Immunopterin: A prospective therapy and preventative to fight COVID-19?

2.1 Cold family incidences

Literature reviews were conducted on “common cold” families incidence studies to determine the likelihood of subjects in the Immunopterin observational prophylactic and therapeutic studies of having been exposed to coronavirus cold viruses.

To accomplish this, we reviewed scholarly articles containing cold virus family incidence data using Google Scholar to measure the likelihood that subjects in the therapeutic and prophylactic efficacy studies were exposed to coronavirus colds. To get a broader understanding of cold epidemiology, we compared incidence data of the most common cold viruses [2,3,4].

Importantly, these statistics were used to determine the extent to which Immunopterin acted prophylactically and therapeutically against coronavirus colds. Further, these statistics were applied to estimate the number of subjects in the cold/flu observational studies who were likely to have been exposed to coronavirus cold viruses and other common cold virus families.

2.2 Immunopterin therapeutic efficacy

Thirty-four (34) subjects voluntarily participated in a non-randomized 5-year observational study to assess the therapeutic outcomes of Immunopterin. Ages ranged from 25 to 73 years of age with varying degrees of health and wellness. While detailed demographic data were not collected, both genders were amply represented as was a broad BMI range. All subjects in this study took Immunopterin therapeutically when they felt cold or flu symptoms, which were reflective of those seen in the general population (fever, runny nose, congestion, headache). Recovery time is defined to be the hourly period from dosing of Immunopterin until subjects became asymptomatic. Self-reported accounts of recovery times were recorded after each cold incidence. Sample therapeutic data containing recovery times were compared to US population mean recovery times for colds. The mean and standard deviations of the recovery times were calculated. A t-test determined the t-score, and the 95% CI. A z-test was used to compare the observational sample mean to the US population mean of reported cold incidence.

2.3 Immunopterin prophylactic efficacy

The authors reviewed the Moheno [1] observational study to understand the efficacy of Immunopterin as a prophylactic against flus and colds. In this study subjects (N = 31) were asked to take one to three capsules of Immunopterin daily. Open-ended questioning was used in informal interviews. Self-reported results were quoted and tabulated contemporaneously. Content analysis was conducted to determine prominent themes. The mean of sample cold incidence data was compared to the US population mean.

2.4 Cytokine study review

The data from the Moheno et al. studies [8,9] was repurposed to understand the relationships between calcium pterin dosing and certain plasma cytokine levels in mice to determine the primary modes of actions of Immunopterin. Originally, the three-fold aims of these studies were to evaluate the antineoplastic actions of various molar concentrations of calcium pterin (CaPterin) and dipterinyl calcium pentahydrate (DCP), the two calcium pterins the researchers investigated, and to compare the antineoplastic efficacies of these calcium pterins to calcium chloride. Further details of the experimental design are described in the Materials and Methods section of Moheno et al. [8,9] including detailed instructions for the synthesis of the studied calcium pterins. A representative example of the methodology is as follows:

Twenty-nine (29) athymic nude mice were inoculated in the right flank with MDA-MB-231 breast cancer cells. Mice were divided in to 5 groups of 5 mice and 1 control group of 4 mice. Tumor sizes (measured with caliper) and animal weights were measured twice weekly. Daily oral doses of CaPterin (calcium pterin for two dosage groups of 5 mice each, N = 10, are given in Figure 3) and DCP were given by gavage until termination. Experimental groups were dosed once daily as indicated in the experimental design. A second control group was untreated to evaluate the effects of animal handling and gavaging on tumor growth. No difference was found.

The mice were not anesthetized but were restrained during dosing. Blood samples were collected via cardiac venipuncture at termination (70–98 days). Samples then were processed to EDTA plasma for analysis.

Murine cytokine levels in plasma were determined by ELISA at Alta Analytical Laboratories (San Diego) using a LINCOplex Kit (Linco Research) [8,9]. Cytokine and chemokines were tabulated as a dosing response curve of the calcium pterins. Anti-tumor measures were charted as a function of calcium pterin dosing. Analysis of variance (ANOVA) models were generated.

2.5 Statistical methods

One sample t-tests and one mean z-tests were conducted to compare the sample mean cold recovery time to the US population’s mean with SPSS PASW Statistics GradPack 18. Analysis of variance (ANOVA) models were generated by StatView SE + Graphics (v 1.03) to study the relationships among calcium pterin dosages and a large set of cytokines [8,9]. Standardized partial regression plots were generated showing interleukin-10 and interleukin-6 levels as functions of CaPterin dosing (Graduate Pack 15.0 for Windows, 2006).

2.6 Drug information

Immunopterin (calcium pterin-6-carboxylate chelate) is the active ingredient produced from 25 mg of calcium chloride dihydrate and 300–375 µg folic acid in 40 mL of water. The U.S. recommended dietary allowances (RDAs) vary with age, pregnancy, and lactation for folic acid and ranges from 400 to 600 µg for ages 14 and older. The U.S. RDA of folic acid for infants to 8-year-olds ranges from 65 to 200 µg. The recommend dietary allowances for calcium vary with age, pregnancy, and lactation for calcium and ranges from 800 to 1,200 mg. The calcium RDA for children ages 9–18 is 1,300 mg. The calcium RDA for infants to 8-year-olds ranges from 210 to 800 mg. The chloride RDA for ages 9–70-year-olds is 2300 mg. The chloride RDA for infants to 8-year-olds ranges from 180 to 1,900 mg.

The pharmacokinetics, pharmacodynamics, and bioavailability of Immunopterin are covered in the Appendix.

2.7 Immunopterin synthesis

In the case of the therapeutic and prophylactic studies of Immunopterin, Arizona Nutritional Supplements, Inc. was contracted to manufacture Immunopterin in a 25 mg capsular form.

Immunopterin can be readily compounded in compounding pharmacies and in a variety of healthcare and laboratory settings worldwide. Its constituent parts can be sourced readily.

Immunopterin can be synthesized in a variety of laboratory settings by dry mixing a ratio of 25 mg of CaCl₂ dihydrate to every 300–375 µg of folic acid, using a mortar and pestle, and adding to water. The calcium chloride serves as a catalyst to acid hydrolyze the folic acid and form the active ingredient, calcium pterin 6-carboxylate chelate, upon addition to water (40 mL) [1,10]. Proper excipients such as tricalcium phosphate, microcrystalline cellulose, gelatin, silicon dioxide, and magnesium stearate are added to accommodate a gelatin capsule. Complete instructions for synthesis of CaPterin and DCP (Figure 2) are referenced in the Moheno et al. [8,9] cytokine studies.

Figure 2

X-Ray crystallographic structure: (a) CaPterin and (b) dipterinyl calcium pentahydrate (DCP) [9].

1. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee. The research was done within compliance of local and federal laws as it pertains to food-marketing observational studies that do not involve randomization of experimental and control groups. The intervention involved a U.S. Food and Drug Administration (FDA) generally recognized as safe (GRAS) designated nutraceutical compound whose active ingredients are folic acid and calcium chloride dihydrate. The folic acid and calcium levels are within FDA recommended dietary allowances per dose. The nutrients are designated food grade.

2. Informed consent: Informed consent has been obtained from all individuals included in this study.

3. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. The treatment of lab animals in murine cytokine studies (Moheno et al.) [8,9] was within accordance with Perry Scientific (San Diego) standard operating procedures, which adheres to the USDA Animal Welfare Act and with the conditions specified in “The Guide for Care and Use of Laboratory Animals” from the Institute for Laboratory Animal Research (ILAR). Perry Scientific was in general compliance with the Food and Drug Administration Good Laboratory Practice Regulations.

3.1 Incidences of viral cold families

From 10 to 34% of cold viruses in hospital-based studies are coronaviruses [2,3,4]. The Coronavirus cold represents the second largest family of colds after rhinoviruses. Rhinoviruses constitute the largest cold family with up to 50% of all hospital-based common cold incidences. Adenoviruses constitute 1% of the cold viruses. Other less common colds include respiratory syncytial viruses, and parainfluenzas.

3.2 Immunopterin therapeutic efficacy

The mean cold recovery time for 34 participants with 129 cold incidences taking Immunopterin was (30 ± 13) h: that is 1 1/4 days. The statistical mode of the Immunopterin sample cold recovery times was 24 h. United States population data show that the mean recovery time for the “common cold” is 7 days or 168 h [2,3,4]. This is an approximate 80% reduction in cold recovery times. Ninety-five percent of the US population cold recovery times range from 4 to 10 days. Assuming two standard deviations lie between 7 days (168 h, the mean) and 10 days (240 h), then 95% of the population recovered within 4–10 days. Thirty-six (36) hours was determined to be the U.S. population standard deviation. Using a one sample t-test (N = 129, number of cold incidences), the therapeutic sample was compared with the US population mean (168 ± 36) h for recovery times for colds. (t = 25.08, p < 0.001); 95.44% CI = [27.93–32.72] h. z-test algorithm yielded a z-score (z = −12.473, p < 0.00001). Based on cold incidence statistics, we estimate conservatively that from 10 to 34% (13–44 of the incidences) (N = 129) of those in the Immunopterin therapeutic efficacy sample should have contracted a coronavirus cold over the 5-year observation period [2,3,4].

3.3 Immunopterin prophylactic efficacy

Subjects taking Immunopterin prophylactically every day had (0%) incidence of colds or flus over a 2-year observational period (N = 31). It has been estimated that 64% of the US population will get a cold each year. We would have expected 20 subjects in the prophylactic sample to get colds each year [11]. Hence, Immunopterin appeared to act as a strong prophylactic against colds. Given that coronavirus cold incidence ranges from 10 to 34%, the authors estimate that (2–7 subjects) should have contracted a coronavirus cold annually over the duration of the 2-year study, yet none did [2,3,4].

3.4 Cytokine study review

Stepwise regression techniques revealed significant direct proportionality between CaPterin dosages and Interleukin-10 [9] (Figure 3). This key finding strongly suggests that calcium pterins act as interleukin-10 agonists. Conversely, another regression plot revealed significant inverse proportionality between CaPterin dosage and Interleukin-6; thus, strongly suggesting that calcium pterins generally act as IL-6 inhibitors within certain dose ranges. Indeed, several studies on CaPterin and DCP effects on plasma IL-10 and Il-6 have borne this out [8,9,10,12,13].

Figure 3

Standardized partial regression plots showing interleukin level changes as a function of CaPterin dosages [9]. Only two [2] groups of 5 mice each (N = 10) received CaPterin at 7 and 21 mg/kg/day and are represented in these graphs. “In statistics, standardized (regression) coefficients, also called beta coefficients or beta weights, are the estimates resulting from a regression analysis where the underlying data have been standardized so that the variances of dependent and independent variables are equal to 1.[1] Therefore, standardized coefficients are unitless and refer to how many standard deviations a dependent variable will change, per standard deviation increase in the predictor variable.” [https://en.wikipedia.org/wiki/Standardized_coefficient]. (a) Interleukin-10 murine plasma levels demonstrate a directly proportional relationship to CaPterin dosing. (b) Interleukin-6 levels demonstrate an inverse proportional relationship to CaPterin dosing.

3.5 SARS-CoV-2 observational case studies

4.1 Viral cold family incidence data

The zero percent incidences of colds and flus with Immunopterin prophylaxis is in marked contrast to US population and hospital-based incidence data. Because Immunopterin shows therapeutic and prophylactic efficacies against all variants of upper respiratory infections, it is reasonable to infer that Immunopterin shows efficacy in treating rhinoviruses, coronaviruses, and influenzas. There were highly significant differences between hospital-based coronavirus cold incidence data and the Immunopterin prophylactic and therapeutic observational samples.

Because of sample size limitations, it is not certain that Immunopterin acted as a prophylactic in populations who were exposed to less common cold viruses like adenoviruses, respiratory syncytial viruses, and parainfluenzas.

4.2 Immunopterin therapeutic efficacy

Immunopterin was found to reduce corona cold recovery times by a factor of approximately 5 over the 5-year observational period. The average statistical measures showed highly significant differences in coronavirus cold recovery times. It is quite remarkable that the mean recovery time for colds for subjects taking Immunopterin was 1 1/4 days.

The coronavirus incidence data are in a range of 10–34% of common colds [2,3,4]. Based on these statistics, we estimate conservatively that from 13 to 44 of the incidences of those in the therapeutic efficacy study (N = 129) were coronavirus colds, over the 5-year period. Because the subjects who were taking Immunopterin therapeutically had highly significant reduced coronavirus cold recovery times, we can surmise that Immunopterin acted therapeutically against coronavirus colds.

4.3 Prophylactic efficacy of immunopterin

The 2-year prophylactic efficacy study on Immunopterin demonstrated considerable broad-spectral antiviral efficacies against cold and flu viruses given that the sample population had 0% incidences of colds and flus [1] and given an estimated 64% of the US population will get a cold each year [11]. We estimate that approximately two-thirds of a sample population of [20] should have caught an upper respiratory infection annually, and that 2–7 of these should be coronavirus colds. Yet, in the case of those using Immunopterin, there were zero incidences of colds or flus over the 2-year period [1].

4.4 Cytokine studies and modes of action

The multivariate analysis of variance data and univariate trends of murine plasma cytokines consistently show that the tested calcium pterins, CaPterin and DCP, increase IL-10 and inhibit IL-6 levels [8,9,10,12,13].

Coronavirus cold incidence data, reduced coronavirus recovery times, evidence of prophylaxis, and the modulation of cytokines levels show that Immunopterin very likely acts as an immunomodulator against the Coronaviridae family of viruses.

Calcium pterins generally inhibit interleukin-6 (IL-6) cytokine levels. IL-6 is a powerful inflammatory cytokine that is part of a larger set of cytokines that take part in inflammatory responses [14,15,16]. IL-6 is a rather well described cytokine which goes usually very much in parallel with CRP but not IFN-gamma or neopterin [17,18,19,20].

IL-10 is the master cytokine controller of the immune response and regulator of the inflammatory response [15,16]. The overarching role of IL-10 as a control on inflammatory cytokines is evidenced by its ability to enhance the activation and proliferation of mast cells, CD8+ T cells, Natural Killer (NK) cells, and B cells.

Cytokine literature and data [8,9,10,12,13] corroborate the likely mechanism of action of calcium pterins, i.e., that IL-10 plasma cytokines are increased, resulting in the downregulation of inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-12, IL-18 and the tumor necrosis factor-α (TNF-α).

Equally important is the fact that an agonized IL-10 activates humoral response which accelerates B lymphocyte (B cell) mitosis [21]. The vast and formidable enterprise of B cell mitosis entails B cell clone selection, differentiation, and the proliferation of B cells and plasma cells that excrete prodigious amounts of targeted antibodies [22]. The agonism of IL-10 and the resultant accelerated B cell mitosis are likely a key driver in the shorter coronavirus cold recovery times of Immunopterin.

4.5 SARS-CoV-2 and coronavirus cold cross-reactivity

Grifoni et al. [5] demonstrated pan-coronavirus cross-reactivity when specialized CD4+ and CD8+ helper T cells that target SARS-CoV-2 were found in 40–60% of subjects who had no exposure to COVID-19. These findings suggest that their subjects had previously contracted a coronavirus cold and that the immune memory of specialized coronavirus helper T cells was retrieved. A similar study showed T cell cross-reactivity across the severe acute respiratory syndrome SARS- CoV-1 and several coronavirus colds [6]. Importantly, T-cell cross-reactivity might help to accelerate COVID-19 recovery times for the 40–60% for those who have previously contracted a coronavirus cold [5].

4.6 SARS-CoV-2 observational case studies

The five observational COVID-19 case studies described in Results 3.5 strongly suggest Immunopterin’s therapeutic and prophylactic efficacy against confirmed COVID-19. A randomized, controlled clinical trial is being planned.