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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
Original article: The long history of Beta-d-N4-hydroxycytidine and its modern application to treatment of Covid-19 in people and FIP in cats.

Beta-d-N4-hydroxycytidine is a small molecule (nucleoside) that was studied in the late 1970s in the former Soviet Union as part of biological weapons research [2]. The weaponization of diseases such as smallpox was a worldwide threat, but the danger of using the smallpox virus for this purpose was too great. Smallpox was eradicated from the world, virtually all stocks were destroyed and further research was banned. This led the US and the Soviet Union to research other RNA viruses as biological weapons and antivirals to defend against them. The Venezuelan equine encephalomyelitis virus (VEEV) was one of the first viruses to be seriously considered for use as a biological weapon. [3]. VEEV is transmitted to humans by mosquito bites and causes high fever, headaches and encephalitis with swelling that can be fatal. Beta-d-N4-hydroxycytidine has been found to inhibit not only VEEV replication but also a wide range of alphaviruses, including Ebola, chikungunya, influenza virus, norovirus, bovine diarrhea virus, hepatitis C virus and respiratory syncytial virus. [3-8]. The first reports of an inhibitory effect of beta-d-N4-hydroxycytidine on human coronavirus NL63 date back to 2006 [9]. Recent studies have confirmed its inhibitory effect on a wide range of human and animal coronaviruses [8].

An important part of the recent history of beta-d-N4-hydroxycytidine is associated with the Emory Institute for Drug Development (EIDD) [1], where he received the experimental designation EIDD-1931. The US government has provided significant financial support for the study of antivirals against alphaviruses in institutions such as Emory since 2004. [10]. In 2014, the Defense Threat Reduction Agency provided institutional support to find an antiviral compound against VEEV and other alpha-coronaviruses. "N4-Hydroxycytidine and its derivatives and antiviral uses" were included in U.S. Patent Application 2016/106050 A1 of 2016 [11]. Additional funding in 2019 was provided by the National Institute of Allergies and Infections for fellowship of the esterified beta-d-N4-hydroxycytidine precursor (EIDD-2801) for the treatment of influenza. [10]. The stated purpose of the chemical changes of EIDD-2801 was to increase its oral bioavailability, which would ultimately allow beta-d-N4-hydroxycytidine to be administered as pills and not as injections. In 2019/2020, the focus of research changed from influenza to SARS-CoV-2 [2]. The commercialization of EIDD-2801 was entrusted to Emory's Ridgeway Biotherapeutics subsidiary, which subsequently worked with Merck on a lengthy and costly FDA approval process. The current version of EIDD-2081 for field testing was named Molnupiravir.

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

Merck's commitment to conditional and full FDA approval of Molnuparivir continues. In its statement, Merck stated: [12] "In anticipation of the results of the MOVe-OUT program, Merck manufactures Molnupiravir at its own risk. Merck expects to produce 10 million therapeutic doses by the end of 2021, with more expected to be produced in 2022. Merck is committed to providing timely access to Molnupiravir worldwide, if authorized or approved, and plans to introduce access to tiered prices based on World Bank admission criteria that reflect countries' relative ability to fund their pandemic health response. As part of its commitment to extend the global approach, Merck has previously announced that it has entered into non-exclusive voluntary licensing agreements for Molnupiravir with established generic manufacturers to accelerate the availability of Molnupiravir in more than 100 low and middle income countries (LMICs) following approval or emergency approval by local regulatory agencies. . " This "generosity" is unlikely to apply to use in animals.

Drugs to inhibit the current Covid-19 pandemic have been the subject of accelerated field trials in the last two years, and one of them, Remdesivir, has been approved for use in hospitalized patients in record time. Last year, Molnupiravir was submitted for conditional approval as an oral medicinal product for home treatment of the infection at an early stage. [12]. However, anti-coronavirus compounds have been developed previously for another common and serious feline disease, feline infectious peritonitis (FIP). These drugs include a protease inhibitor (GC376) [13] and an RNA-dependent RNA polymerase inhibitor (GS-441524), which is an active ingredient of Remdesivir [14]. The success of antiviral drugs in the treatment of FIP prompted a recent study by EIDD-1931 and EIDD-2801 for their ability to inhibit FIPV in tissue cultures. [15]. The effective EC50 concentrations for EIDD-1931 against FIPV are 0.09 μM, EIDD-2801 0.4 μM and GS441524 0.66 μM [15]. The percentage of cytotoxicity at 100 μM is 2.8, 3.8 and 0, respectively. Therefore, EIDD-1931 and EIDD-2801 are slightly more effective at inhibiting viruses, but also more cytotoxic than GS-441524. These laboratory studies suggest that EIDD-1931 and EIDD-2801 are excellent candidates for the treatment of FIP.

Although EIDD-1931 and EIDD-2801 are a great promise for the treatment of FIP, there are several obstacles that will make the legal use of these compounds unlikely in the near future. GS-441524, the active form of Remdesivir and patented by Gilead Sciences, was investigated for use in cats with FIP shortly before the Covid-19 pandemic. FIP research [14] therefore stimulated the potential use of Remdesivir against Ebola and not SARS-like coronavirus [14]. Although these studies were conducted in collaboration with scientists from Gilead Sciences, the company refused to grant GS-441524 rights to treatment in animals as soon as it became clear that there was a much larger market for Covid-19 in humans. [16]. Similarly, my attempts over the past 2-3 years at Emory, Ridgeback Biotherapeutics, and Merck Veterinary Division to investigate EIDD-1931 and EIDD2801 for the treatment of FIP in cats have either remained unanswered or rejected, no doubt for similar reasons why Gilead refused to grant rights for GS-441524. However, the great worldwide need for FIP treatment quickly supported the unapproved market for GS-441524 from China. The same need to treat FIP has recently aroused interest in Molnupiravir, also from China.

Situation with EIDD-1931 vs. EIDD-2801 / Molnupiravir and GS-441524 vs. Remdesivir raises the question of why some medicines are being converted to prodrugs for marketing purposes [17]. Remdesivir was reportedly esterified to increase antiviral activity, although studies in cats showed that GS-441524 and Remdesivir had similar viral inhibitory activity in tissue culture. [18]. However, Remdesivir was found to be poorly absorbed by the oral route and was therefore conditionally approved for injectable use only. EIDD-2801 was designed to increase the oral absorption of EIDD-1931, although previous research has shown that EIDD-1931 is well absorbed orally without esterification. [6]. The motives for the commercialization of Remdesivir instead of GS-441524 for human use have been scientifically questioned, as it appears to be better in several ways without further modification. [17]. Why EIDD-2801 was chosen for commercialization, when EIDD-1931 would be cheaper, 4 times more effective against viruses and one third less toxic than EIDD-2801 [15]? The strength of patent rights and the longevity of patents may be more important factors in these decisions. [16,17,19].

One of the problems in the treatment of FIP in cats is the blood-eye and blood-brain barriers, which become very important when the disease affects the eyes and / or the brain. [13, 14, 20]. This problem has been largely overcome in the treatment of ocular and neurological forms of FIP with GS-441524 by gradually increasing the dose to increase blood levels and thus drug concentrations in the ventricular fluid and / or brain. [20]. GC376, one of the most effective antivirals against FIP virus in culture [17], is not effective against ocular and neurological FIP due to the inability to get enough drug to these sites, even if the dose is increased 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 now occurs in some cats treated with GS-441524, especially in individuals with the neurological form of FIP. Long treatment procedures and difficulties in transporting enough drug to the brain support the development of drug resistance.

The short-term and long-term toxic effects of the drug candidate on the test person or animal are crucial. GS-441524 showed lower toxicity in cell cultures than GC376, EIDD-1931 and EIDD-2801 [15]. Most important, however, is the toxicity that manifests itself in vivo. GC376 is one of the drugs with the highest coronavirus inhibitory effect [15], but slows the development of adult teeth when given to young kittens [13]. No serious toxicity was observed during nearly three years of field use of GS-441524, reflecting the complete absence of cytotoxic effects in vitro at concentrations up to 400 µM. [18]. However, EIDD-1931 and EIDD-2801 show significant cytotoxicity at 100 μM [15]. Therefore, the ability of EIDD-1931 to make fatal mutations in RNA has been raising a number of questions for some time. [8, 21, 22]. This was the main reason why the application for the treatment of diseases was still delayed. However, the current recommended duration of treatment with Covid-19 Molnupiravir is only 5 days at the initial stage of treatment. [10]. However, the recommended duration of FIP treatment with GS-441524 is 12 weeks [14], which represents a much longer time for the manifestation of toxicity. Therefore, close observation of cats during treatment with EIDD-1931 or EIDD-2801, whether short-term or long-term, will be important.

All existing antiviral drugs have led to the development of drug resistance through mutations in the viral genome. Although Remdesivir appears to be less susceptible to such mutations compared to drugs used in viral diseases such as HIV / AIDS, resistance is well documented. [23-25]. Resistance to GS-441524 in cats treated for FIP was observed at a higher frequency, especially in cats with neurological FIP, where it is more difficult to deliver sufficient drug to the brain [13, 14, 20]. Resistance to GS-441524 in cats is also likely to be a major problem, as cats with FIP are often treated for 12 weeks or longer, while Remdesivir (and Molnupiravir) are recommended for only five days during the initial viremic stage of Covid-19. [16]. The problem of drug resistance in HIV / AIDS treatment is effectively addressed by using a cocktail of different drugs simultaneously with different resistance profiles. Mutants resistant to one drug will immediately inhibit other drugs, thus preventing their positive selection during treatment. Inhibition of resistance is particularly strong when the two drugs attack different proteins involved in virus replication. For example, GC376 is a protease inhibitor [13], while GS-441524 acts on an RNA-dependent RNA polymerase [18]. However, GC376 is not as well absorbed across the blood-brain barrier. Although the necessary research has not yet been performed, there appears to be no cross-resistance between GS-441524 and Molnupiravir and is as effective as GS-441524 in crossing the blood-brain barrier. [3]. This makes Molnupiravir (or 5-hydroxycytidine) an important contribution to the future treatment of FIP.

As expected, Molnupiravir has recently been tested on cats with FIP by at least one Chinese retailer, GS-441524, and preliminary results are available on the FIP Warriors website. [26]. Field trials included 286 cats with various forms of naturally occurring FIP observed at pet clinics in the United States, the United Kingdom, Italy, Germany, France, Japan, Romania, Turkey, and China. The 286 cats that participated in the study, including seven cats with ocular (n = 2) and neurological (n = 5) FIP, did not die. Twenty-eight of these cats were cured after 4-6 weeks of treatment and 258 after 8 weeks. All treated cats were healthy after 3-5 months, a period during which relapses would be expected to relapse unsuccessfully. These data provide convincing evidence of the safety and efficacy of Molnupiravir in cats with various forms of FIP. However, we hope that this field study will be written in manuscript form, submitted for review and published. Either way, Molnupiravir is already marketed to owners of cats with FIP. At least one other major retailer of GS-441524 is also interested in using Molnupiravir for FIP, indicating a demand for additional antiviral drugs for cats with FIP.

Safe and effective dosing for Molnupiravir in cats with FIP has not been published. However, at least one vendor from China provided certain pharmacokinetic and field test data for Molnuparivir in cats with naturally occurring FIP in a leaflet for the product Hero-2081. [26]. However, this information does not clearly indicate the amount of Molnupiravir in one of their "50 mg tablets" and the actual dosing interval (q12h or q24h?). Fortunately, the estimated starting dose of molnupiravir for 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 GS-441524 [14,18]. Molnupiravir (EIDD-2801) has an EC50 of 0.4 μM / μl against FIPV in cell culture, while the EC50 of GS-441524 is about 1.0 μM / μl. [18]. Both have a similar oral absorption of about 40-50 %, so the effective subcutaneous (SC) dose for Molnupiravir would be approximately half the recommended 4 mg / kg SC every 24 hours of the initial dose for GS441524. [14] or 2 mg / kg SC q24h. The per-os (PO) dose would be doubled to account for less effective oral absorption at a dose of 4 mg / kg PO every 24 hours. The estimated initial oral dose of molnupiravir for cats with FIP can also be calculated from the available Covid-19 treatment data. Patients treated for Covid-19 are given 200 mg of molnupiravir PO q12h for 5 days. This dose was evidently calculated from a pharmacokinetic study performed in humans, and if the average person weighs 60-80 kg (70 kg), the effective inhibitory dose is 3,03.0 mg / kg PO q12h. The cat has a basal metabolic ratio 1.5 times higher than humans, and assuming the same oral absorption in both humans and cats, the minimum dose for cats according to this calculation would be 4.5 mg / kg PO every 12 hours. Assuming that molnupiravir crosses the blood-brain barrier and the blood-brain barrier as efficiently as GS-441524 [3,18], the dose would be increased ~ 1.5 and ~ 2.0-fold to allow adequate penetration into the aqueous humor and cerebrospinal fluid for cats with ocular (~ 8 mg / kg PO, q12 h) or neurological FIP (~ 10 mg / kg PO, q12h). The treatment will last 10-12 weeks and the monitoring of the response to treatment will be identical to GS-441524 [14, 20]. These recommendations are based on published data assumptions and further experience with Molnupiravir will be required in this area. Molnupiravir is unlikely to be safer and more effective than GS-441524 in the treatment of FIP, but a third antiviral drug may be particularly useful in preventing resistance to GS-441524 (as a cocktail of antivirals with different resistance profiles) or in treating cats that no longer respond. good on GS-441524. It is largely unknown whether Molnupiravir will be without long-term toxic effects, as the active substance N4-hydroxycytidine is an extremely potent mutagen. [21] and the duration of FIP treatment is much longer than with Covid-19 and there is a likelihood of major side effects.

It is a pity that EIDD-1931 (N4-hydroxycytidine), the active substance in Molnupiravir, has not received much attention in the treatment of FIP cats than Molnupiravir. EIDD-1931 has a 4-fold greater inhibitory effect against the virus than Molnupiravir (EC50 0.09 vs. 0.4 μM) and the percentage of cytotoxicity is slightly lower (2.3% vs. 3.8% at 100 μM) [15]. N4-hydroxycytidine is also efficiently absorbed orally [3], which was downplayed in the development of EIDD-2801 (Molnupiravir). This scenario is identical to the GS-441524 vs. Remdesivir, the second of which, Remdesivir, was chosen for commercialization, although current research suggests that GS-441524 would be the best candidate.[17].

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