The 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 the  old 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 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 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 can be  deadly. Beta-d-N4-hydroxycytidine was found not only to inhibit 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 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 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-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 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 as tautomers. In  one form, it mimics cytidine, with a single bond between the carbon and N-OH 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, 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 of  viral 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 2 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 to widespread global access, Merck previously announced that the company has entered  into non-exclusive voluntary licensing agreements for Molnupiravir with established generic  manufacturers 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 drug  for home treatment of early-stage infection [12]. However, effective anti-coronavirus compounds were  developed 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 these  compounds, 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 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 same need 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 and was therefore conditionally approved only for injection. EIDD-2801 was  created 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 of  Remdesivir 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 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 ocular and neurological forms of FIP with  GS-441524 by progressively increasing the dosage to raise 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 not effective 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 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 and  difficulty 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 most  coronavirus 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, EIDD 1931 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]. 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 through mutations in the  viral 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 [23-26]. Resistance to  GS-441524 in cats being treated for FIP has been seen with greater frequency, especially in cats with  neurological FIP, where it is more difficult to get sufficient drug into the brain [13, 14, 20]. Resistance to  GS-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 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 face of 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 and Molnupiravir 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 [27]. 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 that  participated in the trial, including seven cats 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 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 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 antiviral drug 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 field-testing data on  Molnuparivir in cats with naturally occurring FIP in their advertising flier for a product called Hero 2081 [27]. 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, 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 the  recommended 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 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 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 for cats with FIP and no eye or brain involvement. If Molnupiravir crosses  the blood-to-eye and blood-to-brain barrier at equal efficiency to GS-441524, e.g., ~40% [3,18], the  starting dosage would be increased ~2.5 times to ~12 mg/kg PO, q12 h allow for adequate  penetration into aqueous humor and cerebrospinal fluid for cats with ocular or neurological FIP.  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 more experience 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 a cocktail of antivirals with different 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 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, was  not given greater consideration for treating COVID-19 than Molnupiravir. EIDD-1931 is 4 times more  virus inhibitory than Molnupiravir (EC50 0.09 vs 0.4 uM) and the percent cytotoxicity is not  significantly different between the two drugs, ranging from 0-2.8% across the range of 10 to 100  uM [15]. Toxicity appears to rise for both drugs only at 100 uM. GS-441524, by comparison has no  toxicity even at 400 uM [18]. 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 the latter being chosen for commercialization even though  current research indicates the former would have been the best candidate [17].  

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Version updated April 20, 2022