scavenge iron despite pathogen countermeasures, yet non-toxic to normal uninfected cells.
DIBI – a new iron scavenger
DIBI is a highly soluble, 9kDa iron-chelating antimicrobial polymer, designed to sequester only that iron in the host’s body that is required for growth and proliferation of infectious organisms and without seriously altering the body’s essential iron stores. DIBI is conjugate of a GRAS polymer backbone used pharmaceutically since the 1950s plus an iron binding moiety similar to Deferiprone (FDA approved in 2011) (Figure 1).
DIBI is non-toxic
DIBI is non-toxic in animal testing and against normal human cells in culture. OECD-prescribed toxicity testing in rats showed no observable adverse effects (NOAEL) with 14-day repeat dosing at 1,000mg/ kg/ day oral and 500mg/ kg/ day intravenous DIBI (highest doses tested). DIBI required extraordinarily high concentrations to demonstrate cytotoxicity for human fibroblasts (EC50 8,928 μg/mL) and mouse fibroblasts (793 μg/mL) and with human epithelial cells (691 μg/mL).
Figure 1. DIBI polymer structure compared to Deferiprone and schematic representation of a single molecule of DIBI chelating three iron molecules.
DIBI is a potent antimicrobial agent
DIBI has µM MICs against pathogens (MRSA = 4µM/mL, C. albicans = 2µM/mL), between >70 and >500 times more potent than Deferiprone, depending on the pathogen (Table 1).
Table 1. MICs for DIBI and Deferiprone against representative bacterial and fungal pathogens.
Figure 2. DIBI suppresses infection by Gram positive and Gram-negative antibiotic-resistant bacteria (mouse).
a Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300.
Source: Ang et al. 2018
DIBI is effective against Gram positive and Gram-negative antibiotic-resistant bacteria in mouse models of skin and lung infections (Figure 2).
Mice infected topically with antibiotic resistant MRSA S. aureus ATCC 43300 on either skin wounds or on their anterior nares were treated with the chelator DIBI and bacterial burdens after 5 days were compared to sham treated (vehicle treated only) groups. The stars show significant reductions in bacterial burdens compared to the sham treatment. Mice infected intranasally with A. baumannii antibiotic resistant isolate LAC4 to initiate pneumonia followed by septicemia were treated intranasally once with DIBI or sham treated with only vehicle and bacterial burdens after 24 h infection were determined for both lungs and spleens. The stars show significant reductions in bacterial burdens compared to the sham treatment (Parquet et al. 2018).
DIBI enhances other antimicrobial agents
DIBI enhances antibiotic killing and prevents survivor re-growth. When combined, sub-inhibitory concentrations of an antibiotic and DIBI result in pathogen killing with no regrowth (Figure 3).
DIBI has significant benefits as a COVID-19 therapeutic
In response to the COVID-19 pandemic, we are redirecting our research and development efforts to testing ways in which DIBI can reduce infections and mortality in infected individuals. DIBI has properties consistent with 4 different mechanisms by which COVID-19 infection may be inhibited or outcomes improved:
A. baumannii ATCC17978 sensitive to the aminoglycoside Gentamicin (GEN) (Minimum Inhibitory Concentration (MIC) = 1 μg/mL) was exposed to 0.5 μg/mL GEN (i.e., ½ MIC) and this caused only partial killing of the population followed by rapid recovery growth that reached near untreated control Colony Forming Units (CFU) levels by 24 h of exposure. The DIBI concentration used in this experiment was 20μg/mL and this on its own did not affect total CFU by 24 h. It should be noted that a large rapidly growing iron-replete bacterial population had been introduced at 0 h and gross effects of DIBI on bacterial numbers would not be expected under these test conditions. However, this DIBI treatment exhibited effects of iron withdrawal as evident when DIBI was applied in combination with GEN. The GEN/DIBI combination resulted in extensive and continued bacterial killing with no detectable CFU remaining at 24 h exposure (results adapted from Parquet et al. 2019).
Figure 3. A time kill kinetic assay showing chelator enhancement of antibiotic killing and prevention of survivor re-growth.
4 DIBI COVID-19 approaches:
suppression of viral attachment via down regulation of receptor,
interfering with COVID-19 replication to impede and clear the infection,
suppression of cytokine storm in severe complicated COVID-19 cases, and
suppression of secondary bacterial pneumonia in severe COVID-19 cases
The first two direct antiviral effects are rapidly testable and have recently been anticipated by Liu et al. (Liu, Zhang, Nekhai and Liu, Depriving Iron Supply to the Virus Represents a Promising Adjuvant Therapeutic Against Viral Survival 2020, Curr Clin Microbiol Rep.). Chelation Partners has completed and published relevant proof of concept for the second two approaches to improve outcomes and reduce mortality in sepsis and pneumonia models including with antibiotic resistant bacteria. Clinical testing is the next step.
DIBI may suppress initial viral attachment via down regulation of host receptor
Figure 4. Comparison of select hepcidin and coronavirus spike protein sequences.
Figure 5. DIBI-mediated iron withdrawal modulates iron regulatory gene mRNA expression.
MDA-MB-468 cells were cultured for 48 h in the presence of medium alone or the indicated concentrations of DIBI.
Source: Greenshields et al 2019
DIBI may block viral uptake or replication
Various natural flavonoid compounds have been shown to possess antiviral activity to coronaviruses:
Kaempferol reported to block coronaviral release by blocking 3a channels of the virus.
Luteolin has been reported to block SARS-CoV uptake by binding to the S2 viral protein.
The activity of these natural flavonoids has been shown to be possibly related to their iron chelating activities. DIBI shares some structural similarities with flavonoids but is a much more potent iron chelator than natural flavonoids or other synthetic iron chelating agents (Figure 6). DIBI’s size, chemical binding groups and high affinity for iron are consistent with antiviral activity through blocking either uptake or release of COVID-19.
DIBI may suppress cytokine storm
COVID-19 mortality is often the result of a dysregulated inflammatory response in which excessive cytokine release (cytokine storm) causes broad damage to lungs and other organs. This is similar to sepsis when seen in overwhelming bacterial infection. DIBI has been shown to suppress key aspects of such dysregulated inflammatory responses through modulating production and release of key cytokines such as IL-6 (Figure 7).
Figure 6. Structural similarity of DIBI with anti-viral natural flavonoids.
DIBI anti-infective for secondary bacterial pneumonia
Secondary bacterial pneumonia often occurs in ventilated patients and is typically caused by bacteria normally resident in the upper respiratory tract such as Acinetobacter baumannii which can overtake compromised patients. These bacteria are increasingly antibiotic resistant due to widespread antibiotic usage. COVID-19 patients are given antibiotics prophylactically. DIBI has been shown in animal models to suppress pneumonia caused by resistant bacteria and to also improve bacterial killing by antibiotics including organisms that are “drug resistant” (Figure 8).
Sources: Lalani and Poh, 2020, Viruses
Martin-Benlloch, et al., 2018, New J. Chem.
Figure 7. DIBI treatment of macrophages inhibits IFNβ, IL-1β and IL-6 but not TNFα mRNA expression.
Source: unpublished from D.W. Hoskin laboratory
Figure 8. Effect of DIBI, Ciprofloxacin (CIP) or DIBI+CIP treatment on infection in the lungs of mice following intranasal challenge with A. baumannii*.
*these bacteria are resistant to CIP on their own
Effect of DIBI, Ciprofloxacin (CIP) or DIBI+CIP treatment on infection in the lungs and blood of BALB/c mice following i.n. challenge with A. baumannii LAC-4. Mice were infected intranasally with 3.0 x 106 (solid bars) or 1.7 x 107 (open bars) CFU of A. baumannii LAC-4 at 0 h. The mice were administered intranasally 3 and 10 h later with a total of 100 mg/kg (11µmol/kg) DIBI, 100 mg/kg DIBI + 20 mg/kg (60µmol/kg), CIP, 20 mg/kg CIP or diluent (control).
Bacterial burdens in the lung and blood were determined by quantitative bacteriology at 24 h after inoculation.
Source: Parquet et al. 2019 AAC
The COVID-19 spike protein has structural homology with host hepcidin, a protein which regulates iron uptake/export of host cells by binding to Ferroportin receptor on cells (Figure 4). Iron is dysregulated in viral infection and in also in T2DM (Type 2 diabetes mellitus, a major predisposing condition) with lowered hepcidin and possibly upregulated Ferroportin receptor.
DIBI treatment of host cells downregulates Ferroportin expression. DIBI reduces available Ferroportin sites that may be involved in viral attachment/cell entry (Figure 5).