In spite of the numerous repurposed or clinical stage drugs that have been evaluated, one well studied clinical stage candidate drug has apparently not been evaluated regarding its antiviral activity as monotherapy or in combination therapy for the treatment of COVID-19 disease.
In fact, this clinical stage candidate drug possesses the properties that makes it ideal for the rapid treatment of a large population of patients with mild-to-mod- erate COVID-19 disease.
This candidate drug is Tucaresol (Figure 1), or 4-[(2-formyl-3- hydroxyphenoxy) methyl] benzoic acid (IUPAC nomenclature); Molecular Weight = 272; soluble in aqueous base. Tucaresol is an orally active antiviral agent with good oral bio- availability (70%), ambient temperature stability and excellent potency (less than 100 mg daily), thereby permitting treatment of many patients with a minimal amount of drug.
Additionally, Tucaresol is readily manufactured by a two-step proprietary synthesis de- veloped by us (patent pending).
Importantly, Tucaresol is not encumbered by pre-exist- ing patent(s) claiming compound ownership (Composition of Matter), synthesis (process) or treatment of coronavirus, including SARS-CoV-2.
Tucaresol first appeared as a new compound in European patent application EP 00549924, 1982, from The Wellcome Foundation Ltd.
However, per this application, Tucaresol was initially under development by Glaxo Wellcome for the palliation of sickle cell anemia.
Since the drug mechanism required stoichiometric binding of Tucaresol to hemoglobin to prevent red blood cell sickling and increase oxygen affinity, Tucaresol was administered to patients in high doses (grams).
Subsequently, human toxicity and pharmacokinetic data, such as maximum tolerated dose, were available well above that required for clinical work with Tucaresol as an antiviral agent administered at daily doses of up to 100 mg per patient. However, it was during this work with high dose Tucaresol that indications of immune activity were observed, thereby resulting in cessation of the sickle cell anemia project.
Exploration of this newly discovered activity revealed that Tucaresol functions as a host-targeted antiviral agent by protection of antiviral immune cells via controlled co-stimulation of specific T cells (CD4+ T helper cells) in the presence of a pathogenic virus to maintain normal immune status or by reconstitution of the same antiviral immune cells significantly depleted by a pathogenic virus, as was demonstrated in HIV patients and SIV macaques (details below).
Important to note is that Tucaresol does not function as an immunostimulant in normal immune status mammals thereby prevent- ing a hyper-active and potentially fatal immune response such as cytokine storm (cytokine release syndrome). In fact, the ability to stimulate a controlled immune response in an immunodeficient mammal, such as may occur during a viral infection, is a key tenant of the intellectual property of Tucaresol as defined by the granted patents to Glaxo Wellcome Inc..These patents, issued in 1996, 1999 and 2000, respectively, state in their first claim a method for treating an immunodeficient mammal, which exemplifies the use of Tucaresol to treat and eliminate (in one macaque) an SIV infection (US patents 5,508,310; 5,872,151; and 6,096,786).
There are no documented reports, to the best of our knowledge, regarding the eval-uation of Tucaresol in patients with COVID-19 disease. However, as noted above, Tucaresol was evaluated for treatment of immunodeficiency virus in patients and macaques. In view of similarities between Human Immunodeficiency Virus (HIV) and SARS-CoV-2, especially with respect to host CD4+ T helper cell function and subsequent depletion of T cell numbers during infection, it is appropriate to review the published immunodeficiency virus Tucaresol treatment results with consideration to the merit of a similar clinical trial for treatment of patients with COVID-19 disease. HIV and SARS-CoV-2 are RNA viruses that have reached the human population from animals. SARS-CoV-2, along with other viruses such as measles, influenza, viral hepatitis, CMV, RSV, Ebola virus and Marburg virus, is associated with lymphopenia. Similarly, HIV infection can cause a well known lymphopenia associated disease, AIDS (Acquired Immune Deficiency Syndrome).
In both HIV and SARS-CoV-2, the viruses gain entry into the immune system by binding to the CD4+ T helper cells, which is achieved by directly binding to the CD4 cell surface receptor22. Neither virus binds to and enters CD8+ cytotoxic T lymphocytes (CTLs). Since T helper cells play a pivotal role in coordinating the immune response to a viral infection by activating multiple immune cell types, including B cells (antibody produc- tion) and CD8+ CTLs (cell mediated immunity), it is not a surprise that both viruses focus on direct entry, control and subsequent depletion of CD4+ T helper cells. As such, the resultant lymphopenia is an indicator of a poor clinical outcome.
TUCARESOL, CLINICAL STUDIES
In view of the important role played by T cells regarding patient survivability during an HIV infection, as reflected by the appearance of secondary infections that may accompany HIV, and with regard to the selective interaction (binding) of Tucaresol with CD4+ T helper cells, it is appropriate to consider a proof-of-concept (phase 2) clinical trial employing Tucaresol for treatment of patients with mild-to-moderate (non-hospitalized) COVID-19 disease, either in a monotherapy or preferably combination antiviral drug ther- apy setting. However, another option is treatment of patients with severe (hospitalized) COVID-19 disease accompanied by lymphopenia.
In the case of the earlier evaluation by GlaxoSmithKline of Tucaresol for treatment of HIV infected patients in a multicenter, pla- cebo controlled phase 2 trial, a two dose, 25 mg and 50 mg daily, 3 month combination therapy study with HAART (Highly Active Antiretroviral Therapy) was planned with 45 patients already receiving HAART and a study start date of 2004-11-01.
This trial ap- pears lengthy with a study completion verification date of 2008-10-13, an issuance of “His- tory of Changes for Study: NCT00343941” and notification that “Final trial results were not reported”23. However, demonstration of significant modulation of T cell activity by Tucaresol during a phase 1b/2a trial in HIV patients was published in 2004 as a Glax- oSmithKline and University of Milan collaboration24.
This clinical trial consisted of 4 groups of HIV positive patients, a total of 24 patients, in which half of the patients received HAART prior to the trial and the other half were HAART naive. This was a 16 week pulse dose escalation protocol in which Tucaresol was administered as one 25 mg dose during week 1, 25 mg/day for 4 days during week 4, 50 mg/day for 4 days during week 8 and 100 mg/day for 4 days during week 12 with time between doses to permit drug wash out. One of the 4 groups received only Tucaresol while the second group received Tucaresol and HAART at the same time.
Groups 3 and 4, already on HAART prior to the trial, received Tucaresol according to the dose schedule above. The difference between the two groups is that one group consists of patients that are immunologic non-respond- ers as evidenced by a below average CD4+ T helper cell count.
Following administration of Tucaresol, increases in percentages of memory T lymphocytes (CD4+/CD45RO+) were observed in all patients, including those patients treated only with Tucaresol. Following administration of Tucaresol, a significant increase in memory T cells was seen on week 12 in the group already on HAART and on week 13 in the group of immunologic non-re- sponders; P<0.05 in both cases. Any significant P value is of note since each group consists of only 6 patients.
More significant was a sustained increase in percentages of naive T lymphocytes (CD4+/CD45RA+) observed in all patients concomitantly with administration of Tucaresol. Also, an increase in naive CD4+ T cells was observed at approximately week 8 in all groups following the third Tucaresol administration.
Env-stimulated per- forin-expressing CD8+ T cells were increased in all groups. Perforin-expressing p24-stimulated CD8+ T cells were similarly amplified. As regards HIV plasma viremia, there were no changes in HIV RNA levels, including those patients treated only with Tucaresol,
except a decrease in HIV RNA in patients starting simultaneous treatment with Tucaresol and HAART. Therefore, while treatment with Tucaresol alone did not eliminate viremia, it prevented a significant increase in HIV viremia. Administration of Tucaresol was as- sociated with increased interleukin 12 and decreased interleukin 10. In particular, the reduction of interleukin 10 upon administration of Tucaresol was significant at weeks 8, 12 and 16 in the patient group already on HAART; P<0.001.
High interleukin 10 expres- sion, similar to interleukin 6 expression, can predict poor clinical outcomes in COVID-19 patients25. The above phase 2 (planned) clinical trial was preceded by a phase 1 dose escalating safety trial in 24 HIV infected patients already on HAART (ClinicalTrials.gov registry number; NCT00006209).
In conclusion, in patients on HAART with proper viral suppression, treatment with Tucaresol resulted in: (i) stimulation of cytotoxic T lymphocyte activity, (ii) generation of naive T cells and (iii) did not result in any adverse effects or increase in patient viral load.
TUCARESOL, PRECLINICAL STUDIES
The three patents granted to Glaxo Wellcome Inc. (US 5,508,310; 5,892,151; and 6,096,786) demonstrated the host targeted antiviral activity of Tucaresol by exemplifica- tion of the elimination of simian immunodeficiency virus (SIV) in a macaque, as well as reduction of SIV in the second animal.
Two of four macaques of average weight 2.5 kg with established SIV infection were injected ip; 30 mg/kg Tucaresol every other day for nine days (five injections). Viral load was measured on day 11. No virus was detected in the first animal while a ten-fold reduction in SIV was observed in the second animal.
Human pharmacokinetic and pharmacodynamic data, along with other human pre- clinical data, is available in three publications from Wellcome Research Laboratories, later Glaxo Wellcome R & D Ltd.26-28. Interestingly, these studies were not targeted towards clinical evaluation of Tucaresol at a dose of up to 100 mg/daily as noted above but instead were directed to the maximum tolerated dose of Tucaresol, since the preclinical studies were initially focused on Tucaresol as a potential treatment for sickle cell disease.
There- fore, in the first administration of Tucaresol into humans, the pharmacokinetics and phar- macodynamics were studied in healthy male volunteers following oral doses of 200 mg – 3,600 mg. Tucaresol is well tolerated with some gastrointestinal discomfort at oral doses of 1,200 mg or more.
There were no clear effects on routine hematology or biochemistry, platelet aggregation, resting or exercise heart rates or blood pressure. Lymphadenopa- thy was observed in a few individuals at high dose Tucaresol (>2 grams), 7-10 days after beginning administration, which suggests stimulation of the immune system.
At the time of peak whole blood concentration following administration of 3,600 mg of Tu- caresol, approximately 70% of the candidate drug is present in the blood, indicating good oral bioavailability. Food intake and gender apparently have no significant effect on orally administered Tucaresol.
Additional information regarding preclinical evaluation of Tucaresol, including studies undertaken in rodents and larger animals, is available in a Doctor of Medicine thesis based on work at Wellcome Research Labs29. Similar to the three publications noted above, this work was focused on significantly higher doses of Tucaresol as a poten- tial treatment for sickle cell anemia.
Animal toxicology studies included acute oral and iv mouse studies, 14 days oral subacute rabbit studies and a one month oral dose ranging Wistar rat study. The latter study included a hematology, biochemistry and pathology profile. A one month oral toxicology study was undertaken with cynomolgus monkeys. Also, teratogenicity (rats, rabbits) and Ames mutagenicity tests were undertaken.
Pharmacokinetic studies were performed with a single oral dose of Tucaresol in rats, single oral carbon-14 labeled dose in rabbits, three iv carbon-14 labeled doses over three days in two beagles and three iv doses over three days in two cynomolgus monkeys. Another pharmacokinetic study was done with carbon-14 labeled Tucaresol administered orally to two monkeys and administered iv to another two monkeys.
As noted above, Tucaresol is a benzoic acid substituted benzaldehyde small molecule with a unique mechanism of action based on a selective transient covalent bond (Schiff base) formed between its aldehyde moiety and amino groups present on CD4+ T helper cells (lysine side chains).
This T helper cell Schiff base formation with Tucaresol induces sodium/potassium ion channel activation and subsequent tyrosine phosphorylation of cy- toplasmic signaling proteins. This costimulatory signal transduction pathway converges with the signaling events resulting from virus peptide fragment binding (within the con- text of MHC II) to the T cell receptor.
Tucaresol co-stimulation favors enhancement of a Th1 proinflammatory cytokine response by effectively increasing interleukin 2, interleu- kin 12 and interferon-gamma while diminishing a Th2 anti-inflammatory cytokine re- sponse with reduction of interleukin 430. As such, Tucaresol mimics the transient cova- lent bond (Schiff base) formation that occurs between the ‘specialized’ carbonyls (likely arising from glycated proteins) from MHC (major histocompatibility complex) class II bound virus peptide fragment present at the surface of APCs (Antigen Presenting Cells) and amino groups (lysine side chains) from CD4+ T helper cells31. This mimetic activity of Tucaresol enhances antigen-specific T cell activation and cell mediated immunity via the increased Th1 proinflammatory cytokine response.
Perhaps one reason Tucaresol has not received much attention as a clinical stage can- didate drug for treatment of SARS-CoV-2 is the perception that the aldehyde function will covalently bind nonspecifically off-target to lysine(s) present in multiple proteins thereby resulting in drug toxicity. However, this concern was addressed in 2019 with FDA ap- proval of a 2-hydroxybenzaldehyde analog of Tucaresol, Voxelotor (GBT440, Global Blood Therapeutics), for the treatment of sickle cell disease.
Therapy is achieved by high dose stoichiometric binding (and reversible Schiff base formation) with hemoglobin via a mechanism first observed with Tucaresol. Lack of significant toxicity of Voxelotor was supported by the recent $5.4 billion acquisition of Global Blood Therapeutics by Pfizer.
Interestingly, it was published this year that the time spent by Voxelotor correctly bound to hemoglobin (the residence time) was “dramatically enhanced” by a hydroxyl group ortho to the aldehyde moiety of Voxelotor, thereby facilitating a favorable equilibration of the drug correctly bound to hemoglobin32. The lack of toxicity reported for Tucaresol in animal and human studies cited above is in agreement with this lack of toxicity ob- served with Voxelotor.
The above overview of the mechanism of Tucaresol suggests that it offers a unique tool for treatment of viral infections. In summary, Tucaresol represents a hybrid or bridge between antiviral drugs and vaccines. Similar to low molecular weight antiviral drugs, Tucaresol is a small molecule, orally active, relatively fast acting chemical entity while similar to vaccines, Tucaresol will enhance both the antibody (humoral) and T cell (cell mediated) immune response, via interaction with important CD4+ T helper cells.
Antiviral drugs are best administered for therapeutic purposes or treatment of active infections ide- ally as soon as possible while vaccines are best administered for prophylactic purposes especially when the virus is rapidly pathogenic such as Ebola virus. However, Tucaresol may be useful in both a therapeutic setting, for example administered in combination with a virucidal drug, or in a prophylactic setting, for example administered at the same time as immunization with a vaccine and for a few days thereafter, to provide immune protec- tion during the period of immune priming, first dose of vaccine, and commencement of boosting of the immune response, second dose of vaccine.
reference link :https://www.preprints.org/manuscript/202211.0221/v3
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