Abstract
Background. Since the beginning of the novel coronavirus (SARS-CoV-2) disease outbreak, there has been an increasing interest in discovering potential therapeutic agents for this disease. In this regard, we conducted a systematic review through an overview of drug development (in silico, in vitro, and in vivo) for treating COVID-19. Methods. A systematic search was carried out in major databases including PubMed, Web of Science, Scopus, EMBASE, and Google Scholar from December 2019 to March 2021. A combination of the following terms was used: coronavirus, COVID-19, SARS-CoV-2, drug design, drug development, In silico, In vitro, and In vivo. A narrative synthesis was performed as a qualitative method for the data synthesis of each outcome measure. Results. A total of 2168 articles were identified through searching databases. Finally, 315 studies (266 in silico, 34 in vitro, and 15 in vivo) were included. In studies with in silico approach, 98 article study repurposed drug and 91 studies evaluated herbal medicine on COVID-19. Among 260 drugs repurposed by the computational method, the best results were observed with saquinavir (n = 9), ritonavir (n = 8), and lopinavir (n = 6). Main protease (n = 154) following spike glycoprotein (n = 62) and other nonstructural protein of virus (n = 45) was among the most studied targets. Doxycycline, chlorpromazine, azithromycin, heparin, bepridil, and glycyrrhizic acid showed both in silico and in vitro inhibitory effects against SARS-CoV-2. Conclusion. The preclinical studies of novel drug design for COVID-19 focused on main protease and spike glycoprotein as targets for antiviral development. From evaluated structures, saquinavir, ritonavir, eucalyptus, Tinospora cordifolia, aloe, green tea, curcumin, pyrazole, and triazole derivatives in in silico studies and doxycycline, chlorpromazine, and heparin from in vitro and human monoclonal antibodies from in vivo studies showed promised results regarding efficacy. It seems that due to the nature of COVID-19 disease, finding some drugs with multitarget antiviral actions and anti-inflammatory potential is valuable and some herbal medicines have this potential.
1. Introduction
Coronavirus disease 2019 (COVID-19), which was first identified in December 2019, and shortly after, declared a pandemic by World Health Organization (WHO) [1]. As of January 18, 2022, there have been more than 326 million confirmed cases and 5.54 million deaths globally [2]. Coronaviruses belong to the family of Coronaviridae, RNA viruses with crown-like spikes on the surface of the coronavirus particles. According to a meta-analysis of Macedo et al. [3], the mortality rate of COVID-19 was 17.1% for patients admitted to hospitals, whereas WHO estimated a fatality rate of 6.73%, which was much lower than that calculated from published studies. Among the critical cases of COVID-19, the mortality rate reaches 40% [4].
Substantial efforts have been made in the treatment of patients with COVID-19. The WHO recommendations in the treatment of COVID-19 are as follows [5]: molnupiravir (conditional), baricitinib (strong), ruxolitinib and tofacitinib (conditional), sotrovimab (conditional), casirivimab and imdevimab (conditional), IL-6 receptor blockers (tocilizumab and sarilumab) (strong), remdesivir (conditional), and systemic corticosteroids (strong). The WHO recommends not to use ivermectin, lopinavir/ritonavir, hydroxychloroquine, and convalescent plasma.
The pathogenesis of COVID-19 was explained by cytokine storm, reduction in ACE2 expression, and activation of complement pathway-induced microvascular injury and thrombosis [6]. The mechanisms of the recommended agents are focused on the mentioned pathogenesis to improve the clinical outcome of COVID-19, and antiviral therapies are missing. The anticoronaviral strategies include preventing the synthesis of viral RNA, inhibiting virus replication, blocking the virus binding to human cell receptors, or inhibiting the viruses’ self-assembly process [7]. The SARS-CoV-2 contains at least four structural proteins: spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein, and 16 nonstructural proteins (NSPs). Among the translated NSPs, the main protease, also called chymotrypsin-like protease (3C-like protease), and the papain-like protease are two essential proteases for proteolytic processing of the coronavirus replicase polyprotein, therefore generating functional replication complex of the virus, whereas RNA-dependent RNA polymerase is the central enzyme for RNA synthesis. These three NSPs play crucial roles in coronavirus replication, making them attractive targets for anticoronaviral drug design [8].
The S protein, a surface-located trimeric glycoprotein of coronaviruses, promotes the attachment of viruses to host cells through binding to angiotensin-converting enzyme 2 (ACE2) and virus-cell membrane fusion during viral infection. Thus, the S protein has been considered as a major target for the development of vaccines and drug [9].
The development of a new therapeutic agent is a complex, lengthy, and expensive process, which can take 2–4 years of preclinical development and 3–6 years of clinical development and over 500 million dollar cost. There are three critical steps to develop a new drug including discovery and development, preclinical research, and clinical development [10].
Drug discovery involves screening hits, medicinal chemistry, and optimization of hits to reduce potential drug side effects. For drug discovery, two different complementary approaches can be applied: classical pharmacology, also known as phenotypic drug discovery, which is the historical basis of drug discovery, and reverse pharmacology, also known as designated target-based drug discovery. Screening methods based on phenotypic drug discovery have been used to discover new natural products mainly from the terrestrial origin [11]. These two strategies have advantages and disadvantages and promote very different screening assays. The frequent re-discovery of the same compounds, the technical difficulties associated with the isolation of compounds from extracts, and the incompatibility of natural product extracts with high-throughput screening (HTS) campaigns were the disadvantages of phenotypic drug discovery. On the other hand, natural product structures have the characteristics of high chemical diversity, biochemical specificity, and other molecular properties that make them favorable as lead structures for drug discovery, which serve to differentiate them from libraries of synthetic and combinatorial compounds [12]. Overly simplified assays, acting of drug on more than one target, the multifactorial nature of diseases, and challenges to identify a single molecular target are some limitations of target-based drug discovery. Therefore, a comprehensive screening strategy will incorporate both targeted and phenotypic assays, with one format designated as the primary screen and the other as a secondary or follow-up assay. During the spread of COVID-19 outbreak, great efforts have been made in therapeutic drug discovery against the virus. Because COVID-19 is a new, acute, severe infectious disease, the anti-SARS-CoV-2 drug development strategies are to screen existing drugs to identify potentially effective drugs, to expand indications, and to develop a vaccine [12]. The safety of conventional drugs has been mostly verified; if effective, they can be quickly applied in clinical practice (repurposing of existing drugs). The recent rises of several high transmissible strains sounded alarms for currently used vaccines and drugs. Therefore, developing broad-spectrum antiviral drugs not only to combat COVID-19 but also to provide protective arsenals against future viral outbreaks is a requirement. Scientists continue the development of broad-spectrum antiviral drugs from natural or chemical sources, which have the potential advantages of broad-spectrum therapeutic effect and insensitivity to viral evasion. Given the urgency of the SARS-CoV-2 outbreak, here we discuss the discovery and development of new therapeutics for SARS-CoV-2 infection based on the strategies from preclinical drug discovery.
2. Methods
We report this systematic review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [13].
2.1. Data Sources and Searches
Studies published in PubMed, Scopus, Web of Science, EMBASE, Google Scholar, and DrugBank were searched from December 2019 to March 2021 using the following search terms: “Coronavirus,” “Covid-19,” “SARS-CoV-2,” “Drug design,” “Drug development,” “In silico,” “In vitro,” and “In vivo” alone or in combination without language restrictions. The keywords were selected using expert opinions, mesh, and related article titles.
All articles with full text or in the absence of full text with abstract are included in the screening of this study. Studies were excluded if studies were comments, editorial, letters, review, and preprints.
2.2. Data Extraction
Two researchers independently extracted data from included studies using a predefined data extraction form. All disagreement was discussed and solved after rechecking the source data with a third investigator. The data extracted, including the last name of the first author, type of the study (in silico, in vitro, in vivo), and name of the agent (chemical compound, drug, herb, etc.), studied the mechanism and efficacy of the agent according to study design that specified according to the following definition.
Computer-aided drug design can be divided into three different categories. All are based on ligands and receptors, which are briefly described [14] as follows:(1)Dock Receptor-Based Approach [15]. Once the three-dimensional structure of the ligand molecules and their receptor is known, the receptor-based method is a good candidate for identifying or optimizing drugs. Due to the presence of three-dimensional structures of compounds and receptors, the nature of the interaction between the ligand and the receptor and the type of structure that the ligand can have to interact with them in favorable conditions can be identified using this method. The compound is simulated on the active site of the dock (meaning anchoring) and on the interaction of the ligand with the receptor by molecular mechanics and molecular dynamics. In this method, due to the ligation of the ligand in the active position, the ligand changes in terms of conformity and changes its position in different conditions and shows interaction with the receptor in different types of situations. To determine the type of ligands that can be docked into the receptor site, the matching of the shape and the complementarity of the hydrophobic, hydrophilic, and charged parts must be considered. Various software packages such as AUTODOCK-Glide-LUDI and LigandFit are used to design the drug based on the structure of the receptor.(2)Ligand-Based Approach [16]. This method is used where the three-dimensional structures of the receptor are unknown and instead the structure of the ligands is known, which is one of the common methods. In this method, by indirectly studying compounds that react with biomolecules, they seek to design compounds that are pharmacologically active. In ligand-based drug design methods, in the absence of biomolecule structure, by studying specific ligands, it seeks to identify the structural and physicochemical properties of the compounds so that the desired compound can be designed based on data extracted from the study of previous compounds. This method is a kind of drug design based on pharmacophore (pharmacophore refers to the part of the drug to which the effect of the drug depends on that part of the molecule), and by studying the quantitative relationship between structure and their activity, drugs can be designed by this method. It can be said that it is a method for designing the pharmacophores of drugs.(3)Denovo Design-Based Approach [17]. This method is used when the structure of the ligand is unknown but the structure of the receptor is known. In this method, there is information about the structures of the receptor or quasi-receptors, but there is no structure of the main composition that can interact with the active site of the receptor. One of the functions of drug design based on this method is to suggest and present the main composition that is complementary to the active site. The basis of the method is that the database of existing 3D structures is used to find small molecules that can interact with the active site of the receptor in terms of size, geometry, and functional groups. Software packages such as GROW and LEGEND are used to design drugs by this method.
Drug design methods in the computer include quantitative structure-activity relationship (RASQ), docking, molecular dynamic simulation, and computational modeling. In these studies, the efficacy is evaluated based on the function of the drug or compound agent and the mechanism of action. Figure 1 shows the methods of computer-aided drug design (CADD).
2.2.1. Computational Methods in Drug Design
Quantitative Structure-Activity Relationship (QSAR). QSAR provides studies on the relationship between chemical structure and biological activity or other biological activities that are important in selecting or removing a compound before synthesis and testing. QSAR [18] is especially important to predict the result, especially when it is not possible to experiment with a compound. Molecular descriptors, which are the most important components of QSAR, can be obtained experimentally or through mathematical formulas from various theories such as quantum mechanics, chemical graph theory, and study theories. QSAR seeks to establish a statistically significant relationship between structure and performance. It also explains the specific effect of a drug and can ultimately predict the effect of newly synthesized chemical compounds. QSAR model is also an equation that predicts a property through molecular descriptors and their coefficients. Evaluation of the effectiveness of new compounds that have been studied using this method can be reported as a percentage of enzyme inhibition if the modeling has been done and mentioned in the article.
2.2.2. Docking
In this technique, to achieve a combination with a pharmacological effect and increase the pharmacological activity of the drug, different formulations of a drug interact with the receptor, and the structure that has the best interaction with the receptor and the lowest energy level is selected for laboratory steps [19]. In this way, possible structures that have a stronger interaction with the receiver can be isolated at this stage. The same issue is considered and reported as an effectiveness measure.
2.2.3. Molecular Dynamic Simulation
In this technique, which is based on the simulation of drug-receptor interaction in the body, docking problems are solved and in fact play a complementary role in this technique [20]. Due to the time-consuming work with this technique, effective structures cannot be achieved directly through it, and the final stages of the study of drug-receptor interactions should be evaluated before starting laboratory work by having effective compounds from the previous stages. Molecular dynamic simulations produce information at the microscopic level (position and velocity of atoms). The conversion of these data into macroscopic values (pressure, energy, etc.) is done using statistical mechanics. In fact, molecular dynamics and statistical mechanics link microscopic concepts and macroscopically observable quantities. Molecular dynamic simulations are only able to predict the thermodynamic behavior and stability of the ligand binding mechanism at the active site of the target enzyme. This is reported as a criterion.
2.2.4. In Vitro
Study of drug in cell culture medium: effectiveness in these studies means inhibition of the replication of COVID-19 by the compound or drug under study [21].
The half-maximal inhibitory concentrations (IC50) are a measure of the effectiveness of a compound in inhibiting biological function [22].
In vivo studies are those in which the effects of drugs are tested on whole living organisms or cells usually animals as opposed to a tissue organism or dead organism. In vivo testing is better studied for observing the overall effects of an experiment on living subjects [23].
3. Results
In this review, we reported a significant number of articles with in silico, in vitro, and in vivo approaches for drug development of COVID-19. We retrieved a total of 2538 articles from the initial database search. After the removal of duplication and screening, 317 studies were selected for inclusion in this review. Figure 2 shows the PRISMA diagram.
The analysis of article contents indicated that 266 studies performed in silico approaches against viral targets; 34 studies used in vitro approaches against SARS-CoV-2; and 15 studies used in vivo (animal) models.
3.1. Results from In Silico Drug Discovery
From 267 studies used in silico approaches, 98 article studies repurposed approved drugs with a new mechanism of action and 91 studies evaluated natural products (e.g., herbal medicine) on COVID-19. The characteristics of these studies are summarized in Tables1 and 2. Also, Table 3 shows the characteristics of the remaining studies (N = 87).
In silico studies used the following component of novel coronavirus as targets: main protease (N = 154), spike glycoprotein (N = 62), nonstructural protein (N = 45), RNA-dependent RNA polymerase (N = 21), papain-like protease (N = 19).
About 260 drugs were repurposed by the computational methods for COVID-19 therapy such as about 120 drugs candidate against the main protease, 52 drugs against the spike glycoprotein, 14 drugs against RNA-dependent RNA polymerase, and 28 drugs against other nonstructural proteins.
Among the studied repurposed drugs, the best results (regarding efficacy) were observed with saquinavir (N = 9 study), ritonavir (N = 8 study), lopinavir (N = 6 study), remdesivir (N = 3 study), and amikacin, danoprevir, favipiravir, and telaprevir.
Table 1 shows target-based synthesis of data for COVID-19 drug repurposing. As presented, at least two studies show the efficacy of aprepitant, cobicistat, dipyridamole, and dihydroergotamine against the main protease and tegobuvir (N = 2) against spike protein. The following list of drugs had multitarget action: avapritinib, famotidine, bictegravir, ziprasidone, capmatinib, pexidartinib, amprenavir, zafirlukast, cilostazol, paromomycin, lopinavir, and remdesivir.
A total of 91 studies used in silico methods to evaluate the effects of natural products including herbal medicine against SARS-CoV-2. Among them, 54 studies used the main protease as main target, in which eucalyptus (N = 3), Tinospora cordifolia (N = 3), and flavonoids (e.g., hesperidin, rutin, and herbacetin) were the most studied and effective. Other studied targets were as follows: spike (N = 22) and multitarget (N = 20). From the plant metabolites, oleanolic acid, hesperidin, epigallocatechin gallate, jensenone, tinosponone, and anistone show promising results in computational methods against COVID-19. Aloe, green tea, eucalyptus, curcumin, and many Chinese, Indian, and African plants were also effective on COVID-19 in silico.
The derivatives of pyrazoles, oxadiazoles, phenyltriazolinones, triazoles, benzoylpinostrobin, benzoic acid, benzylidenechromanones, coumarin, and selenium show efficacy in computational methods and could be considered as lead molecules for drug design and synthesis against COVID-19. Melatonin, ebselen, phenyl furoxan, thimerosal, isatin, romidepsin, phenyl mercurin, pleconaril, and tyrosine kinase inhibitors (such as nilotinib and imatinib) are examples of other drugs that are effective in in silico methods.
3.2. Results from In Vitro and In Vivo Studies
Screening of in vitro studies leads to finding 34 studies. Different compounds were evaluated and most studies focused on the inhibition of viral replication, which was assessed by the quantification of viral RNA by PCR and IC50 value reported. Most of the studies use the Vero E6 cell line for the assessment of replication of SARS-CoV-2 (Table 4). In particular, main protease, papain-like protease, RNA-dependent RNA polymerase, NSP-14, NSP-15, spike protein, and TMPRSS2 were evaluated in vitro as targets. Also, three studies focused on in vitro inhibition of inflammatory markers such as interleukins and the effects of the cytopathic effect on infected cell.
Nitric oxide, ginkgolic acid, anacardic acid, troxerutin, bisindolylmaleimide derivatives, small molecules (GRL-172, and 5 h), baicalin and baicalein (phytochemicals), and bepridil were effective drugs in vitro against the main protease of COVID-19.
The following drugs could inhibit the spike/ACE2-mediated cell entry of COVID-19 in vitro: romidepsin, panobinostat, givinostat, sirtinol, saquinavir, lipopeptides, hydroxyzine, azelastine, heparin, and glycyrrhizic acid.
Alkenyl sulfonylurea derivatives [7], an IMU-8381 inhibitor of human dihydroorotate dehydrogenase, pyrazole derivatives, and phillyrin, regulated the expression of inflammatory cytokines (e.g., IL, TNF-α, and NF-κB) induced by SARS-CoV-2 markedly and could be considered as adjuvant treatment of COVID-19 severe disease.
Doxycycline, chlorpromazine, azithromycin, heparin, bepridil, tannic acid, and glycyrrhizic acid are well-known drugs that show both in silico and in vitro inhibitory effects against SARS-CoV-2 and should be considered for this purpose.
Also, in vitro studies show that lopinavir/ritonavir, sofosbuvir, and favipiravir have no antiviral effects against SARS-CoV-2 (huge gap between in vitro IC50 and free plasma concentration).
We included 16 in vivo studies in our final analysis. Most of them use a combination of in vitro and in vivo methods for the evaluation of novel drugs. Table 5 shows the detail of these studies.
Human monoclonal antibodies were the most evaluated drugs (N = 4), promote the reduction in viral load (in vitro), and prevent infection in animal models of SARS-CoV-2.
A prodrug of hydroxycytidine (molnupiravir) improved pulmonary function and reduced viral titer in vivo and was introduced as a potential broad-spectrum antiviral agent against SARS-CoV-2.
Chloroquine and chlorpromazine did not inhibit viral replication in mouse lungs, but protected them against clinical disease.
Dalbavancin shows significant inhibitory ability in both in vitro and in vivo models of COVID-19. The drug binds directly to ACE2 and blocks its interaction with the SARS-CoV-2 spike protein.
4. Discussion
Drug development is a multistep process, typically requiring more than five years to assure the safety and efficacy of the new compound. There are several strategies in antiviral drugs for coronaviruses including empirical testing of known antiviral drugs, large-scale phenotypic screening of compound libraries, and target-based drug discovery. To date, an increasing number of drugs have been shown to have anticoronavirus activities in vitro and in vivo, but only remdesivir and several neutralizing antibodies have been approved by the US FDA for treating COVID-19. However, remdesivir’s clinical effects are controversial and new antiviral drugs are still urgently needed. Given the urgency of the SARS-CoV-2 outbreak, here we discuss the discovery and development of new therapeutics for SARS-CoV-2 infection, which have been conducted in basic research (in silico) and preclinical study (in vitro, in vivo). Our database search identified about 3000 studies, which means a global effort for drug development in the current COVID-19 pandemic. Although the chance of successful drug development is very low (less than about 10%) and till today, there are no approved drugs. Many potential candidates (at least 420 drugs) are in clinical trials.
As summarized in the results, many compounds are in the development process for COVID-19 disease. Some of these compounds are completely new and could serve as seeds (or leads) for developing antiviral drugs against COVID-19, but as we need therapeutics as soon as possible, half of the studies focused on drug repurposing (repositioning), which is a process of investigation of existing drugs for new therapeutic purposes. With the emergence of a growing COVID-19 pandemic, the drug repurposing process was being accelerated. Clinical trials using repurposed drugs may take less time and have a lower overall cost of manufacturing and could have a wide distribution of drugs. According to our results, 260 drugs repurposed by the computational methods for COVID-19, among them saquinavir, ritonavir, and lopinavir, showed the best efficacy in in silico environment. These drugs can be rapidly repurposed for clinic application for treating COVID-19 patients given their proven safety.
Many trials are performed using a combination of ritonavir-lopinavir. The results of a systematic review and meta-analysis showed that this drug combination has no more treatment effects than other therapeutic agents in COVID-19 patients and is currently not used anymore [134]. We could not find any clinical trial on saquinavir, which is the most studied drug in silico and show high potency against COVID-19. Saquinavir could be a suggestion for further clinical research.
Given that the development of synthetic chemicals for therapeutic use is a random process that might result in serendipitous discovery, many pharmaceutical companies are now focused on the development of plant-derived drugs. Natural products and their structural analogs have historically made a major contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless, natural products also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization, and optimization. In recent years, several technological and scientific developments—including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances—are addressing such challenges and opening up new opportunities. Consequently, interest in natural products as drug leads is being revitalized. Medicinal plants have attracted significant attention to treat infectious diseases. Complex molecular structures and a wide variety of natural compounds make medicinal plants an excellent biological resource for drug discovery. Our results show that various plants have potential antiviral activities and could use or be a basis for drug development against COVID-19.
Some of the studied plants are among what used by people on a daily basis, such as green tea, aloe, curcumin, and eucalyptus. A systematic review and meta-analysis of RCTs on herbal medicine in the management of COVID-19 show the significant effects of the combined therapy of herbal medicine in treating COVID-19 without any significant side effects [135].
Our results retrieved 91 in silico studies of natural products including herbal medicine. The results of this in silico approach showed that some of these studied active ingredients have a high affinity for each of the four important viral proteins compared with the inhibitors previously reported for each of these proteins. They could possibly have an inhibitory effect on the SARS-CoV-2 and COVID-19. Most evaluated chemical compounds had inhibitory effects against one or two proteins of SARS-CoV-2. In addition to having a great affinity to attach to the viral proteins, these herbal compounds have antioxidant, vasoprotective, anticarcinogenic, and antiviral properties. Thus, they can be applied as extremely safe therapeutic natural compounds and clinical assessments might have notable outcomes for controlling COVID-19.
Due to the nature of preclinical (in vitro and in vivo) studies, the number of drug development studies in this area was less than in silico studies. Again, repurposed drugs were the most studied drugs in vitro (both herbal and synthetic). Some of them such as saquinavir, heparin, glycyrrhizic acid, and chlorpromazine show efficacy in both in vitro and in silico environments. Chlorpromazine is the single agent that was found to have efficacy in in silico, in vitro, and in vivo areas.
In terms of mechanism of action, different targets from the structural and nonstructural proteins of COVID-19 were evaluated. Most of the studies focused on the main protease, papain-like protease, and spike glycoprotein. Apart from the specific protein that leads to viral replication, SARS-CoV-2 causes a surge of pro-inflammatory cytokines and chemokines, which cause damage to lung tissue and deterioration of lung function. Therefore, the design of a drug with multitarget of action against different proteins of COVID-19 and also anti-inflammatory potential could be valuable.
Currently, there is no highly efficacious and specific treatment for SARS-CoV-2. Herein, we provided data on novel compounds in therapeutic drug discovery and development. Due to the nature of SARS-CoV-2 and the rises of several high transmissible strains, repurposing existing drugs has demonstrated power by bringing several drugs to approval for treating COVID-19 patients, such as remdesivir. Our results confirmed that a large number of repurposed agents are currently being explored for treating SARS-CoV-2 infection. However, these drugs still suffer from suboptimal therapeutic effect or known strong side effect. To accelerate drug discovery and development, especially during the current pandemic, natural products capture attention again. Our results showed that various natural bioactive compounds are being investigated in the preclinical step of drug development for COVID-19. In addition to having high affinity, these herb active ingredients have antioxidant, vasoprotective, anticarcinogenic, and antiviral properties. Therefore, they can be used as extremely safe therapeutic compounds in drug design studies to control COVID-19. However, the pharmacological effects and adverse reactions of some drugs under development are still unclear, and hence, well-designed high-quality studies are needed to further study the effectiveness and safety of these potential drugs to accelerate drug development targeting SARS-CoV-2 and thus promote progress towards ending the pandemic.
Data Availability
All data generated or analyzed during this study are included in this published article.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This manuscript was financially supported by a grant from the Research and Technology Department of Isfahan University of Medical Sciences (grant no. 340068), Isfahan, Iran.