1The long history of Beta-d-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 late 1970s in theold Soviet Union as part of biological weapons research [2]. Weaponization of 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 researchforbidden. This led to research by both the US and the Soviet Union into other RNA viruses as biologicalweapons and antivirals to defend against them. Venezuelan equine encephalomyelitis virus (VEEV) was
one of the first viruses seriously considered as a biological weapon [3]. VEEV is transmitted to humans
through mosquito bites, and causes high fever, headaches, and encephalitis, and swelling of the that canbe deadly. Beta-d-N4-hydroxycytidine was found not only to inhibit VEEV replication, but a broad rangeof 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-N4hydroxycytidine on a 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 from the Emory
Institute for Drug Development (EIDD) [1], and its experimental designation was EIDD-1931. Significant
financial support for studies of antivirals against alphaviruses at institutions like Emory was provided by
the US government as far back as 2004, with considerable financial support to present [10]. The Defense
Threat Reduction Agency provided institutional support in 2014 with the goal to find an antiviralcompound against VEEV and other alphacoronaviruses. “N4-Hydroxycytidine and derivatives and antiviral 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-N4-hydroxycytidine to be administered as pills rather than injections. A
switch of research emphasis from influenza to SARS-II CoV came in 2019/2020 [2]. Commercialization of
EIDD-2801 was given to an Emory Affiliate called Ridgeway Biotherapeutics, which then partnered withMerck for the lengthy and expensive FDA approval process. The current field-testing version of EIDD2081 has been named Molnupiravir.
Beta-d-N4-hydroxycytidine, the active ingredient of Molnupiravir, exists in two forms as tautomers.
In one form, it mimics cytidine, with a single bond between the carbon and N-OH group. In its otherform, 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, the viral RNA-dependent RNA polymerase reads it as uridine
instead of cytidine and inserts an adenosine instead of guanosine. Switching between forms causes
mismatching during transcription, resulting in numerous mutations in the viral genome, and cessation ofviral replication [8].
Advancement of Molnupiravir by Merck to conditional and full approval by the FDA is on an accelerated
course. In a corporate statement, Merck has stated the following [12] - “In anticipation of the results

2from 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. ……. . Merckis committed to providing timely access to Molnupiravir globally, if it is authorized or approved, andplans 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 to widespread global access, Merck previously announced that the company has enteredinto non-exclusive voluntary licensing agreements for Molnupiravir with established genericmanufacturers to accelerate availability of Molnupiravir in more than 100 low- and middle-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 drugfor home treatment of early-stage infection [12]. However, effective anti-coronavirus compounds weredeveloped earlier for another common and highly disease of cats, feline infectious peritonitis (FIP).
These drugs include a protease inhibitor (GC376) [13] and an RNA dependent RNA polymerase inhibitor(GS-441524) that is the active moiety of Remdesivir [14]. The success in treating FIP with antiviral drugs
has prompted a recent study of both EIDD-1931 and EIDD-2801 for their ability to inhibit 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 thesecompounds, respectively. Therefore, EIDD-1931 and -2801 are slightly more virus inhibitory but also
more cytotoxic than GS-441524. 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 are several
obstacles that make legal use of these compounds unlikely any time soon. GS-441524, the active formof 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 weredone in collaboration with scientists from Gilead Science, the company refused animal rights for GS441524 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 GS441524 out of China. This same need to treat FIP has recently fueled interest on Molnupiravir as atreatment for FIP, also out of China.
The situation with EIDD-1931 and EIDD-2801/Molnupiravir and GS-441524 and Remdesivir brings toquestion 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 and was therefore conditionally approved only for injection. EIDD-2801 wascreated to enhance oral absorption of EIDD-1931, even though earlier research indicated that EIDD-1931
is well absorbed orally without esterification [6]. The motives behind the commercialization ofRemdesivir instead of GS-441524 for use in humans has been scientifically questioned, as the latter
appears to be superior in several ways without further modifications [17]. Why was EIDD-2801 was put

3forward for commercialization when EIDD-1931 would be cheaper, 4 times more virus inhibitory andone third less toxic than EIDD-2801 [15]? Strength of patent rights and patent longevity may be morecompelling 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 ocular and neurological forms of FIP with
GS-441524 by progressively increasing the dosage to raise blood levels and therefore the concentrationof drug in the aqueous humor and/or brain [20]. GC376, one of the most potent antivirals against FIPvirus in culture [17], is not effective against ocular and neurological FIP because of the inability to getenough 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 in horses 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 neurological form of FIP. The long treatment courses anddifficulty in getting sufficient drug into the brain favors the development of drug resistance.
The short- and long-term toxic effect of a candidate drug on the test person or animal is of prime
importance. GS-441524 has a lower toxicity than GC376, EIDD-1931 and EIDD-2801 in cell cultures [15].
However, it is the toxicity that occurs in vivo that is most important. GC376 is among the mostcoronavirus inhibitory drug known [15], but it will retard 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 lack of cytotoxic effects in vitro at concentrations as high as 400 uM [18]. However, EIDD1931 and EIDd-2801 demonstrate significant cytoxicity at 100 uM [15]. Therefore, it is the ability of
EIDD-1931 to create fatal mutations in RNA has been of greatest concern for some time [8, 21, 22]. Thishas 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 muchgreater 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 through mutations in theviral genome. Although Remdesivir has appeared less susceptible to such mutations than other drugs
used in viral diseases like HIV/AIDS, drug resistance has been well documented [23-25]. Resistance toGS-441524 in cats being treated for FIP has been seen with greater frequency, especially in cats withneurological FIP, where it is more difficult to get sufficient drug into the brain [13, 14, 20]. Resistance toGS-441524 in cats 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 five days during theinitial 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 resistanceprofiles. Drug resistant mutants to one drug will be immediately inhibited by the other drugs, preventing
their positive selection in the face of treatment. Inhibition of resistance is particularly strong when thetwo drugs attack different proteins involved in virus replication. For instance, GC376 is a proteaseinhibitor [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, itappears that there will be no cross-resistance between GS-441524 and Molnupiravir but is as effective
as GS-441524 in crossing the blood-to-brain barrier [3]. These things would make Molnupiravir (or 5hyroxycytidine) and important addition to future FIP treatment.

4
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, and China. No deaths occurred among 286 cats thatparticipated in the trial, including seven cats with ocular (n=2) and neurological (n=5) FIP. Twenty-eightof these cats were cured after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats remained
healthy 3-5 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 catswith 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 ofcats with FIP. At least one other major seller of GS-441524 is also interested in using
Molnupiravir for FIP, indicating a demand for additional antiviral drug treatments to cats withFIP.
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 field-testing data on
Molnuparivir in cats with naturally occurring FIP in their advertising flier for a product called
Hero-2081 [26]. However, this information does not clearly state the amount of Molnupiravir
in one of their “50 mg tablets” and the actual dosing interval (q12h or q24h?). Fortunately, anestimated starting dosage for Molnupiravir in cats with FIP can be obtained from published invitro 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 the recommended 4 mg/kg SC q24h starting dosage for GS441524 [14], or 2 mg/kg SC q24h. The per-os (PO) dosage would be doubled to account for lessefficient oral absorption to a dosage of 4 mg/kg PO q24h. An estimated starting oral dosage for
Molnupiravir in cats with 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 anaverage 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 blood-to-brain barrier at
equal efficiency to GS-441524 [3,18], the dosage would be increased ~1.5 and ~2.0 times toallow for adequate penetration into aqueous humor and cerebrospinal fluid for cats with ocular(~8 mg/kg PO, q12 h) or neurological FIP (~10 mg/kg PO, q12h). The duration of treatment
would be 10-12 weeks and monitoring of treatment response identical to GS-441524 [14, 20].
These recommendations are based on presumptions from published information and moreexperience with Molnupiravir in the field 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 prove extremely helpful in preventing GS-441524 resistance (as acocktail of antivirals with different resistance profiles) or in treating cats that no longer respondwell to GS-441524. The big unknown is whether Molnupiravir will be free from long term

5

toxicities, as the active ingredient, N4-hydroxycytidine, is an extremely potent mutagen [21]and the treatment time for FIP is much longer than for Covid-19 and chances of side-effects
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. EIDD1931 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]. N4-hydroxycytidine
is also efficiently absorbed by the oral route [3], something downplayed in the development of
EIDD-2801 (Molnupiravir). This scenario is identical to that of GS-441524 and Remdesivir, with thelatter being chosen for commercialization even though current research indicates the former would
have been the best candidate [17].
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