THE LONG HISTORY OF BETAD-N4-HYDROXYCYTIDINE AND
ITS MODERN APPLICATION TO
TREATMENT OF COVID-19 IN
PEOPLE AND FIP IN CATS.
Niels C. Pedersen DVM, PhD
October 20, 2021
Beta-d-N4-hydroxycytidine is a small molecule (nucleoside) that was studied in the late1970s in the old Soviet Union as part of biological weapons research [2]. Weaponizationof diseases like smallpox was a worldwide fear, but the dangers of using smallpox virus
for this purpose was just too great. Smallpox had been eradicated from the world, virtually
all stocks destroyed, and further research forbidden. This led to research by both the US
and the Soviet Union into other RNA viruses as biological weapons and antivirals to
defend against them. Venezuelan equine encephalomyelitis virus (VEEV) was one of thefirst viruses seriously considered as a biological weapon [3]. VEEV is transmitted tohumans through mosquito bites, and causes high fever, headaches, and encephalitis, andswelling of the that can be deadly. Beta-d-N4-hydroxycytidine was found not only toinhibit VEEV replication, but a broad range of alphaviruses, including Ebola, chikungunya,
influenza virus, norovirus, bovine diarrhea virus, hepatitis C virus and respiratory syncytial
virus. [3-8]. The earliest report of the inhibitory effect of beta-d-N4- hydroxycytidine ona human coronavirus NL63 was in 2006 [9]. Recent studies have confirmed its inhibitory
effect on a broad range of human and animal coronaviruses [8].

An important part of the more recent history of beta-d-N4-hydroxycytidine comes fromthe Emory Institute for Drug Development (EIDD) [1], and its experimental designation was
EIDD-1931. Significant financial support for studies of antivirals against alphaviruses atinstitutions like Emory was provided by the US government as far back as 2004, withconsiderable financial support to present [10]. The Defense Threat Reduction Agency

provided institutional support in 2014 with the goal to find an antiviral compound against
VEEV and other alphacoronaviruses. “N4-Hydroxycytidine and derivatives and anti viral
Uses related thereto” was covered by US Patent Application 2016/106050 A1 in 2016[11]. Additional funding came in 2019 from the National Institute of Allergy and Infectious
to partner research into an esterified prodrug of beta-d-N4-hydroxycytidine (EIDD-2801)
for treatment of influenza [10]. The stated intent of the chemical alterations to EIDD-2801
was to enhance its oral bioavailability, which would ultimately allow beta-d-N4hydroxycytidine to be administered as pills rather than injections. A switch of researchemphasis from influenza to SARS-II CoV came in 2019/2020 [2]. Commercializationof EIDD-2801 was given to an Emory Affiliate called Ridgeway Biotherapeutics, whichthen partnered with Merck for the lengthy and expensive FDA approval process. The
current field-testing version of EIDD 2081 has been named Molnupiravir.

Beta-d-N4-hydroxycytidine, the active ingredient of Molnupiravir, exists in two forms astautomers. In one form, it mimics cytidine, with a single bond between the carbon and NOH group. In its other form, which mimics uridine, it has an oxime with a double bond
between the carbon and N-OH group. In the presence of beta-d-N4-hydroxycytidine, theviral RNA-dependent RNA polymerase reads it as uridine instead of cytidine and inserts
an adenosine instead of guanosine. Switching between forms causes mismatching duringtranscription, resulting in numerous mutations in the viral genome, and cessation of viral
replication [8].

Advancement of Molnupiravir by Merck to conditional and full approval by the FDA is onan accelerated course. In a corporate statement, Merck has stated the following [12] – “In
anticipation of the results from MOVe-OUT, Merck has been producing Molnupiravir at risk.
Merck expects to produce 10 million courses of treatment by the end of 2021, with more doses
expected to be produced in 2022. ……. . Merck is committed to providing timely access to
Molnupiravir globally, if it is authorized or approved, and plans to implement a tiered pricing
approach based on World Bank country income criteria to reflect countries’ relative ability
to finance their health response to the pandemic. …… . As part of its commitment towidespread global access, Merck previously announced that the company has entered into

non-exclusive

voluntarylicensingagreements

forMolnupiravir

withestablishedgeneric manufacturers to accelerate availability of Molnupiravir in more than 100 low- andmiddle-income countries (LMICs) following approvals or emergency authorization by local
regulatory agencies.” It is unlikely that this “generosity” will also apply to animal use.

Drugs to inhibit the causative agent of the current Covid-19 pandemic, have been rapidly
field tested over the last two years and one of them, Remdesivir, was approved in record
time for hospitalized patients. Molnupiravir has been moved toward conditional approval
within the last year as an oral drug for home treatment of early-stage infection [12].
However, effective anti-coronavirus compounds were developed earlier for anothercommon and highly disease of cats, feline infectious peritonitis (FIP). These drugs include
a protease inhibitor (GC376) [13] and an RNA dependent RNA polymerase inhibitor (GS441524) that is the active moiety of Remdesivir [14]. The success in treating FIP with antiviraldrugs has prompted a recent study of both EIDD-1931 and EIDD-2801 for their ability toinhibit FIPV in tissue culture [15]. The effective concentration-50% (EC50) for EIDD-1931
against FIPV is 0.09 uM, EIDD-2801 0.4 uM and GS-441524 0.66 Um [15]. The percent
cytotoxicity at 100 uM is 2.8, 3.8 and 0 for these compounds, respectively. Therefore,
EIDD-1931 and -2801 are slightly more virus inhibitory but also more cytotoxic than GS441524. These laboratory studies indicate that both EIDD-1931 and EIDD-2801 are also
excellent candidates for FIP treatment.

Although EIDD-1931 and EIDD-2801 hold great promise for the treatment of FIP, there areseveral obstacles that make legal use of these compounds unlikely any time soon. GS441524, the active form of Remdesivir, and patented by Gilead Sciences, was researched
for use in cats with FIP shortly before the Covid-19 pandemic occurred. Therefore, it was
the potential use of Remdesivir against Ebola virus and not SARS-like coronaviruses that
prompted research on FIP [14]. Even though these studies were done in collaboration with
scientists from Gilead Science, the company refused animal rights for GS 441524 once it
became obvious that there was a much larger market for Covid-19 in humans[16]. Similarly, my attempts over the last 2-3 years to Emory, Ridgeback Biotherapeutics,
and the Veterinary Division of Merck to research EIDD-1931 and EIDD-2801 for FIP in cats

have either gone unanswered or rebuffed, undoubtedly for similar reasons to why Gilead
refused to grant animal rights for GS-441524. However, the great worldwide need for an
FIP treatment rapidly fueled an unapproved market for GS 441524 out of China. This sameneed to treat FIP has recently fueled interest on Molnupiravir as a treatment for FIP, also
out of China.

The situation with EIDD-1931 and EIDD-2801/Molnupiravir and GS-441524 and Remdesivir
brings to question why some drugs are converted to prodrugs for marketing [17].
Remdesivir was reportedly esterified to increase antiviral activity, although studies in cats
showed that GS-441524 and Remdesivir had similar virus inhibitory activity in tissue
culture [18]. However, Remdesivir was found to be poorly absorbed by the oral route andwas therefore conditionally approved only for injection. EIDD-2801 was created toenhance oral absorption of EIDD-1931, even though earlier research indicated that EIDD1931

is well absorbed orally without esterification [6]. The motives behind thecommercialization of Remdesivir instead of GS-441524 for use in humans has beenscientifically questioned, as the latter appears to be superior in several ways without
further modifications [17]. Why was EIDD-2801 was put forward for commercialization
when EIDD-1931 would be cheaper, 4 times more virus inhibitory and one third less toxic
than EIDD-2801 [15]? Strength of patent rights and patent longevity may be
more compelling factors in these decision [16, 17, 19].

One of the problems in the treatment of FIP in cats is the blood-to-eye and blood-to brain
barriers, which become of great importance when the disease affects the eyes and/or
brain [13, 14, 20]. This problem has been overcome in large part in the treatment of ocularand neurological forms of FIP with GS-441524 by progressively increasing the dosage toraise blood levels and therefore the concentration of drug in the aqueous humor and/or
brain [20]. GC376, one of the most potent antivirals against FIP virus in culture [17], is noteffective against ocular and neurological FIP because of the inability to get enough drug
into these sites even when increasing the dosage several times [14]. Fortunately, it
appears that EIDD-1931 can reach effective levels in the brain as indicated by studies inhorses with VEEV infection [3]. Drug resistance is another problem that is now being seen

in some cats being treated with GS-441524, especially individuals with the neurologicalform of FIP. The long treatment courses and difficulty in getting sufficient drug into thebrain favors the development of drug resistance.

The short- and long-term toxic effect of a candidate drug on the test person or animal isof prime importance. GS-441524 has a lower toxicity than GC376, EIDD-1931 and EIDD2801 in cell cultures [15]. However, it is the toxicity that occurs in vivo that is mostimportant. GC376 is among the most coronavirus inhibitory drug known [15], but it willretard adult dentition when given to young kittens [13]. No serious toxicity has been
observed over almost three years of field use of GS-441524, mirroring the complete lackof cytotoxic effects in vitro at concentrations as high as 400 uM [18]. However, EIDD 1931
and EIDd-2801 demonstrate significant cytoxicity at 100 uM [15]. Therefore, it is the abilityof EIDD-1931 to create fatal mutations in RNA has been of greatest concern for some time[8, 21, 22]. This has been a big reason why it has been slow to be applied to disease.
However, the current recommended treatment of Covid-19 with Molnupiravir is for only
5 days at the early stage of treatment [10]. However, the recommended treatment with
GS-441524 for FIP is 12 weeks [14], allowing much greater time for toxicity to manifest
itself. Therefore, it will be important to carefully observe cats on EIDD-1931 or EIDD-2801
treatment for both short- and long-term effects.

All antiviral drugs to date have yielded to the development of drug resistance throughmutations in the viral genome. Although Remdesivir has appeared less susceptible tosuch mutations than other drugs used in viral diseases like HIV/AIDS, drug resistance has
been well documented [23-25]. Resistance to GS-441524 in cats being treated for FIP has
been seen with greater frequency, especially in cats with neurological FIP, where it ismore difficult to get sufficient drug into the brain [13, 14, 20]. Resistance to GS-441524 incats is also likely to be more of a problem because cats with FIP are often treated for
12 weeks or more, while Remdesivir (and Molnupiravir) are recommended for only fivedays during the initial viremic stage of Covid-19 [16]. The problem of drug resistance has
been effectively managed in HIV/AIDS treatment by using a cocktail of different drugs at
the same time with different resistance profiles. Drug resistant mutants to one drug will

be immediately inhibited by the other drugs, preventing their positive selection in the faceof treatment. Inhibition of resistance is particularly strong when the two drugs attack
different proteins involved in virus replication. For instance, GC376 is a protease inhibitor[13], while GS-441524 acts on the RNA dependent RNA replicase [18]. However, GC376 is
not as well absorbed across the blood-to-brain barrier. Although sufficient work has not
yet been done, it appears that there will be no cross-resistance between GS-441524 andMolnupiravir but is as effective as GS-441524 in crossing the blood-to-brain barrier [3].
These things would make Molnupiravir (or 5- hyroxycytidine) and important addition to
future FIP treatment.

As anticipated, Molnupiravir has recently been tested in cats with FIP by at least one
Chinese sellers of GS-441524, and preliminary results reported on the FIP Warriors CZ/SK
website [26]. The field trial consisted of 286 cats with various forms of naturally occurring
FIP seen in pet clinics in US, UK, Italy, Germany, France, Japan, Romania, Turkey, andChina. No deaths occurred among 286 cats that participated in the trial, including sevencats with ocular (n=2) and neurological (n=5) FIP. Twenty-eight of these cats were cured
after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats remained healthy 35 months later, a period during which relapses would be expected in cats not
successfully cured. This data provides compelling evidence for the safety and efficacy of
Molnupiravir for cats with various forms of FIP. However, it is hoped that this field trial will
be written in manuscript form, submitted for peer review, and published. Nevertheless, it
is now being sold to owners of cats with FIP. At least one other major seller of GS-441524
is also interested in using Molnupiravir for FIP, indicating a demand for additional antiviraldrug treatments to cats with FIP.

A safe and effective dosage for Molnupiravir in cats with FIP has not been published.
However, at least one seller out of China has provided some pharmacokinetic and fieldtesting data on Molnuparivir in cats with naturally occurring FIP in their advertising flier fora product called Hero-2081 [26]. However, this information does not clearly state theamount of Molnupiravir in one of their “50 mg tablets” and the actual dosing interval (q12hor q24h?). Fortunately, an estimated starting dosage for Molnupiravir in cats with FIP canbe obtained from published in vitro cell culture studies of EIDD-1931 and EIDD-2801 [15]and laboratory and field studies of GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50
of 0.4 uM/ul against FIPV in cell culture, while the EC50 of GS-441524 is around 1.0 uM/ul[18]. They both have similar oral absorptions of around 40-50%, so an effective
subcutaneous (SC) dosage for Molnupiravir would be approximately one-half therecommended 4 mg/kg SC q24h starting dosage for GS 441524 [14], or 2 mg/kg SC q24h.
The per-os (PO) dosage would be doubled to account for less efficient oral absorption toa dosage of 4 mg/kg PO q24h. An estimated starting oral dosage for Molnupiravir in catswith FIP can also be calculated from available data on Covid-19 treatment. Patients being
treated for Covid-19 are given 200 mg of Molnupiravir PO q12h for 5 days. This dosage
was obviously calculated from a pharmacokinetic study done on people, and if
an average person weighs 60-80 kg (70 kg), the effective inhibitory dosage is ~3.0 mg/kg
PO q12h. A cat has a basal metabolic rate 1.5 times a human, and assuming equal oral
absorption for both people and cats, the minimum cat dosage by this calculation would
be 4.5 mg/kg PO q12 hr. Assuming that Molnupiravir crosses the blood-to-eye and bloodto-brain barrier at equal efficiency to GS-441524 [3,18], the dosage would be increased~1.5 and ~2.0 times to allow for adequate penetration into aqueous humor andcerebrospinal fluid for cats with ocular (~8 mg/kg PO, q12 h) or neurological FIP (~10mg/kg PO, q12h). The duration of treatment would be 10-12 weeks and monitoring oftreatment response identical to GS-441524 [14, 20]. These recommendations are based onpresumptions from published information and more experience with Molnupiravir in thefield will be needed. It is doubtful that Molnupiravir will prove safer and any more effective
than GS-441524 for the treatment of FIP, but a third antiviral drug may could proveextremely helpful in preventing GS-441524 resistance (as a cocktail of antivirals withdifferent resistance profiles) or in treating cats that no longer respond well to GS-441524.
The big unknown is whether Molnupiravir will be free from long term toxicities, as theactive ingredient, N4-hydroxycytidine, is an extremely potent mutagen [21] and thetreatment time for FIP is much longer than for Covid-19 and chances of sideeffects greater.

It is unfortunate that EIDD-1931 (N4-hydroxycytidine), the active ingredient of Molnupiravir,
has not been given greater consideration for treating cats with FIP than Molnupiravir. EIDD
1931 is 4 times more virus inhibitory than Molnupiravir (EC50 0.09 vs 0.4 uM) and thepercent cytotoxicity is somewhat less (2.3% vs. 3.8% at 100 uM), respectively [15]. N4hydroxycytidine is also efficiently absorbed by the oral route [3], something downplayedin the development of EIDD-2801 (Molnupiravir). This scenario is identical to that of GS441524 and Remdesivir, with the latter being chosen for commercialization even thoughcurrent research indicates the former would have been the best candidate [17].

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