Research into the new therapeutic properties of already known pharmaceutical drugs is conducted constantly and everywhere. Sometimes it leads to surprising discoveries that subsequently save countless lives.
Let’s recall that the well-known drug acetylsalicylic acid, marketed under the brand name “Aspirin,” was registered by Bayer in 1899 as an alternative to sodium salicylate with fewer side effects in the treatment of rheumatism. Today, 120 years after the widespread use of acetylsalicylic acid in medicine began, its range of applications has expanded incredibly. Acetylsalicylic acid is included in the WHO’s List of Essential Medicines. The organization recommends using acetylsalicylic acid to treat: mild to moderate pain, acute migraines, transient musculoskeletal pain, dysmenorrhea, to reduce fever, as well as in cases of acute myocardial infarction and ischemic stroke.
The history of pharmacology is replete with examples of existing drugs literally being given a “new lease on life” after the discovery of their previously unknown therapeutic properties. For example, zidovudine, a nucleoside reverse transcriptase inhibitor for the human immunodeficiency virus (HIV), was originally developed as an anticancer agent. It was only 20 years later that it was discovered to block HIV-1 replication. Sildenafil (“Viagra”) was originally synthesized as a means of improving myocardial blood flow in the treatment of angina and coronary artery disease. Metformin, described nearly 100 years ago as an antidiabetic agent, “resurfaced” only at the end of the last century, becoming one of the most popular drugs for treating type 2 diabetes. Its recently discovered hepatoprotective properties are currently being actively researched.
Sometimes, traditional medicine recipes can also be helpful. A prime example is the work of Chinese researcher Yu Yu Tu, a specialist in traditional Chinese medicine, whose team screened more than 100 single-component and combined herbal extracts in less than a year in 1971. They discovered that the extract of sweet wormwood (Artemisia annua) was 95–100% effective against malaria in mouse and monkey models. A year later, the active compound artemisinin was isolated, which subsequently helped save millions of lives. In 2015, Youyou Tu was awarded the Nobel Prize in Physiology or Medicine for her discoveries regarding a new therapy against malaria.
Notably, artemisinin and its derivatives have also been found to possess anticancer activity; clinical trials on this topic are currently underway. Anticipating the reader’s next question, I will answer: yes, its derivatives have already been tested against SARS-CoV-2, and the results of in vitro studies suggest that “the outlook is promising.” Very recently, a study was even published on the effect of a two-component drug containing artemisinin on the course of COVID-19 in humans. The results are encouraging, although the study itself was conducted at a low level of evidence from an evidence-based medicine perspective: the sample size was small (23 patients versus 18 in the control group, all with a confirmed diagnosis of COVID-19); patients were not randomized; and the study was open-label (participants knew what they were taking).
It is important to note that all such preliminary positive results should be viewed with a great deal of caution. One might recall the story of another antimalarial drug, chloroquine, and its derivative, hydroxychloroquine, which were initially considered effective treatments for COVID-19. Thus, in mid-June, the U.S. Food and Drug Administration (FDA) revoked authorization for their use in treating COVID-19. The official statement cited clinical trial data indicating that chloroquine and its derivative hydroxychloroquine have no effect on mortality or on accelerating recovery from COVID-19.
This and other examples clearly demonstrate that research into drugs does not stop once they appear on pharmacy shelves. Today, in response to the pandemic, scientists in laboratories around the world are tirelessly testing hundreds of long-standing pharmaceuticals for activity against SARS-CoV-2. Without exaggerating too much, one could say that just about everything that could theoretically work is currently being tested against the coronavirus. Therefore, when evaluating yet another claim of efficacy, it is worth paying attention first and foremost to the quality of the studies conducted: adherence to the standards of evidence-based medicine in the experimental design. One of the long-standing drugs in the pharmaceutical industry that is currently being actively researched in this regard is vitamin D. We will discuss it further below.
Basic Information About Vitamin D
Vitamin D refers to a group of biologically active fat-soluble substances, the most common of which are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D2 can only enter the human body through food. For example, mushrooms are rich in it, especially those that have been exposed to sunlight. This is because ergosterol, found in the cell membranes of mushrooms, is converted into ergocalciferol when exposed to ultraviolet light. Sources of vitamin D3 include fatty fish such as mackerel, sardines, and, in particular, salmon. Unlike vitamin D2, which can only be obtained from food, vitamin D3 is synthesized in the skin under the influence of ultraviolet B radiation (290–315 nm). The light is absorbed by 7-dehydrocholesterol to form previtamin D3, which is then spontaneously converted into vitamin D3.
A small study conducted on women aged 55–70 found that sunlight was their primary source of vitamin D. Spring and summer accounted for >80% of their total annual intake (vitamin D, like all fat-soluble vitamins, can be stored in the body in adipose tissue and the liver). The authors, however, note that this ratio may vary significantly depending on the region and the diet of a specific group of people.
Chemically, ergocalciferol and cholecalciferol differ in the presence of a double bond in the molecule between 22 and 23 carbon atoms and a methyl group at 24 carbon atoms. In the human body, both of these substances have similar biological activity. Regardless of the source of origin, once in the bloodstream, vitamin D binds to the vitamin D-binding protein, and once in the liver, it undergoes hydroxylation (addition of an OH group) by 25 carbon atoms. This reaction is catalyzed by one of the cytochrome P450 CYP2R1 enzymes, and the output is 25(OH)D (calcifediol). Exactly 25(OH)D is the main circulating form of vitamin D in the bloodstream, the concentration of which is measured to determine a person's vitamin D status. Calcifediol needs another transformation to become the active form. It occurs mainly in kidney cells, where another cytochrome P450 enzyme, CYP27B1, attaches a hydroxyl group to 1 carbon atom in the mitochondria. The biologically active form of vitamin D 1,25(OH)2D (calcitriol) mediates its main function: maintaining calcium homeostasis in the body. In the cytoplasm of small intestine cells, calcitriol interacts with its receptor (VDR, vitamin D receptor). Complex 1.25(OH)2D–VDR, in turn, already forms a complex in the nucleus with the retinoic acid receptor X (RXR, Retinoid X receptor) and the protein nucleoporin p62. Complex 1.25(OH)2D–VDR–RXR binds to its sensitive DNA sequences (VDRE) of a number of genes, regulating their expression. One of these genes is the epithelial calcium channel gene. Its increased expression allows more calcium to enter the cell, where the vitamin D-dependent calcium-binding protein calbindin mediates the transfer of calcium into the bloodstream. Acute vitamin D deficiency in the body primarily affects the condition of bones, leading to the development of rickets in children and osteomalacia in adults. Childhood rickets is characterized by stunted growth, muscle weakness, skeletal deformity, hypocalcemia, and periodic seizures. In osteomalacia, demineralization of bone tissue is observed, leading to deformation of the bones of the skeleton and, as a result, frequent fractures.
Further studies have shown that the expression of vitamin D receptors is far from limited to cells of the small intestine: they have been found in almost all body tissues. There are now over 200 known genes whose expression can be influenced by vitamin D. These facts could not but lead researchers to the idea of other, not limited to maintaining calcium homeostasis and unknown effects of vitamin D on human health.
Is it easier to get sick with vitamin D?
Today, most authors agree that the concentration of calcifediol in the blood below 75 nmol/l (30 ng/ml) indicates its deficiency in the human body, and a drop in concentration below 25-30 nmol/l (10-12 ng/ml) is fraught with a serious risk of osteomalacia. Unfortunately, in the second decade of the 21st century, acute vitamin D deficiency is far from uncommon even in developed countries. For example, one in ten Europeans suffers from it, and the proportion of people with moderate vitamin D deficiency in European countries reaches 40%. At the same time, a daily intake of 1000 IU of vitamin D can completely eliminate its deficiency in the body.
1 IU (International unit) for vitamin D (cholesterol or ergocalciferol) is equal to 25 ng.
Since the 1920s, when the key role of vitamin D in preventing rickets was established, researchers' interest in the vitamin's effect on human health has not waned. To date, a large number of studies have accumulated indicating its connection with a wide range of diseases, including heart disease and certain types of cancer. In a systematic review published in The Lancet Diabetes & Endocrinology in early 2014, the authors analyzed 290 prospective cohort and 172 randomized intervention trials that evaluated the effects of vitamin D on health outcomes, excluding skeletal diseases, in individuals aged 18 years and older. Many prospective cohort studies have shown a link between low serum vitamin D concentrations and a wide range of acute and chronic diseases (cardiovascular, metabolic, infectious, and even psychiatric). However, randomized intervention studies have not confirmed that an increase in the concentration of vitamin D in the blood can change the likelihood or clinical course of these diseases. The authors concluded that the association between vitamin D deficiency and health disorders reported by prospective cohort studies is not causal, and its low serum levels are more likely a marker of poor health. However, it is noted that the daily intake of 20 micrograms of vitamin D by the elderly seems to slightly reduce mortality from all causes. The researchers explain this by the fact that due to aging and lifestyle changes in the elderly, the recovery of its deficiency caused by poor health is disrupted. In this regard, the addition of low doses of vitamin D to the diet leads to a slight increase in the survival rate of this group of people.
Experts from the International Non-profit Organization for the Study of the Effectiveness of Evidence-based Medicine (Cochrane) are a little more optimistic in their conclusions. In the same year, 2014, their meta-analysis was published on the effect of vitamin D on mortality among adults. The authors concluded that taking vitamin D3 reduces mortality from all causes, and this effect is most pronounced in the elderly. At the same time, the combined intake of other forms of vitamin D, calcifediol and calcitriol, does not affect mortality and is even fraught with the risk of hypercalcemia. It is emphasized that there is no significant difference in mortality in people taking vitamin D3 in doses greater than 800 IU per day. It was also not possible to identify any effect of vitamin D intake on mortality among people who did not initially suffer from vitamin deficiency.
In 2017, another large meta-analysis reanalyzed individual patient data (IPD) from eight independent studies from Norway, Germany, Iceland, Denmark, and the Netherlands. A special feature of IPD meta-analysis is the re-processing of "raw" data obtained directly from researchers who have published original papers. Collectively, data from ~27,000 patients collected over 10.5 years of follow-up, during which 6,802 patients died, were reanalyzed. It turned out that the ratio of the risks of death from all causes in relation to the group with minimal mortality (vitamin D concentration 75-99.99 nmol/l) in groups with its concentrations of 40-49.99, 30-39.99 and <30 nmol/l, they average 1.15, 1.33 and 1.67, respectively. This means that people with acute vitamin D deficiency (<30 nmol/L) died on average 1.67 times more often during the follow-up period than people from the control group with the lowest mortality rate.
The authors also note that, despite the limited statistical ability to assess the relationship between vitamin D and mortality in people with its concentration above 125 nmol / l, there were no clear indications of the toxicity of high concentrations of vitamin D in the blood.
Two years later, another team of authors came up with similar results after investigating mortality over a period of 10.4 years and the concentration of vitamin D in the blood serum of ~10,000 people from Iceland, Germany and Norway. The observed risk ratio of death from all causes was highest (1.76) in people with its deficiency (<30 nmol/l) compared with the control group.
A serious limitation of these studies is that they carried out only one measurement of vitamin D concentration at one time. As we know, this indicator can vary significantly depending on the diet or the level of solar insolation. Also, despite the high quality of data analysis (large samples and taking into account many different factors, including age, gender, body mass indexes, blood collection season, etc.), these studies are correlational. We cannot unequivocally state that low vitamin D levels are the cause of increased mortality. Perhaps there is some kind of X factor that leads to a parallel decrease in the concentration of vitamin D in the blood and significantly increases the chance of dying. To clarify this, another kind of research is needed, namely interventional ("intervention studies"). In such studies, a registered medicinal product is used in a way other than that described in the registered instructions, for example, to treat another disease.
One of them (a randomized, double-blind, placebo-controlled trial) evaluated the effect of taking vitamin D3 at a dose of 2,000 IU and 1 g of unsaturated omega-3 fatty acids daily on the incidence of cardiovascular disease and cancer in men and women over 50 years of age in the United States. In total, about 26,000 people participated in the experiment. During follow-up, cancer was diagnosed in 1,617 experimental participants (793 cases in the vitamin D group and 824 in the placebo group), and the main cardiovascular event (most often myocardial infarction or stroke) occurred in 805 participants (396 in the vitamin D group and 409 in the placebo group). After statistical processing of the results, the authors concluded that taking vitamin D and omega-3 did not reduce the incidence of cancer or cardiovascular diseases compared with placebo. There were no statistically significant differences in mortality between the groups from breast cancer, prostate cancer and colorectal cancer, as well as from myocardial infarction and stroke. There is one detail worth paying attention to in this study. Among the ~16,000 participants who agreed to blood sampling before the experiment, the average serum vitamin D concentration was 77 nmol/L, and only 12.7% of participants had levels below 50 nmol/L. Thus, the vast majority of the subjects were not deficient in vitamin D. And as we remember from other work, the probability of death from all causes increases significantly only with severe vitamin D deficiency (<30 nmol/l). Starting from 40 nmol/l and above, this probability practically does not change.
Today, in the COVID-19 pandemic, while waiting for vaccination, people are in urgent need of a drug that effectively reduces the severity of the transmitted disease. The researchers' attention was once again attracted by the "sunny" vitamin, and interest in it flared up with renewed vigor.
Vitamin D and COVID-19
Along with the alleged association of vitamin D levels with cancer and cardiovascular diseases, its possible connection with the development of respiratory tract infection has been discovered. IPD meta-analysis of 25 randomized controlled trials (approximately 11,000 participants aged 0 to 95 years) He found that taking vitamin D significantly reduced the risk of acute respiratory tract infection in all participants. At the same time, the effects of vitamin D were significantly more pronounced in people with its initial deficiency.
COVID-19 is also an acute infectious respiratory disease. A logical step would be to look at how the course of this disease is related to a person's vitamin D status. Observations also played a role here — the groups with the highest risk of developing severe COVID-19, as a rule, correspond to risk groups for acute vitamin D deficiency in the body. Here we are talking about elderly people suffering from obesity and/or hypertension, cardiovascular diseases, and diabetes. Also, both of these groups include ethnic subpopulations of people whose skin is naturally rich in melanin, living outside the tropical zone.
Most of the available data on vitamin D and COVID-19 are preliminary. To date, many papers have been published without peer review in the medRxiv open access preprint repositories. And the few that have been published in peer-reviewed scientific journals are retrospective and correlative. Here are a few of them.
A correlation analysis between the average concentrations of vitamin D in the blood and the number of COVID-19 cases (including deaths) in European countries revealed a negative correlation between them. The same pattern was observed for the number of deaths caused by COVID-19.
The authors honestly point out the limitations of the work: for example, the number of detected cases in a country depends on the number of tests performed, as well as on the measures taken by each country to prevent the spread of infection. In addition, the assessment of mortality from COVID-19 can be strongly influenced by differences in medical standards in different European countries.
In another small paper published in early September, the authors assessed whether the vitamin D status before testing for COVID-19 was related to the test results. We used data from 489 patients who had the results of measuring vitamin D levels no earlier than a year before testing for COVID-19. It was revealed that 25% of the participants had a vitamin deficiency before the test. Among the 172 participants whose last vitamin D level measurement showed vitamin D deficiency (<20 ng/ml), 19% tested positive for COVID-19. Whereas in the other three groups with vitamin D deficiency (>20 ng/ml), the proportion of people with a positive test was 12%. A positive test result was also associated with age over 50 and dark skin color.
Another study, based on an analysis of data from the British Biobank for over half a million people, found no potential link between vitamin D concentrations and the risk of contracting COVID-19. It has also not been revealed that vitamin concentration can explain racial differences in morbidity. But this study has one major limitation: data on vitamin D concentrations in serum were collected between 2006 and 2010. How informative can the data from fourteen years ago be? During this time, many of the participants could have radically changed their lifestyle, which would have changed not only the concentration of vitamin D in the blood, but also many other parameters that potentially affect the incidence of COVID-19.
Another recent study also evaluated the relationship between vitamin D concentrations in the blood and the incidence of COVID-19 in the period March-June 2020 in ~190,000 patients in the United States. The authors found that a positive COVID-19 test was more common in patients with vitamin D deficiency (12.5%) than in patients with adequate vitamin values and those with elevated vitamin D levels (8.1% and 5.9%, respectively). On this basis, it is concluded that the incidence of COVID-19 strongly and inversely correlates with vitamin D status, and this relationship persists regardless of latitude, race, gender, and age. Again, the blood vitamin D concentration data included in the study was collected in the 12 months prior to testing for COVID-19.
After reviewing these new results, many people probably have the same logical question: is vitamin D deficiency one of the factors in the development of COVID-19, or is there a decrease in vitamin D levels in seriously ill people due to general poor health? Unfortunately, there is no definite answer to this question. However, there are now known ways to activate both the innate and adaptive immune systems, in which vitamin D is a key player. The mechanisms by which the vitamin can reduce the incidence of infectious diseases in humans will be discussed below.
How it might work: suggested mechanisms
In 2006, it was shown that activation of Toll-like receptors in human macrophages increases the expression of the vitamin D receptor and vitamin D-1-hydroxylase (we discussed it above — this enzyme is responsible for the formation of the active form of vitamin D, calcitriol). Calcitriol, in turn, triggers the expression of the antimicrobial peptide cathelicidin, which damages the membrane of the microbe and thereby destroys it (Mycobacterium tuberculosis pathogens were used in this study). Toll-like receptors are receptors of the plasma membrane of immunocompetent cells that recognize conserved molecular fragments of bacteria, viruses, fungi, and parasitic protozoa and activate the innate immune system. Previously, it was noted that the black population of the United States has an increased susceptibility to tuberculosis along with a reduced concentration of vitamin D in the blood serum. The authors showed that the addition of serum from African Americans to the culture of monocytes (one of the types of immune cells), unlike serum obtained from Europeans, does not lead to effective expression of cathelicidin. Whereas the introduction of vitamin D into this serum from the outside significantly increased the expression of this antimicrobial peptide by monocytes.
These were the first results that directly demonstrated a causal relationship between innate immunity and vitamin D levels in the blood. Apparently, this path of immune activation is our very recent evolutionary acquisition. A comparison of the sequences of the cathelicidin gene in several mammals revealed that the promoter necessary for the induction of its expression by calcitriol is present only in humans and chimpanzees and is absent in the genomes of mice, rats and dogs. It is also worth noting that vitamin D sensitivity is an extremely flexible trait in mammalian evolution. For example, lethal doses in case of overdose of this vitamin for animals of different species may differ several times.
It has also been shown that calcitriol in the culture of monocytes and epithelial cells stimulates the expression of the Nod-like receptor type 2 gene. The NOD2 receptor binds bacterial peptidoglycans, in particular muramyl dipeptide, which is an integral part of bacterial cell walls. Its activation by muramyl dipeptide through the transcription factor NF-kB induces the expression of a gene encoding another antimicrobial peptide, β-defensin 2. This peptide, like cathelicidin, disrupts the integrity of the pathogen's membrane, leading to its death.
In 2018, Korean researchers demonstrated the ability of human β-defensin 2 to stimulate antiviral immunity upon presentation of the S-protein of the coronavirus that causes Middle East respiratory syndrome (MERS). The authors observed an increase in the expression of interferon-β and interferon-γ, ribonuclease L, antiviral proteins and protein kinase-R in the culture of human monocytes, which suppresses mRNA translation in the cell, preventing the synthesis of viral proteins. According to them, β-defensin 2 can activate not only the innate antiviral immune response, but also mediate the induction of an effective adaptive immune response.
More recently, a scheme has been proposed describing the effect of vitamin D on the activation of antiviral protection of macrophages by stimulating autophagy. Autophagy, literally self―eating, is an active process of destroying large molecular complexes and even entire organelles that have failed. This garbage collection mechanism is also a powerful means of fighting the cell against viruses that have entered it. Viruses, interacting with membrane toll-like receptors, enhance the expression of the already known CYP27B1 hydroxylase and the vitamin D receptor. By binding to its receptor, calcitriol mediates the suppression of the m-Tor pathway, a powerful inhibitor of autophagy, and also stimulates key participants in its activation. Along with these processes, concentrations of Ca2+ and NO ions increase in the cell, which, in turn, additionally stimulates autophagy. In addition to directly regulating the pathways associated with the induction of autophagy, vitamin D also stimulates the expression of the antimicrobial peptides β-defensin 2 and cathelicidin, which were mentioned above.
The role of vitamin D in adaptive immunity has also been demonstrated. It is known that vitamin D receptors are not expressed in naive (not in contact with the antigen) human T cells. However, they began to be expressed after stimulation of the T-cell receptor recognizing the antigen. Vitamin D, having bound to its receptor, triggered a cascade of reactions that eventually led to the activation of T cells. Researchers have found that chronic vitamin D deficiency affects their proliferative ability. T cells isolated from people with low serum concentrations of the antigen had a reduced ability to proliferate after exposure to the antigen, compared with T cells from people with normal concentrations. The observed violation of the T-cell response disappeared when vitamin D was added to the culture medium from the outside.
The authors suggested an evolutionary advantage of such an alternative vitamin D-dependent T cell activation pathway for humans. It takes about two days after the presentation of the antigen. This delay may give the innate immune system the ability to quickly deal with the infection. If this is successful, the start of T cell division occurs in a relatively non-inflammatory microenvironment with a small amount of antigen and continues to a limited extent. If the innate immune system cannot cope with the infection, then T-cell division occurs in a pro-inflammatory microenvironment with a large amount of antigen. The proliferative response of T cells in this case will be much stronger. It is known that T cells are capable of explosive division, and this delay in their maturation, associated with the activation of the vitamin D receptor, can reduce the risk of undesirable immunopathology, "holding the horses of immunity."
As we have seen, vitamin D directly affects both innate and adaptive immune responses. In all likelihood, these mechanisms mediate the possible effect of its acute deficiency on mortality from infectious diseases, in particular, in the elderly. Following this logic, an adequate concentration of vitamin in the blood is one of the factors determining the balance between the two systems, when, on the one hand, effective pathogen destruction occurs, and on the other hand, fatal hyperactivation of the immune system, the infamous cytokine storm, does not develop.
In conclusion, I would like to emphasize once again that vitamin D, of course, is not a panacea. Apparently, taking it has a positive effect only in the case of acute deficiency, mainly in the elderly. Further large-scale studies are needed to establish the cause-effect relationship between vitamin deficiency and the development/course of various diseases (excluding childhood rickets and osteomalacia in adults, where its direct role has been proven). In the near future, I hope we will get an unambiguous answer to the question: did vitamin D deficiency affect the incidence of COVID-19 in particular? If it turns out to be positive, then, among other factors (age over 65 years, overweight, diabetes, etc.), vitamin deficiency will be the factor that can be easily corrected quickly. In addition, in 2026 and beyond, COVID or other viral respiratory diseases will not leave us, and the feasibility of clinical use of vitamin D remains unresolved and not fully clear. So far, we don't have a definite answer. But even today, if you are at risk of developing acute vitamin D deficiency, this is a significant reason to correct your eating habits, including adding vitamin D supplements to your diet, as well as spending time outdoors. Evolutionarily, our metabolic pathways evolved in completely different conditions than the current ones. Ancient people had a different diet, skin insolation levels, and other physical activity. Perhaps, in order to preserve our own health in the modern world, our evolutionary past must be taken into account.