The first U.S. Ebola patient, Thomas Eric Duncan, has died despite being administered the experimental nucleoside-type antiviral pro-drug Brincidofovir. How is this possible? Well , the first problem is that Duncan was not treated until it was far too late for antiviral therapy to work.
Let's take this opportunity to look at some nucleoside antivirals, incuding the drug that was administered to Mr. Duncan in Dallas, TX (Brincidofovir).
The first thing to know is remember Brincidofovir and Cidofovir were developed to work against dsDNA Viruses, though Ebola is a negative-sense ssRNA virus. The analogy is similar to the previous Operon Labs post on the potential action of HIV RT inhibitor Lamivudine and our Ebola Virology Overview.
Broncidofovir, Cidofovir, Lamivudine: Background
 |
 |
| Cidofovir | Brincidofovir (Cidofovir pro-drug) |
| Cidofovir, is a nucleoside-derived antiviral that is active against a variety of DNA Viruses including Cytomegalovirus (CMV), Adenovirus, HPV, Herpes Virus, and Orthopox viruses. | Brincidofovir is a highly-fat soluble orally active form of Cidofovir. Brincidofovir is orally absorbed and circulates as the lipid-conjugate above; it enters cells through diffusion, and is cleaved intracellularly into Cidofovir by Phospholipase enzymes. |
|
In both examples above, intracellular Cidofovir is converted to it's active form , Cidofovir Diphosphate , by intracellular protein kinases. These enzymes phosphorylate Cidofovir, resulting in its active DP form. *NOTE: Oral Brincidofovir is useful because it can result in Cidofovir-DP intracellular concentrations that are approximately 10 to 100 times higher than can intravenous Cidofovir, with far fewer side effects. |
|
Brincidofovir, the drug to the right, is orally active and was administered to Thomas Eric Duncan as an anti-Ebola therapy. Cidofovir , the drug to the left , is not orally active.
Brincidofovir (right molecule) is eventually converted into Cidofovir (left molecule) inside human cells by enzymes.
Cidofovir's phosphonate group limits its bioavailability, due to the negative charge of the phosphonate group at physiological pH, which prevent G.I absorption. Thus, Cidofovir can only be administered via intravenous infusion. Conversely, Brincidofovir is orally absorbed from the G.I. tract into the lymphatic system, due to it's long hydrophobic chain,
source: http://www.chimerix.com/c/discovery-clinical-trials/technology-discovery.php
The active antiviral compound produced in either case -- Cidofovir-Diphosphate -- is the same for both the above drugs.
Cidofovir DP -- Intracellular Antiviral active form of both Brincidofovir and Cidofovir.
Broncidofovir, Cidofovir, Lamivudine: Mononegavirales RnRp Inhibition
Now what's interesting is that Cidofovir has zero published test data showing that it has high affinity for RdRp. Here is an example on a study where Cidofovir was tested against Measles Virus -- a good virus to compare to as an Ebola reference. Measles, like Ebola, is a negative-sense single-stranded virus in the same Genus as Ebola -- Mononegavirales.
In the Measles assay, Cidofovir has very low affinity for Measles RNA Dependent Rna Polymerase (RdRp) -- Cidofovir has zero anti-Measles activity up to 70uM (meaning it's IC50: far above >100 uM) ... This is in the same range as HIV RT drug Lamivudine (IC50: 180 uM for Lamivudine-TP against Measles RdRp). Lamivudine actually might be more potent than Cidofovir against Measles RdRp.
Perhaps both these drugs work against Ebola for another reason not identified... Perhaps the accumulation of high levels of the active drug in PBMCs during conditions of high cellular stress? Perhaps Ebola RnRp is particularly susceptible due to it's high polymerase error rate , or it's biomechanical structure? Or perhaps one, both, or neither of these drugs work against RNA viruses like Ebola. In terms of Cidofovir (the ultimate active drug administered to Thomas Duncan)... Let's see how it does against Measles, a virus comparable to Ebola...
"The [antivirals] were tested versus wild type measles virus in B95a cells (Figure 1) [...] Cidofovir, a DNA polymerase inhibitor, shows no effect in this assay [against Measles negative-sense ssRNA Virus Polymerase]." (McGuigan et al, 2013). This is suggesting that Cidofovir (the active component in Broncidofovir) has very poor activity against Measles RnRp.
Now, granted, perhaps the doses weren't high enough. Or perhaps Ebola RnRp is more susceptible. Perhaps something else is in play. But regardless, this suggests that we absolutely need more published scientific data from Chimerix (manufacturer of Broncidofovir) before we conclude that Brincidofovir (or even Lamivudine for that matter) is the latest and greatest Ebola antiviral -- since Cidofovir does not even inhibit Measles at 70 uM. Keep in mind that Measles is susceptible (IC50) to Lamivudine at approx 180 uM. Results for Cidofovir against Measles are relatively poor.
Tissue Culture Infective Dose: Lower value means more potent Measles antiviral
So suffice to say, just looking at the public data, it does not look promising that Cidofovir or Brincidofovir would be active against Ebola. Pharmacology studies indicate Brincidofovir has a Cmax blood concentration of 623.1084 nanomolar, or 0.623 micromolar. If blood Cidofovir-DP levels are normalized to Cmax, this results in intracellular concentrations of CDV-DP of 3.4 uM. To normalize for the 10x to 100x greater bioavailability of Brincidofovir, we multiply to yield a Cidofovir-DP effective intracellular concentration range of 34 uM to 340 uM of in human PBMCs treated with Brincidofovir. The long story short is that Cidofovir is not a particularly potent drug to begin with, with only 1/100 of the extracellular drug being converted to the active form intracellular.
BUT: With Brincidofovir having great bioavailablity and pharmacokinetics, we reach Cidofovir-DP intracellular concentrations from 34uM to 340uM. . . The active level of Cidofovir-DP in the Measles assay is at minimum 0.7uM. So Brincidofovir has potential.
But Brincidofovir has not been tested on RNA Viruses. There is no published research that I can find besides press releases from Chimerix. Hopefully, further data is forthcoming.
Any nucleoside-type antivirals likely must be administered soon after onset of symptoms, or they probably will not be effective in containing disease mortality. Most all nucleoside antiviral must be activated by intracellular host kinases to their DP or TP (Triphosphate) Form, which can take 24h to 48h to distribute into tissue compartments, and for the drug to encounter the rate-limiting enzymatic conversion steps. This is particularly true for pro-drugs like Brincidofovir and T-705.
Here is an example of how Brincidofovir is activated by host cellular kinases. This process is very similar for most all nucleoside related antiviral drugs.
Favipiravir (T-705): The Best Ebola Antiviral currently Approved
A better antiviral Ebola therapy that inhibits most all ssRNA Viruses is an RdRp (RNA-Dependent RNA Polymerase) Inhibitor called T-705 -- (similar in mechanism of action to Brincidofovir) -- T-705 only works in mice and other small animals if administered within the first 6 to 8 days Post-Infection (P.I.). The same seems true in humans, based on it's short clinical testing in Europe against Ebola infected individuals.
T-705 (aka Famipiravir) has been tested in murine models of Ebola, and has been shown to have potent anti-Ebola activity when administered at 150mg/kg B.I.D. This drug has been used to save several patients' lives in Europe. The European treatment protocols are not yet published, but using standard FDA Mouse/Human dose scaling, a 65kg human probably needs to be administered (at minimum) 750 mg of Famipiravir twice per day to treat Ebola Virus Disease (EVD).
Fortunately, Famipiravir has the benefit of being tested in animal models against Ebola, and was designed and intended to substitute as a purine nucleotide in viral RdRp. Famipiravir causes chain-termination of the enzyme after two anti-viral are incorporated sequentially. We have strong data to show Famipiravir will treat Ebola in humans. This is not the case for Brincidofovir (yet). The experimental drug Brincidofovir was designed as a Cidofovir pro-drug, which is ultimately active against some dsDNA viruses. There are no in vivo tests (even in animals) to show Brincidofovir is active against Ebola.
Thus, it is probably too early to evaluate Brincidofovir as an Ebola therapy -- whether positively or negatively. It does have some promise, and should be investigated. However, given that other existing therapies tested in animals are promising (Famipiravir, BCX-4430, Clomiphene, etc), these should be the first-line therapies against Ebola -- not Brincidofovir, which has yet to be tested in animal models. Famipiravir production should be immediately scaled up, and selected as front-line treatment against Ebola. BCX-4430 should immediately enter 'right to try' Phase I trials. Clomiphene (already FDA approved) should immediately be tested on patients willing to give Informed Consent based on Clomiphene's 90% protection rate in murine models.
Brincidofovir and the first U.S. Ebola Fatality: Too Late for Lethal Host Genes
Most likely Duncan received Brincidofovir too late, even if the drug is effective in vivo. Duncan should have been administered therapy sooner by the medical authorities. By the time he received any antiviral, most likely the vascular and organ dysfunction (Severe Sepsis / DIC) was just too severe. . . Even if the viral replication were slowed or stopped, there is still circulatory system and organ damage to deal with -- not to mention being over 14 days into a severe Ebola infection. Why did Duncan take so long to be identified, receive treatment? If this was done sooner, he may have survived. By the time Brincidofovir was started, he was probably in DIC and viral sepsis.
Part of Ebola's lethality is likely due to a DIC/Septic Shock like syndrome that many patients progress to during the terminal phases of the disease. Fatal strains of Ebola up-regulate a whole constellation of host genes. One of the most damaging of these are the MMP class of genes -- specifically MMP-3 (Matrix Metalloproteinase 3).
"Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix and during tissue remodeling"
http://en.wikipedia.org/wiki/MMP3
Host MMP-3 upregulation has been studied and identified (among other genes) to correlate with the activity of lethal Ebola strains. Most likely, MMP-3 is involved in the destruction of surrounding collagen networks around Ebola-infected Fibroblasts. Interestingly (and of potential theraputic note) is that Doxycycline is one of the few 'broad-spectrum' human MMP inhibitors that is FDA-approved.
"Of particular relevance is our finding that metalloproteinase genes were upregulated only in lethal [Ebola] infections and not in response to the nonlethal wild-type or single-mutant viruses. Furthermore, the metalloproteinase gene MMP3 was one of 7 genes associated with lethality that was induced by the double mutant but not by the virus carrying the VP24 mutation. Metalloproteinases control chemokine activity (14, 34, 37, 43) and can regulate inflammation by controlling the activity of chemokines (13, 28), and MMP3-deficient mice are prone to severe inflammation "
.gif)
Molecular signature associated with [Ebola] lethality. Biological network analysis determined by Ingenuity Pathways Analysis to identify functional relationships between the differentially regulated inflammatory genes associated with lethality. This analysis highlights five different subsets of genes that showed a direct functional relationship. These genes are involved in chemotaxis (green), apoptosis (yellow), acute-phase signaling (orange), cytotoxicity of leukocytes (blue), and leukocyte extravasation (purple). All these genes were strongly upregulated at 72 h postinfection as a consequence of lethal Ebola virus infection, with the exception of four chemotaxis genes (CCL21, CD209, CLEC4M, and FCER2) that were downregulated.
http://jvi.asm.org/content/85/17/9060.full