Safety and efficacy of 5-aminolevulinic acid phosphate/iron in mild-to-moderate coronavirus disease 2019: A randomized exploratory phase II trial

Abstract

Background:

5-aminolevulinic acid (5-ALA), a natural amino acid that is marketed alongside sodium ferrous citrate (SFC) as a functional food, blocks severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proliferation in vitro and exerts anti-inflammatory effects. In this phase II open-label, prospective, parallel-group, randomized trial, we aimed to evaluate the safety and efficacy of 5-ALA in patients with mild-to-moderate coronavirus disease 2019.

Methods:

This trial was conducted in patients receiving 5-ALA/SFC (250/145 mg) orally thrice daily for 7 days, followed by 5-ALA/SFC (150/87 mg) orally thrice daily for 7 days. The primary endpoints were changes in SARS-CoV-2 viral load, clinical symptom scores, and 5-ALA/SFC safety (adverse events [AE] and changes in laboratory values and vital signs).

Results:

A total of 50 patients were enrolled from 8 institutions in Japan. The change in SARS-CoV-2 viral load from baseline was not significantly different between the 5-ALA/SFC (n = 24) and control (n = 26) groups. The duration to improvement was shorter in the 5-ALA/SFC group than in the control group, although the difference was not significant. The 5-ALA/SFC group exhibited faster improvement rates in “taste abnormality,” “cough,” “lethargy,” and “no appetite” than the control group. Eight AEs were observed in the 5-ALA/SFC group, with 22.7% of patients experiencing gastrointestinal symptoms (decreased appetite, constipation, and vomiting). AEs occurred with 750/435 mg/day in 25.0% of patients in the first phase and with 450/261 mg/day of 5-ALA/SFC in 6.3% of patients in the second phase.

Conclusion:

5-ALA/SFC improved some symptoms but did not influence the SARS-CoV-2 viral load or clinical symptom scores over 14 days. The safety of 5-ALA/SFC in this study was acceptable. Further evaluation using a larger sample size or modified method is warranted.

1. Introduction

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in December 2019 and rapidly developed into a global pandemic.^[1,2] The therapeutic management of COVID-19 has changed drastically over the past 2 years. When the COVID-19 outbreak began, treatment was almost exclusively symptomatic and supportive, and the condition of many patients transitioned from mild-to severe disease. However, the advent of anti-SARS-CoV-2 vaccines has markedly improved the outcome of severe disease.^[3–5] Nevertheless, proper management of severe COVID-19 remains difficult, with many challenges persisting. Several drugs that reduce the risk of disease progression in patients with mild disease have been introduced.^[6–9] Remdesivir, used in the treatment of COVID-19-associated pneumonia since the start of the pandemic, is effective in patients with mild disease and is currently being administered in clinical settings.^[10] However, the efficacy of each drug, especially those involving monoclonal antibodies, is influenced by the unique characteristics of each mutant strain, making it important to develop oral drugs that are effective against such variants.^[11,12] Most oral drugs that are currently approved for treatment are expensive antivirals; therefore, selection of appropriate candidates for administration is generally limited to patients at risk of severe infection. However, in cases of high infectivity, wherein it is necessary to reduce the incidence and severity of the disease among multiple contacts, or in cases of large numbers of individuals being infected, a low-cost treatment alternative is urgently needed.

One potential candidate is 5-aminolevulinic acid (5-ALA), a naturally occurring amino acid that is an essential precursor for the synthesis of porphyrin compounds.^[13] Notably, 5-ALA phosphate combined with sodium ferrous citrate (SFC) is marketed as a dietary supplement with low production costs. The mechanisms of action of 5-ALA in humans include: Protoporphyrin IX accumulation in cancer cells; Activation of mitochondrial function; Induction of heme oxygenase-1, and; Increase in abnormal gene expression by binding to the guanine quadruplex structure. The benefits of 5-ALA include: Applications in cancer diagnosis and treatment; Hypoglycemic effects; Anti-inflammatory effects, and; Improved cognitive function in ATR-X syndrome.^[14–19] Recent applications of 5-ALA have focused on mitochondrial activation in diabetes mellitus^[20] and mutations in the nuclear gene associated with mitochondrial dysfunction (Koga et al, unpublished observation). In vitro experiments have revealed the antiviral effect of CoPP (Cobaltic Protoporphyrin IX Chloride) against the influenza virus. CoPP has been shown to inhibit viral proliferation and protect vascular endothelial cells through heme oxygenase-1 (HO-1)-mediated anti-inflammatory effects.^[21] The HO-1 may prevent severe disease by suppressing the production of inflammatory cytokines.^[22–25] Recently, the in vitro antiviral effects of 5-ALA against feline infectious peritonitis virus as well as the in vitro and in vivo antiviral effects of 5-ALA against swine fever virus have been reported.^[26,27] 5-ALA/SFC has been shown to decrease ACE2 expression and suppress SARS-COV-2 infection, and a new mechanism has been proposed for the effect of 5-ALA in controlling this infection.^[28] In Plasmodium falciparum, 5-ALA is also thought to target G-quadruplex (G4), and elimination of the parasites in an in vivo efficacy evaluation model has been observed upon 5-ALA/SFC treatment.^[29] Investigations of the effects of 5-ALA in regulating viral infections are gaining increasing attention. Moreover, the 5-ALA metabolite, protoporphyrin IX, inhibited viral proliferation in vitro in a SARS-CoV-2 infection model and inhibited viral S-protein binding to the host ACE2, as reported in an in silico molecular docking study.^[30] Above a specific concentration, we found the potent capability of protoporphyrin IX to entirely suppress the growth and infection of SARS-CoV-2, including the Omicron variant, which was isolated from infected individuals.^[31,32] 5-ALA/SFC is a potential promising therapeutic agent for COVID-19 due to its growth inhibitory and anti-inflammatory activities against SARS-CoV-2. Furthermore, SFC enhances the anti-inflammatory effect of 5-ALA by inducing HO-1 expression.^[33] Based on our projections, it is anticipated that SFC possesses the ability to attenuate the “cytokine storm” associated with COVID-19. Therefore, we are administering SFC in the form of a combined capsule to achieve this therapeutic effect.

The intake of 5-ALA/SFC in addition to conventional symptomatic therapy could lead to faster SARS-CoV-2 eradication and clinical improvement. Nonetheless, it is important to note that the safety and efficacy of 5-ALA/SFC in human participants still require confirmation and further investigation. Therefore, we conducted a phase II trial in patients with mild-to-moderate COVID-19, aiming to confirm the safety of 5-ALA/SFC (a dietary supplement) and to further explore its clinical efficacy.

2. Methods

2.1. Trial registration

The trial was registered with the Japan Registry of Clinical Trials (jRCTs071200048). The full trial protocol can be accessed at https://jrct.niph.go.jp/en-latest-detail/jRCTs071200048.

2.2. Ethical approval

The protocol was approved by the Clinical Research Review Board of Nagasaki University (Approval No. CRB20-019-9), which served as the central review board for all participating centers: Nagasaki Harbor Medical Center, Sasebo City General Hospital, Isahaya General Hospital, Yokohama Municipal Citizen’s Hospital, Kobe City Medical Center West Hospital, Shonan Fujisawa Tokushukai Hospital, and Nagahama Red Cross Hospital. The authors certify that this study received ethical approval from the appropriate ethics committee of each participating hospital. All procedures were conducted in accordance with the Declaration of Helsinki and the Clinical Research Act (Japan). Written informed consent was obtained from all participants.

2.3. Patients

We conducted a phase II open-label, prospective, parallel-group, randomized trial of 5-ALA/SFC involving 50 patients diagnosed with mild (symptomatic and oxygen saturation [SpO₂] ≥ 96%) or moderate (SpO₂ < 96%, non-intensive care unit management, non-ventilator management) COVID-19. Patients were sequentially enrolled from 8 institutions in Japan between February 4 and July 29, 2021. The inclusion criteria were as follows: ≥ 20 years of age; Males and females, inpatient or outpatient; Positive SARS-CoV-2 test result (confirmed via either gene-based nucleic acid or antigen test); and Symptomatic with mild or moderate disease.

The main exclusion criteria were as follows: Asymptomatic with a positive SARS-CoV-2 test result or presence of severe COVID-19; History of porphyria, hemochromatosis, or viral hepatitis; Pulmonary disease capable of leading to severe COVID-19, poorly controlled hypertension, or diabetes mellitus; Photosensitivity and/or prior administration of drugs known to cause photosensitivity at the time of enrollment; or Receipt of therapeutic agents for COVID-19 (except steroids) since the onset of COVID-19 symptoms.

2.4. Randomization and masking

Patients were randomly assigned to the 5-ALA/SFC or control group in a 1:1 ratio using the sealed envelope technique. The Contract Research Organization (EPS Corp, Tokyo, Japan.) was contracted to support this research. The envelopes were folded in triplicate; the contents were invisible from the outside. Because the envelopes and allocation list are prepared by a department of the Contract Research Organization not involved in recruitment, and neither the investigators nor the recruiters of the Contract Research Organization are notified of the allocation list, the groups to which the participants will be allocated is undisclosed prior to allocation. The Contract Research Organization checked the unopened envelopes at the end of the study to confirm the proper use of the envelope method at the implementing institution. Patients assigned to the 5-ALA/SFC group received oral 5-ALA acid phosphate/SFC (250/145 mg) thrice daily for 7 days (Days 1–7), followed by oral 5-ALA acid phosphate/SFC (150/87 mg) thrice daily for 7 days (Days 8–14). Follow-up assessments (SARS-CoV-2 polymerase chain reaction [PCR] test, laboratory blood test, and physical examination) were conducted on Days 21 and 28. Upon the occurrence of adverse events (AE) or exacerbation of the primary disease, the dose was reduced or the administration was stopped at the discretion of the investigators. For both the groups, non-anti-COVID-19 medications, such as nonsteroidal anti-inflammatory drugs, acetaminophen, or antitussives, were administered as needed.

2.5. Primary and secondary endpoints

The primary endpoints were changes in SARS-CoV-2 viral load in saliva, clinical symptom scores, and 5-ALA/SFC safety (AEs and changes in laboratory values and vital signs). Clinical symptom scores were obtained daily from patient diaries from Day 1 to Day 14 and on Days 21 and 28. Cough and phlegm were rated as follows: 0 = “none,” 1 = “sometimes,” 2 = “often,” or 3 = “always”; breathlessness, headache, languor, arthralgia, and myalgia were rated as follows: 0 = “none,” 1 = “slightly painful,” 2 = “painful,” or 3 = “very painful”; abnormal taste, abnormal sense of smell, appetite, and diarrhea were rated as 0 = “no” or 1 = “yes.” SARS-CoV-2 viral load was measured using PCR centrally at Nagasaki University Hospital on Days 1, 3, 7, 14, and 21 using saliva samples collected with a dedicated saliva collection kit (Saliva RNA Sample Collection Kit; Xiamen Zeesan Biotech Co. Ltd., China). Clinical symptom scores were based on the criteria listed in Table 1. Laboratory evaluation items and vital signs for safety events are listed in Table S1, Supplemental Digital Content, http://links.lww.com/MD/J548.

Table 1.

Clinical symptom category for scoring.

Cough	Yes (□ Sometimes □ Often □ Always) □ No
Phlegm	Yes (□ Sometimes □ Often □ Always) □ No
(Feeling of) breathlessness	Yes (□ Slightly painful □ Painful □ Very painful) □ No
Languor	Yes (□ Slightly tired □ Tired □ Very tired) □ No
Headache	Yes (□ Slightly painful □ Painful □ Very painful) □ None
Arthralgia	Yes (□ Slightly painful □ Painful □ Very painful) □ None
Myalgia	Yes (□ Slightly painful □ Painful □ Very painful) □ None
Abnormality of taste	□ Abnormality exists □ No abnormality exists
Abnormal sense of smell (olfaction)	□ Abnormality exists □ No abnormality exists
Appetite (for food)	□ Yes □ No
Diarrhea	□ Yes □ No

The secondary endpoints were as follows: Days to negative SARS-CoV-2 test; Changes in blood laboratory data from baseline (at screening); SpO₂ and partial pressure of arterial oxygen; Improvement in chest X-ray or computed tomography images/number of days until imaging findings disappeared; Number of days until the end of oxygen administration (only for patients receiving oxygen); Duration of hospitalization; Whether a transition occurred from mild-to moderate/severe disease or from moderate-to severe disease; Number of days until the fever resolved (≤37°C) (only for patients with fever > 37°C on Day 1); and Changes in body temperature from baseline (Day 1) (only for patients with a fever > 37°C on Day 1).

2.6. SARS-CoV-2 detection

SARS-CoV-2 was detected using nucleic acid amplification assays following the manual published by the National Institute of Infectious Diseases.^[34,35] Briefly, total nucleic acids were extracted from saliva samples using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (ThermoFisher Scientific, Waltham, MA), with 200 μL of saliva eluted in 100 μL of elution buffer. For SARS-CoV-2 RNA detection, 5 μL of RNA template was evaluated using real-time reverse transcription PCR. The following primers were used: forward 5′-AAATTTTGGGGGACCAGGAAC-3′, reverse 5′-TGGCAGCTGTGTAGGGTCAAC-3′; the probe was 5′-FAM- ATGTCGCGCATTGGCATGGA-BHQ1-3. PCR was conducted using the Thunderbird Probe 1-Step qRT-PCR Kit (Toyobo) and QuantStudio 6 Pro (ThermoFisher Scientific). The PCR cycle consisted of 95°C for 5 minutes followed by 50 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Viral copies were quantified using a standard curve, and SARS-CoV-2 viral load was expressed as copies/5 μL.

2.7. Data analysis

The population used for the safety analysis set (SAS) included all participants who consumed the supplement at least once in the 5-ALA/SFC intake group, along with all participants assigned to the control group. The largest analysis set (full analysis set [FAS]) was the population of participants with SAS values for at least Days 1 and 2 that could be evaluated for any of the primary endpoints. The eligible population that met the study requirements (per protocol set [PPS]) was the portion of the FAS with no negative result for SARS-CoV-2 on Day 1 and no significant deviations that would affect the assessment of efficacy. This was an exploratory study; therefore, no hypothesis testing was performed, and point and interval estimates were used for interpretation. A 95% 2-sided confidence coefficient was used for interval estimation, with no adjustment made for multiplicity; 0 was assigned if the SARS-CoV-2 test result was negative, with no detectable levels of SARS-CoV-2. Other missing values were not subjected to any special completion process and were treated as missing. All data were analyzed using SAS (version 9.4; SAS Institute Inc., Cary, NC).

To determine the degree of change in SARS-CoV-2 viral load, summary statistics were calculated for the measured values of SARS-CoV-2 viral load at each time point, and the degree of change from baseline (Day 1) at each time point. 95% confidence intervals (CIs) based on t-distribution were estimated for the degree of change. For between-group comparisons, an analysis of covariance (adjusted for the number of days from onset to Day 1 as a covariate) was performed to estimate the differences in means and 95% CIs for changes at each time point. Summary statistics were calculated for each allocation group for the total clinical symptom score (cough, sputum, breathlessness, fatigue, headache, taste, smell, appetite, and bowel movement), the measured value at each time point for each item, and the change from baseline (Day 1) at each time point. A frequency table for the clinical symptom score (cough, phlegm, breathlessness, languor, headache, arthralgia, myalgia, abnormal taste, abnormal sense of smell, appetite, and diarrhea) and the degree of improvement in clinical symptoms at Days 7 and 14 was created. 95% CIs for the changes were estimated based on t-distribution. For between-group comparisons, an analysis of covariance was performed (adjusting for the number of days from onset to Day 1 as a covariate), and the differences in means and 95% CIs for the change at each time point were estimated. The median and 95% CI were estimated using the Kaplan–Meier method for the number of days from Day 1 until the first improvement in clinical symptom score (cough, sputum, breathlessness, fatigue, headache, taste, smell, appetite, and bowel movement; the last observation date was the cutoff date for patients who did not improve). Hazard ratios and 95% CIs were estimated using Cox regression analysis (adjusting for the number of days from onset to Day 1 as a covariate).

3. Results

3.1. Study population

A flowchart of the study population is shown in Figure 1. A COVID-19 diagnosis was confirmed in all participants via SARS-CoV-2 PCR. None of the participants were diagnosed by antigen testing. Among the 50 participants enrolled in the study, 24 were assigned to the 5-ALA/SFC group and 26 to the control group. A total of 13 participants completed the study in the 5-ALA/SFC group (11 participants discontinued) and 23 participants completed the study in the control group (3 participants discontinued). A total of 22, 22, and 17 participants were recruited for SAS, FAS, and PPS in the 5-ALA/SFC group, respectively, and 26, 25, and 19 in the control group, respectively.

Figure 1.

CONSORT flowchart. AE = adverse event, PI = principal investigator, SAE = serious adverse event.

The main demographic characteristics of the participants were recorded (Table 2). The mean ± SD age was 49.8 ± 16.8 years in the 5-ALA/SFC group (n = 22), with 72.7% (n = 16) of the participants being < 60 years of age, whereas the mean ± SD age was 42.8 ± 13.5 years in the control group (n = 25), with 88.0% (n = 22) of the participants being < 60 years of age. The mean ± SD number of days from onset to Day 1 was 5.8 ± 3.5 days in the 5-ALA/SFC group (n = 22), with 50.0% (n = 11) of the participants having an onset of ≤ 5 days, whereas the mean ± SD number of days from onset to Day 1 was 5.4 ± 2.6 days in the control group (n = 25), with 60.0% (n = 15) of participants having an onset of ≤ 5 days. In the control group, 88.0% (n = 22) of participants had mild disease and 12.0% (n = 3) had moderate disease. The SARS-CoV-2 viral load (Ct) on Day 1 was < 30 in 35.0% (n = 7) and 36.8% (n = 7) of participants in the 5-ALA/SFC and control groups, respectively, and 30 to 36 in 55.0% (n = 11) and 36.8% (n = 7) of participants in the respective groups; 10.0% (n = 2) and 26.3% (n = 5) of participants had viral loads of ≥ 37 in the 5-ALA/SFC and control groups, respectively. No risk factors for severe disease (age ≥ 65 years, diabetes mellitus, hypertension, or smoking) were present in 54.5% (n = 12) of participants in the 5-ALA/SFC group and 40.0% (n = 10) of participants in the control group. The main concomitant medications were acetaminophen in 54.5% (n = 12), probiotics in 27.3% (n = 6), and antitussives in 22.7% (n = 5) of participants in the 5-ALA/SFC group, and acetaminophen in 42.3% (n = 11), antitussives in 34.6% (n = 9), and probiotics in 19.2% (n = 5) of participants in the control group. Inhaled steroids were used by 9.1% (n = 2) of participants in both the 5-ALA/SFC and control groups; the 2 patients in the control group were given inhaled steroids for complications not associated with COVID-19 treatment.

Table 2.

Patient demographics (categorical variables) (FAS).

(Data) item		5-ALA/SFC group	Control group
		(n = 22)	(n = 25)
Age (yr)	Mean	49.8	42.8
Sex	Male	11 (50.0%)	13 (52.0%)
Baseline (Day 1) severity
	Mild	18 (81.8%)	22 (88.0%)
	Moderate	4 (18.2%)	3 (12.0%)
Number of days from onset to Day 1 (two categories)	Average (d)	5.8	5.4
	≤5 d	11 (50.0%)	15 (60.0%)
	>5 d	11 (50.0%)	10 (40.0%)
SARS-CoV-2 vaccination history (dose)	None	22 (100.0%)	24 (96.0%)
	One	0 (0.0%)	0 (0.0%)
	Two	0 (0.0%)	1 (4.0%)
Inpatient/outpatient	Hospitalization	12 (54.5%)	15 (60.0%)
	Admitted to a convalescent home	9 (40.9%)	10 (40.0%)
	Outpatient	1 (4.5%)	0 (0.0%)
History of smoking	Smoker	4 (18.2%)	8 (32.0%)
	Previous smoker	3 (13.6%)	1 (4.0%)
	Non smoker	15 (68.2%)	16 (64.0%)
Day 1 viral load (Ct value)	<30	7 (35.0%)	7 (36.8%)
	≥30 and ≤ 36	11 (55.0%)	7 (36.8%)
	≥37	2 (10.0%)	5 (26.3%)
Risk factors for severe disease*	None	12 (54.5%)	10 (40.0%)
	Any	10 (45.5%)	15 (60.0%)

5-ALA = 5-aminolevulinic acid, FAS = full analysis set, SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2, SFC = sodium ferrous citrate.

^*Age ≥ 65 yr, diabetes mellitus, hypertension, or history of smoking.

3.2. Primary endpoints

Figure 2 presents data on changes in SARS-CoV-2 viral load (copies/5 μL; FAS). There was considerable variation in the change from baseline in SARS-CoV-2 viral load (copies/5 µL) over time, and there was no difference between the 5-ALA/SFC and control groups. The analyzed data are shown in •Table S2, Supplemental Digital Content, http://links.lww.com/MD/J549.

Figure 2.

Change in SARS-CoV-2 viral load (copies/5 μL, FAS). Viral load at each time point (Days 0, 3, 7, 21, and 28). FAS = full analysis set, SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

Changes in clinical symptoms are shown in Figures 3 and 4. Higher rates of symptom resolution were observed in the 5-ALA/SFC group than in the control group with regards to cough, languor, abnormal taste, and appetite on Day 14. The estimated between-group differences in the change in total clinical symptom score in the FAS analysis was 1.7 (95% CI: −0.2–3.6) for Day 3, 2.5 (95% CI: 0.4–4.5) for Day 7, 0.8 (95% CI: −1.4–3.0) for Day 14, 2.5 (95% CI: 0.1–4.8) for Day 21, and 3.7 (95% CI: −1.6–9.1) for Day 28. The differences were not statistically significant.

Figure 3.

Change in clinical symptom frequency (FAS). Improvement rates of the 11 clinical symptoms analyzed in the FAS group on Days 7 and 14 compared with baseline (Day 0). Each panel represents a symptom: (A) cough, (B) phlegm, (C) breathlessness, (D) headache, (E) languor, (F) arthralgia, (G) myalgia, (H) abnormal taste, (I) abnormal sense of smell, (J) appetite, and (K) diarrhea. The 5-ALA/SFC group had higher rates of symptom improvement (resolution) in cough, fatigue, dysgeusia, and appetite on Day 14 compared with the control group. 5-ALA = 5-aminolevulinic acid, FAS = full analysis set, SFC = sodium ferrous citrate.

Figure 4.

Change in clinical symptom frequency (PPS). Improvement rates of the 11 clinical symptoms analyzed in the PPS group on Days 7 and 14 compared with baseline (Day 0). Each panel represents a symptom: (A) cough, (B) phlegm, (C) breathlessness, (D) headache, (E) languor, (F) arthralgia, (G) myalgia, (H) abnormal taste, (I) abnormal sense of smell, (J) appetite, and (K) diarrhea. The 5-ALA/SFC group had higher rates of symptom improvement (resolution) in cough, fatigue, dysgeusia, and appetite on Day 14 compared with the control group. 5-ALA = 5-aminolevulinic acid, PPS = per protocol set, SFC = sodium ferrous citrate.

FAS analysis did not reveal a significant difference in the number of days to improvement between the 2 groups (Fig. 5). Improvement of clinical symptoms (based on the physician’s assessment) in the 5-ALA/SFC and control groups occurred in 16 and 24 patients, respectively, with a median of 10.0 (95% CI: 7.0–13.0) and 13.0 (95% CI: 3.0–13.0) days from Day 1 to the time of the first considered improvement, according to FAS analysis, respectively. The hazard ratio (95% CI) was 0.94 (0.47–1.88). In the PPS analysis, there was a trend toward a lower number of days to improvement in the 5-ALA/SFC group compared with the control group. The Cox regression analysis data are shown in Table S3, Supplemental Digital Content, http://links.lww.com/MD/J550.

Figure 5.

Kaplan–Meier curves of the number of days from Day 1 to the time of the first considered improvement. (A) FAS. (B) PPS. FAS = full analysis set, PPS = per protocol set.

3.3. Safety assessment

The safety assessment (AEs and changes in laboratory values and vital signs) revealed that AEs after Day 1 (Table 3) occurred in 31.8% (n = 7; 11 AEs) of participants in the 5-ALA/SFC group and 7.7% (n = 2; 2 AEs) of participants in the control group. AEs occurred in 22.7% (n = 5; 8 AEs) of participants in the 5-ALA/SFC group: Grade 1 in 13.6% (n = 3; 5 AEs: decreased appetite [n = 1], constipation [n = 2], increased ALT [n = 1], and increased γ-GT [n = 1]), Grade 2 in 9.1% (n = 2; 2 AEs: vomiting [n = 2]), and Grade 3 in 4.5% (n = 1; 1 AE: decreased appetite [n = 1]). The patient who experienced a Grade 3 AE required prolonged hospitalization owing to loss of appetite; however, symptoms were alleviated with dose reduction. Among the patients who were administered 750/435 mg/day of 5-ALA/SFC during the first phase, 25% (n = 5) showed adverse reactions, whereas among the patients who were administered 450/261 mg/day of 5-ALA/SFC during the second phase, AEs appeared in 6.3% (n = 1) of the participants. One event each of increased ALT and increased γ-GT occurred in the 5-ALA/SFC group. No notable changes or trends were observed in the control group. No significant changes or trends in vital signs were observed in the 5-ALA/SFC group compared with the control group.

Table 3.

Adverse events occurring after day 1 (SAS).

		5-ALA/SFC group (n = 22)		Control group (n = 26)
		n	(%)	n	(%)
Adverse event		7	31.8	2	7.7
Metabolic and nutritional disorders		2	9.1	0	0.0
	Decreased appetite	2	9.1	0	0.0
Mental disorder		0	0.0	2	7.7
	Insomnia	0	0.0	2	7.7
Vascular disorder		1	4.5	0	0.0
	High blood pressure	1	4.5	0	0.0
Respiratory, thoracic, and mediastinal disorders		1	4.5	0	0.0
	Asthma	1	4.5	0	0.0
Gastrointestinal disorder		4	18.2	0	0.0
	Abdominal pain	1	4.5	0	0.0
	Constipation	2	9.1	0	0.0
	Vomiting	2	9.1	0	0.0
Clinical examination		1	4.5	0	0.0
	Increased ALT	1	4.5	0	0.0
	Increased γGT	1	4.5	0	0.0

5-ALA = 5-aminolevulinic acid, SAS = safety analysis set, SFC = sodium ferrous citrate.

The number of days until SARS-CoV-2 PCR showed negative result is demonstrated in Figure 6. The difference in the number of days between the groups was not significant. Other results of secondary endpoints were also not significant (Figure S1–S4, Supplemental Digital Content, http://links.lww.com/MD/J551 and Table S4, Supplemental Digital Content, http://links.lww.com/MD/J552).

Figure 6.

Kaplan–Meier curves of the number of days until SARS-CoV-2 PCR showed negative result. (A) FAS. (B) PPS. FAS = full analysis set, PCR = polymerase chain reaction, PPS = per protocol set, SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

4. Discussion

This is the first study to examine the dose setting and efficacy of 5-ALA in humans with COVID-19. Significant decrease of the SARS-CoV-2 viral load in the 5-ALA group and improvement of clinical symptom scores could not been achieved compared to the control group. However, a higher incidence of symptomatic relief of cough, lethargy, abnormal taste, and loss of appetite on Day 14 was observed. Although the 5-ALA group manifested more AEs compared to the control group, this trend fell in the acceptable range. Albeit some of our results were inconclusive, some promising outcomes related to the regulation of infection immunity by the 5-ALA supplement were observed. Further studies are needed to plan for additional clinical trials or to determine the appropriate use of 5-ALA as prophylaxis medication.

This study confirmed that the maximum tolerated dose of 5-ALA phosphate administered for 7 days is 820 mg/day. For safety, the high-dose group in this study was administered 750 mg/day for 7 days. In addition to its direct effects, such as inhibition of viral entry and proliferation, the drug is expected to have anti-inflammatory activity (by inducing HO-1) and other in vivo effects. This study was an exploratory study aimed at examining the dose setting, and a high-dose of 250 mg t.i.d. was set as the induction dose, with the expectation of producing viral inhibitory and anti-inflammatory effects. Subsequently, a maintenance dose of 150 mg t.i.d. was established, which was expected to have an effect in vivo.

No significant difference was observed between the 5-ALA/SFC and control groups in terms of change in SARS-CoV-2 viral load (copies/5 μL) from baseline to each time point. On Days 3, 7, and 28, the 5-ALA/SFC group showed a greater decrease in SARS-CoV-2 viral load, whereas on Days 14 and 21, a larger decrease was observed in the control group based on FAS analysis; however, the difference was not significant, and the data showed large variations. Several factors may have contributed to this variation. Considering the level of transmission of SARS-CoV-2 when the present study was undertaken, it was considered acceptable for enrolled participants to have previously received a positive test result from the health department at the time of consent. In addition, the study did not set a limit on the number of days from disease onset to study enrollment. Consequently, some participants with a negative viral load on Day 1 (at baseline), and some participants who were not negative but had values close to 0, were enrolled. However, in vitro studies at Nagasaki University have shown that 5-ALA inhibits the growth of SARS-CoV-2^[31,32]; therefore, a review of the dose setting, establishing participant uniformity by implementing inclusion and exclusion criteria, and reexamining the PCR-based system could provide more rigorous results that accurately reflect changes in virus loads, if any, with 5-ALA/SFC treatment.

Evaluation of changes in clinical symptom scores (per patient self-reported) did not yield significantly different results between the groups. The 5-ALA/SFC group exhibited a lower percentage of improvement in the total score; however, the percentage of improvement was higher in terms of the individual symptoms of abnormal taste, abnormal sense of smell, and appetite in the 5-ALA/SFC group than in the control group. Cough, lethargy, abnormal taste, and lack of appetite were also more frequently resolved in the 5-ALA/SFC group at Day 14 compared with that in the control group. An interim report of clinical studies of patients with mild COVID-19 reported that variability may occur when analyzing clinical symptom data^[36,37]; this could have occurred in this study, given the large number of patients with mild disease. Based on the high rate of improvement in some symptoms and the high rate of symptom resolution on Day 14, we believe that further review of this intervention is warranted to better assess the possibility of reducing prolonged clinical symptoms. Similar to the change in viral load, the number of days from Day 1 to the time of the first improvement was not consistent between the FAS and PPS. The PPS group tended to have fewer days until improvement than the control group. For comparison, a phase II trial of 5-ALA/SFC in post-COVID-19 sequelae showed significant improvements in fatigue, anxiety, and mood-related conditions.^[38] A growing body of evidence and experience has demonstrated that symptoms persist long after SARS-CoV-2 infection (“long COVID-19”). They include various symptoms associated with fatigue, memory problems, cognitive impairment (brain fog), dyspnea, sleep problems, joint pain, headache, cardiac abnormalities, and concentration problems. The global prevalence of post-COVID-19 conditions is 0.43.^[39] Experiencing such symptoms for several months and the associated disease burden can exert stress on social activities of affected patients and the healthcare system.^[39,40] Based on the potential merit of 5-ALA/SFC in COVID-19 treatment, further analysis with homogenization of the participants at Day 1 could reveal benefits of this treatment for the clinical symptoms of both acute and long COVID-19.

In this study, more AEs occurred during the first 7 days of treatment, which involved administration of the higher dose of 750/435 mg/day (250/145 mg thrice daily). The safety of 5-ALA/SFC demonstrated in this study was within an acceptable range due to the absence of serious AEs. The occurrence of AEs with the higher 5-ALA/SFC dose suggests that it is essential to determine the optimal dose. Gastrointestinal symptoms were the most frequent symptoms among the observed AEs. SFC is an iron-containing product; therefore, the effects are more likely to be caused by excess iron ingestion than by 5-ALA itself. Data from domestic package inserts for prescription iron supplements indicate that gastrointestinal symptoms occur in > 5% of patients. The amount of iron that enters the body through oral administration of 5-ALA/SFC is almost equivalent to the amount contained in iron tablets.^[41] Gastrointestinal symptoms in combination with fever and malaise associated with COVID-19 may worsen the detrimental effects on patients’ physical condition. Therefore, to ensure the efficacy of 5-ALA/SFC against COVID-19 and to reduce side effects, it is necessary to evaluate whether iron should be included in the formulation, and to optimize the ratio of iron within the formulation. We strongly believe that the primary cause of side effects is associated with the presence of iron. Therefore, we are actively working on developing a supplement or investigational drug that aims to mitigate these side effects by reducing the iron dosage. Additionally, our future plans involve including a dose of 600/348 mg/day, which falls between the 2 doses used in the current study, in the treatment group to investigate the potential of maintaining therapeutic effectiveness while minimizing AEs.

This study has some limitations. First, the approval of drugs for the treatment of COVID-19 (especially remdesivir for moderate-to-severe disease) and the availability of vaccine coincided with the course of the study. Therefore, we encountered a scarcity of eligible patients with moderate disease, as the majority of eligible patients only presented with mild disease. Second, we conducted evaluations on a considerable number of patients with mild disease; however, the subset of patients who exhibited fever (≥37°C) or SpO₂ < 96% on Day 1 was limited in size. This could have potentially impacted the results of certain outcome measures in a negative manner. Third, the immunologic response such as cytokine response evaluation was not performed. Adding this assessment in this study could have been enriched the interpretation of the effect of SARS-CoV-2 infection to human.

Vaccine-based strategies are extremely important for the global control of COVID-19; however, they are limited by the high probability of new mutant strains and the need for reevaluation of vaccine efficacy in each case.^[11,12] The products of 5-ALA, protoporphyrin IX, and HO-1 not only bind to S-protein and angiotensin converting enzyme II to inhibit viral entry but also bind to the guanine quadruplex structure of viral genes to inhibit their function and have multiple targets. Therefore, their effects remain unchanged against problematic strains, such as S-protein mutant strains. It is unlikely that several mutations of multiple targets occur simultaneously. To support this statement, we have published the corresponding in vitro results.^[31,32] Recent studies have revealed the effectiveness of 5-ALA in regulating infectious immunity. Several reports have demonstrated a decrease in IL-6 levels in both in vivo and in vitro experiments, wherein inflammation was induced using lipopolysaccharide.^[42–44] These findings support the potential role of 5-ALA in exerting an anti-inflammatory effect. The more generalized effects of 5-ALA, which are unaffected by mutation status, could serve as a promising solution against COVID-19.

This was an exploratory study, with a small sample size, for remediating the medical and social detriments caused by the spread of COVID-19. The interpretation of the data is therefore limited by the fact that the study was open rather than placebo-controlled, with a non-stratified patient population. Because the main purpose of this phase IIA trial was to confirm the safety and tolerability of 5-ALA in patients with COVID-19, it was not necessary to show a statistically significant difference. In addition to its potential use for symptoms relief in long COVID-19 patients, our results suggest that this supplement holds potential as a prophylactic for preventing severe disease in SARS-CoV-2 infection. Its use as a functional food would allow mass administration to cluster facilities or populations in close contact, as an intervention strategy (for postexposure prophylactic use). This could be useful as an add-on therapy to currently approved anti-COVID-19 drugs in cohorts that were restricted from participating in this study.

5. Conclusion

The 5-ALA/SFC group did not show any significant differences in SARS-CoV-2 viral load or clinical symptom scores (self-reported) with treatment over 14 days compared with the control group, for patients with mild-to-moderate COVID-19. In particular, the overall clinical symptom scores were not significantly different between the groups; however, the 5-ALA/SFC group had a higher rate of improvement in abnormal taste, abnormal sense of smell, and appetite than the control group, with a higher percentage exhibited symptomatic relief while considering cough, lethargy, abnormal taste, and loss of appetite on Day 14. The safety of 5-ALA/SFC in this study was acceptable. The results of this study are inconclusive; however, the data could form the basis for further research on the potential of 5-ALA/SFC in minimizing COVID-19 severity or disease onset in a larger study population.

Acknowledgements

The authors wish to thank all the participants for their contribution to the study. We would like to thank Editage (www.editage.com) for English language review and EPS Corp. for performing the statistical analysis.

Author contributions

Conceptualization: Takeshi Tanaka, Yusuke Kobayashi, Kiyoshi Kita, Koichi Izumikawa.

Data curation: Takeshi Tanaka, Masato Tashiro, Kenji Ota, Ayumi Fujita, Toyomitsu Sawai, Junichi Kadota, Yuichi Fukuda, Makoto Sumiyoshi, Shotaro Ide, Natsuo Tachikawa, Hiroshi Fujii, Makoto Hibino, Hisanori Shiomi, Mai Izumida, Kohsuke Matsui, Momoko Yamauchi, Kensuke Takahashi, Hirotomo Yamanashi, Takashi Sugimoto, Shogo Akabame, Masataka Umeda, Masumi Shimizu, Naoki Hosogaya, Kosuke Kosai, Kazuaki Takeda, Naoki Iwanaga, Nobuyuki Ashizawa, Tatsuro Hirayama, Takahiro Takazono, Kazuko Yamamoto, Yoshifumi Imamura, Taiga Miyazaki, Koya Ariyoshi, Katsunori Yanagihara, Koichi Izumikawa.

Funding acquisition: Koichi Izumikawa.

Investigation: Masato Tashiro, Kenji Ota, Toyomitsu Sawai, Yuichi Fukuda, Makoto Sumiyoshi, Shotaro Ide, Natsuo Tachikawa, Hiroshi Fujii, Makoto Hibino, Hisanori Shiomi, Mai Izumida, Kohsuke Matsui, Momoko Yamauchi, Kensuke Takahashi, Hirotomo Yamanashi, Takashi Sugimoto, Shogo Akabame, Masataka Umeda, Masumi Shimizu, Naoki Hosogaya, Kosuke Kosai, Kazuaki Takeda, Naoki Iwanaga, Nobuyuki Ashizawa, Tatsuro Hirayama, Takahiro Takazono, Kazuko Yamamoto, Yoshifumi Imamura, Taiga Miyazaki.

Methodology: Yusuke Kobayashi, Katsunori Yanagihara.

Project administration: Takeshi Tanaka, Naoki Hosogaya, Yusuke Kobayashi, Koichi Izumikawa.

Supervision: Junichi Kadota, Yusuke Kobayashi, Hiroshi Mukae, Kiyoshi Kita, Koichi Izumikawa.

Validation: Takeshi Tanaka, Kenji Ota, Yusuke Kobayashi, Katsunori Yanagihara, Koichi Izumikawa.

Writing – original draft: Takeshi Tanaka.

Writing – review & editing: Yusuke Kobayashi, Kiyoshi Kita, Koichi Izumikawa.