ALA-Porphyrin Science
Phase 2 randomized clinical trial of 5-Aminolevulinic acid plus sodium
citrate chloride vs placebo for Covid-19 infected patients recovered
with sequelae
Kyoko Imamura,*a,b Hidemitsu Sugiharaa,b and Koichi HirahatacaSocial Cooperation Program of IT Healthcare, Graduate School of Pharmaceutica Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo, Japan. Fax: 03-5841-0281; Tel: 03-5841-0286; E-mail: kimamura@mol.f.u-tokyo.ac.jp
bJapanese Institute for Public Engagement, 1-21-23 Nagatsuta, Midori-ku, Yokohama, Japan, Tel: 080-3523-9261; E-mail: Info@ji4pe.tokyo
cHirahata clinic, 1-24-6 Shibuya, Shibuya-ku, Tokyo, Japan, Tel: 03-3400-3288; E-mail: koichi@hirahata.com

Received in 20th March 2022 , Accepted 24th March 2022

Summary.
Due to Covid-19 pandemic, development of safe and effective vaccines and medications has
become accelerated. Public interests are also on surge and increasing number of food supplements
and nutritional products are used, which alarms safe use of these products usually taken without
supervision by healthcare professionals such as physicians and pharmacists.
5-Aminolevulinic acid with sodium ferrous chloride (5-ALA/SFC) has been on the market for users
with fatigue, less of sleep, and difficulty in sugar control. Its safety profile is already longestablished, but it’d be important to see if it can be safely used for those patients infected by
Covid-19 with sequelae.
In our study, continuous biometric monitoring by smartwatch and applications installed on
smartphone are used by patients taking either placebo or 5-ALA/SFC for 28 days, in addition toroutine clinical and laboratory check up prior to the screening and at the end of observation period.
Despite the limited sample size, differences before and after use of 5-ALA/SFC were followed inboth groups. During 28 days’ administration, little changes were observed in placebo group whereas
significant weekly changes were observed in 5-ALA/SFC group. Future study with larger sample
size is expected to confirm the observation from this exploratory study.
Keyword:5-aminolevulinic acid, porphyrin, Covid-19, sequelae, safety, fatigue
Introduction
Recently health promotion has become much closer to people’s daily life thanks to the advancementof monitoring devices and programs to help them identify their biomedical information [1, 2]. Easy
measurement and visual presentation of the results are readily available at any moment and help
them better manage their health continuously by setting alarms to keep up timely measurement andtake any necessary actions such as taking pills or going to see their doctors. A variety of medical
devices are on the market with or without regulatory approvals, and the programs delivered as
applications for smart-phone users are also widely adopted. The data obtained can be integrated
with their electronic health records [3]. As such, these devices have contributed to drastic change in
ALA-Porphyrin Science, Vol. 10 No 1 2021, 15-22

clinical research by inviting shift to remote monitoring and decentralized clinical trials, which could
be of important option in the wake of Covid-19 [4].
Due to Covid-19 pandemic, healthcare resources such as hospital beds for patients and healthcare
workers and equipment for treatment and prevention are severely constrained and even affected
delivery of usual healthcare [5]. Public health measurements such as travel restriction, hands
washing and keeping social distances are now fundamental part of our daily life, and the use of foodsupplement and nutritional products are on rise for those who feel uneasy to visit clinic in fear ofgetting infected. As these products are not controlled by healthcare professionals, their safety profile
should be shared with the users.
5-Aminolevulinic acid with sodium ferrous chloride (5-ALA/SFC) has been marketed as
supplements for those who care for their health in expectation of maintaining their activity level [6].
5-ALA/SFC has also been known as inhibitory to SARS-CoV-2 infection in vitro [7], and examinedin clinical trial with Covid-19 patients to seek its efficacy and safety in infection [8. In our study, weused 5-ALA/SFC for those who recovered from Covid-19 with sequelae primarily to seek its safety
information in this population. We also measured biometric status of their health status using
smart-watch and smart-phone to explore possibility of identifying any changes.
Experimentals (Methods）
Study Design
This single-center, double-blinded, placebo-controlled phase-2 randomized clinical trial wasconducted from May 31, 2021, to Mar 31, 2022, at Tokyo Center Clinic and led by Dr Hiroki
Nagashima. The trial protocol is available in jRCT database [9]. The trial compared 5-ALA/SFC
with placebo in patients with Covid-19 sequelae, feeling with fatigue. To participate, the siterequired approval by the Certified Review Board. Written informed consent was required from all
patients before registration. This study followed the Consolidated Standards of Reporting Trials(CONSORT) reporting guideline [10].
Eligibility
Patients must have been within six months since infected by Covid-19, 20 to 75 years old inclusiveat the time of informed consent. Their level of fatigue should be 40 mm or more when measured by
Visual Analogue Scale (VAS). They are requested to use smart-watch and smart-phone. Patientsallergic to 5-ALA/SFC, or ingredient in the study drug, or porphyrin should not be able toparticipate, as well as those who are photosensitive or taking drugs known to cause photosensitivity.
They must have normal liver function test (AST and ALT being more than twice the upper normallimit), not serious anemia (hemoglobin level less than 10g/dl), and hypoxic (SpO2 less than 95%).
Patients and their families are diagnosed as porphyria should be excluded. Those with past historyof hemochromatosis or viral hepatitis, allergic to smart-watch band should also be excluded. They
must not be participating other clinical studies. Regarding vaccination, patients who received

Covid-19 vaccines within six months of informed consent must be excluded from this study. Any
patients as judged inappropriate by the investigator should not be included in this study.
Biometric Analysis
In addition to the usual physical examinations and a battery of lab tests, patients are asked to use
smart-watch and smart-phone in the first week after screening visit, to get familiar with the devices
and reporting method of their measurement. Garmin Vivosmart 4 [11] is used as smart-watch tomeasure level of sleep, and iPhone as smart-phone to which applications are installed such as
EQ5D5L Japanese version [12], electronic questionnaire asking symptoms, and VAS asking level offatigue (standardized by Japanese Society of Fatigue Science) by positioning “no fatigue” at level 0
and “worst fatigue” at level 100 on horizontal line on smart-phone display [13]. Patients are also
provided pulse oximeter to measure and report their SpO2 every day. Pittsburgh sleep quality index
Japanese version [14] is used before and after the test agent administration.
Randomization and Treatment
Patients were randomized in a 1:1 ratio to receive either 5-ALA/SFC or placebo. Randomization
was stratified by sex (men vs women).
For patients randomized to 5-ALA/SFC group, three capsules of 50 mg 5-ALA and 29mg SFC weretaken twice a day. For patients randomized to placebo group, matching number of placebo weregiven in the same manner. Disease assessments occurred at the start and end of treatment. Aftercompletion of treatment, patients were observed for further two weeks to evaluate safety oftreatment.
End Points
The primary end point for the trial was the safety of the treatment, which was defined as the typeand frequency of adverse events, diseases, and adverse drug reactions. Reported cases werecategorized by frequency, seriousness, and expected or unexpected. Secondary endpoints included
change in patient-reported level of fatigue by VAS; change in scores by Pittsburgh sleep quality
index; change in sequelae reported at the start of treatment; change in quality of life measured by
EQ5D5L; change in values of activity, body battery, and stress; sleep analysis as measured bysmart-watch. Any other changes in lab test were also analyzed.
Statistical Analysis
Summary statistics were calculated on the number of patients, mean, standard deviation, median,interquartile range. Statistical significance was tested at 2-sided 0.05 level. 95% Confidence interval
was calculated. For primary endpoint, frequency of occurrence of adverse events, diseases, andadverse drug reactions was analyzed in full analysis set. For secondary endpoints, Wilcoxon rank
sum test was used for inter-group comparison. To compare weekly changes in fatigue VAS withbaseline assessment, for example, Wilcoxon signed-rank test was used. Also changes in values andratio were summarized and presented in 95% confidence interval of mean was calculated. Otherendpoints were analyzed in the same manner.
For analysis, aggregate, and table presentation, SAS ver 9.4 was used. For line listing, figures, andtables, Microsoft Excel and Microsoft PowerPoint were used.
Results and discussion
Patient Characteristics and Treatment
From May 31 to Sept 30, 2021, 46 patients were enrolled, of whom 42 patients were eligible fortreatment. 21 patients were randomized to placebo group and 21 to 5-ALA/SCF group. After 2
patients withdrew from the study, the number of patients who completed 28 days’ treatment was 21in 5-ALA/SFC group and 19 in placebo group. Details of patient enrollment, eligibility, andtreatment are provided in Figure 1, and baseline characteristics are described in Table 1. There wasno urgent key-open, and data were fixed until all patients were assessed as valid for analysis.

SafetyFrequency of adverse events, diseases, and adverse drug reactions are tabulated in Table 2.

There was no statistically significant differences between groups in both full analysis set and per
protocol set, Three adverse events were found in 2 patients of 5-ALA/SFC group (acutegastroenteritis 1, diarrhea 1, wheal 1). Whereas one adverse event in one patient of placebo group(arthralgia of left foot).
Efficacy
Patients were asked to score their level of fatigue every day to standardize their skill of assessment.
After this exercise for 7 days, they started to receive treatment. The VAS values of each group over
4 weeks’ treatment are shown in Figure 2. Compared to VAS value at Day 0 as baseline score, nostatistical difference was found between the two groups. If looked at intra-group changes, however,
5-ALA/SFC group showed significant differences from baseline in consecutive weeks of week 3
and week 4.
Patient’s quality of life was measured using EQ5D5L, in which statistical significance (p=0.033)
was observed in the level of anxiety/depression in 5-ALA/SFC group during 4 weeks’ treatment
whereas no significant change in placebo group (Figure 3). Other measures by EQ5D5L (mobility,self-care, usual activities, pain/discomfort) were all statistically significant during 4 week’streatment, although not significant between the groups.

Limitations
As discussed, the main limitation of our study is that due to the arrival of vaccines in Japanesemarketplace and the national policy to get as many vaccinated as soon as possible, recruiting time
was limited (in our study, one patient stopped study participation to get vaccinated) to maintainstudy eligibility to achieve homogeneous study population. Another limitation is a short of funding
resulted in call for participation limited in Tokyo area. When decentralized clinical trial becomesfeasible in Japan, this limitation may be overcome by calling multiple sites to join the study so thatpatients can participate wherever they are.
Conclusion
5-ALA/SFC was safely used in the examined dose by patients recovered from Covid-19 withsequelae. Despite limited number of target participants, statistically significant change wasobserved in the level of fatigue as well as anxiety/depression. As the number of infected patientsgrows rapidly with Omicron variant, more number of patients are left with sequelae and further
study is needed with larger sample size and a range of dosing levels.
Acknowledgement
A special gratitude to Dr Hiroki Nagashima, Director of Tokyo Center Clinic, for his continuous
support throughout the study.
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