Currently prescribed drugs in the UK that could up or downregulate ACE2 in COVID-19 disease: A systematic review

Objective: To review evidence on routinely prescribed drugs in the UK that could up or downregulate Angiotensin Converting Enzyme 2 (ACE2) and potentially affect COVID-19 disease Design: Systematic review Data


INTRODUCTION
The coronavirus SARS-CoV-2 which causes the COVID-19 disease is a global public health emergency.It has been reported in 190 countries with 4,310,786 confirmed cases and 290,455 deaths as of 12 th May 2020.Walker and colleagues from the World Health Organization Collaborating Centre for Infectious Disease Modelling predicted that in the absence of mitigation strategies, the virus would infect seven billion people and account for 40 million deaths this year alone. 1Efforts to shield the elderly (60% reduction in social contacts) and interrupt transmission (40% reduction in social contacts for the wider population) have reduced this number but further deaths are still expected. 1There is an urgent need for solutions.In the absence of a vaccination or effective treatment, there is growing interest in repurposing existing drugs for mitigation.
In particular, drugs affecting the Renin-Angiotensin System (RAS) have been highlighted as potential candidates for further investigation. 2,3This is because the SARS-CoV-2 virus utilises Angiotensin-Converting Enzyme 2 (ACE2) receptors within the RAS for entry into lung alveolar epithelial cells. 4ACE2 has previously been shown to correlate with susceptibility to the SARS-CoV-1 virus, and the spike (S) glycoprotein of this new virus binds to ACE2 with even higher affinity. 5,6Theoretically, altered ACE2 activity could therefore lead to a greater susceptibility to SARS-CoV-2.It could also cause greater severity of the infection. 7Previous studies suggest that dysregulation of ACE2 activity in the lungs could promote early neutrophil infiltration and subsequent uncontrolled activation of the RAS system. 8In mice models, acute lung injury was observed in response to SARS-CoV-1 spike protein, so it is plausible that similar responses will be observed with SARS-CoV-2. 9This is particularly problematic in organs containing high ACE2 such as the lungs as it may contribute to cytokine release syndrome (cytokine storm) and the subsequent respiratory failure that has been observed in those who have died from the disease. 7any prescribed drugs in common use are known to mediate effects through the RAS pathway.Over 45 million of these prescriptions were issued in the UK last year alone, and of these, 15 million were for Angiotensin Converting Enzyme Inhibitors (ACE-I) and Angiotensin Receptor Blockers (ARBs).Acting through the RAS pathway, these drugs may impact ACE2 regulation but their role in the COVID-19 pandemic is not clear.Given the number of people that are potentially on these drugs, it has caused substantial public concern and clinical uncertainty about continuation or cessation of prescribed medications during the pandemic.Accordingly, we reviewed all existing evidence on routinely prescribed UK drugs that might alter ACE2 regulation.Understanding the drug effects on ACE2 given its role in COVID-19 disease could help reassure clinicians and the public in these uncertain times, or direct research on drugs that might attenuate or exacerbate transmission.

METHODS
Our review was conducted in accordance with preferred reporting for systematic reviews and meta-analyses (PRISMA) guidelines and our protocol was submitted for open-access publication prior to commencing our study (in press).We have also made it available in pre-print through the medRxiv manuscript processing system.

Search Strategy
A systematic search in MEDLINE, EMBASE, CINAHL, the Cochrane Library, Web of Science was conducted from inception to the 1 st April 2020.The search strategy is shown in Table 1 below.The reference lists of recent reviews and included studies were screened.We also spoke to topic experts and screened OpenGrey for additional texts.No language limits or study design filters were applied.

Study selection, inclusion and exclusion criteria
The COVID-19 disease is still relative new and there is limited research on drug therapies specific to the virus.In the interest of being comprehensive about potential drugs acting through ACE2, we were as inclusive as possible within our study selection.We included both animal and human models (in vivo and in-vitro).Studies had to meet the following eligibility criteria: i) measures ACE2 levels, activity or gene expression, ii) includes a drug that is currently available on a UK prescription according to the British National Formulary, and iii) measures the effect of that drug against a placebo, control or sham group in an experimental design.Review articles were excluded but their reference lists were screened.Conference abstracts were included if sufficient detail could be elicited.We did not include studies in children under 18 years, or those examining drug effects in utero.

Data extraction
Four members of the team reviewed titles and abstracts for eligibility (AA, HDM, CW, SH).Fulltext review, data extraction and quality assessment were carried out in duplicate using a piloted sheet.Any disagreement between authors was resolved by discussion.Data on the following study characteristics were extracted: i) drug class, ii) drug name, iii) duration of treatment, iii) effect on ACE2 level (upregulation, downregulation, no effect), iv) model (human/rat etc), v) site of ACE2 reception (lung, renal, cardiac etc;), vi) study design, vii) study population, viii) sample size and ix) country.Given the urgency of our research question during the current pandemic, we extracted information from only what was available to us in the published text.

Quality assessment
Our review includes both animal and human models therefore quality assessment was carried out separately for these studies.Human studies were evaluated using the Cochrane risk of bias tool which includes the following domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other sources of bias. 10 Each domain was scored as low risk, unclear risk, or high risk of bias.We classified the overall risk of bias as low if all domains were at low risk of bias, as high if at least one domain was at high risk of bias, or as unclear if at least one domain was at unclear and no domain was at high risk of bias.Although this tool is specific to trials, all included studies were experimental designs and we therefore felt it appropriate to use.The methodological quality of animal studies was assessed using the SYRCLE's risk of bias tool which is based on the Cochrane risk of bias tool. 11SYRCLE's tool includes selection bias, performance bias, detection bias, attrition bias, reporting bias and other biases.

Data analysis
Owing to the mix of study designs and models, a meta-analysis was not appropriate.Narrative synthesis methods were used.We reviewed the meta-data by tabulating the studies according to our inclusion/exclusion criteria, human/animal model, drug classes and effects on ACE2.The consistency in the number of studies and direction of any effects were considered.Where inconsistencies were identified in the effect of a drug between studies, we looked at additional data such as methods, quality and outcome measurement for potential explanatory factors.

Patient and public involvement
It was not possible to involve patients or the public in the design or conduct of our work due to the rapid timelines, but we have invited PPI representatives to help us with drafting a lay summary and in the dissemination of our findings.

Results
We retrieved 6821 studies and screened 3,360 after removing duplicates.Following title and abstract screening, 233 studies were screened by full text.We included 112 studies in the final review.The flow of studies is shown in a PRISMA diagram in figure 1 including the reasons for study exclusion at each stage.

Study characteristics
Table 2 shows the characteristics of the included studies.These originated from 17 different countries with the most common being China (n= 36), the USA (n= 22) and Japan (n=18).There were 10 studies in humans (7 in-vitro and 3 in-vivo) (Table 3) and 102 in animal models (13 invitro and 89 in-vivo).Animal models included rats (n=94), mice (n=7) and canines (n=1).The sample sizes for in-vivo animal models ranged from 6 to 117.For in-vivo human studies (Table 3), sample sizes ranged from 8 to 375 but were not always reported.Participants were predominantly male and white with hypertension or diabetes, although the condition was not always stated.Most models examined ACE2 receptors in the heart or kidneys, only 5 of the 112 included studies reporting ACE2 levels in the lungs; these were all in animal models as shown in Table 2.

Effects of drugs
There were 21 different drug classes examined in the included studies as shown in Table 2. Table 3 tabulates only those that have been examined in human models.The mean drug exposure period ranged from 30 mins for in-vitro studies, to 15 weeks for in-vivo studies.The most common drug classes were ARBs (n= 55) and ACE-I (n= 22).Of the 55 studies that examined ARBs, 43 reported upregulation of ACE2 levels.Most of these studies were in rat models (n= 34) and examined cardiac ACE2 levels (n= 27).For ACE-I, 17 out of 22 studies reported upregulation of ACE2.These were also mainly in rat models (n= 16) and measured cardiac ACE2 levels (n= 14).Of the five studies that assessed statins, these were all within rat models; 3 reported upregulation of ACE2, 1 reported downregulation and 1 reported no effect.
Similarly, oestrogens were examined in 5 studies; 3 reported upregulation, 1 reported downregulation and 1 reported no effects.For calcium channel blockers; 2 out of the 3 studies reported upregulation of ACE2 levels and these were both in-vivo rodent models.The third study was an in-vitro human model that showed downregulation of ACE2 with a calcium channel blocker.There were 3 studies on aldosterone antagonists; all reporting increases in renal ACE2 levels within rat models.
Several diabetes drugs were evaluated and found to increase ACE2.For insulin, 6 out of 8 studies reported upregulation of ACE2 (in mice and rat models).For thiazolidinediones, 5 out of 7 studies reported upregulation (6 mice/rate models and 1 of cerebral human cells in-vitro).For GLP-1 agonists both included studies reported increases in ACE2.Similarly, for the one study examining DPP4 inhibitors, it also reported an increase in ACE2.The only study measuring the effect of SGLT2 inhibitors reported a decrease in ACE2.

Quality assessment
Figure 2 shows the risk of bias across the studies using the SYRCLE's risk of bias tool.In general, studies lacked blind allocation and outcome assessor blinding.They also frequently omitted information needed to make a thorough judgement on the risk of bias.

Discussion
To our knowledge, this is the most comprehensive review of drugs prescribed in the UK that could act on ACE2 receptors and thus potentially affect COVID-19 disease.The ACE2 receptor is reported to be an essential contributor to SARS-CoV-2 entry into the nasopharynx and lungs and the subsequent inflammation that leads to severe acute respiratory distress syndrome. 12,13ur review examined drugs across human and animal models, and we found a number of studies reporting upregulation of ACE2 levels in response to ACE-I (n=22), ARBs (n=55), insulin (n=8), thiazolidinedione (n=7) aldosterone agonists (n=3), statins (n=5), oestrogens (n=5) calcium channel-blockers (n=3) GLP-1 agonists (n=2) and NSAIDs (n=2).However, these drugs were poorly studied in vivo within the lungs or nasopharynx of humans, where they are likely to matter most in influencing severity of outcomes of COVID-19 disease.
We observed that the most frequent drugs to upregulate ACE2 are also those prescribed in people with diabetes or cardiovascular disease.5][16] Notably, these are also conditions with a high prevalence amongst Black, Asian and Minority Ethnic (BAME) groups who have had disproportionally high mortality rates from COVID-19 disease. 17To date, much of this evidence has been limited to clinical commentaries or case reports.Larger cohorts are emerging but have not yet adequately considered a range of potential confounders including co-morbidities, age, sex, deprivation or household numbers which might be more important than prescribed medication in the spread, susceptibility and severity of the disease.For example, in a cohort of 191 people who were infected with the virus in Wuhan, 87% of those who died had coronary heart disease and 47% had diabetes. 18These conditions are associated with an increased risk of death but were not considered as covariates in the analysis.Irrespective of ACE2, people with diabetes are more susceptible to worse infection as the low-grade chronic inflammation and hyperglycaemia associated with the condition results in impaired immune responses with lower IL-1, IL-6, TNF-a, and delayed mobilisation of immune cells in response to pathogens. 19This comorbidity like many other confounders, are highly relevant when examining the risk of death with COVID-19 disease.
This lack of adequate adjustment for existing conditions is highlighted by Sommerstein and colleagues in their editorial on ACE-I and ARBs in COVID-19. 15They also propose that existing co-morbidities such as heart failure may be independently linked to SARS-CoV-2 transmission and severity, and the subsequent poor pulmonary outcomes that are observed in these patients.Indeed, in mice models, arterial hypertension, atrial fibrillation and type 2 diabetes have been shown to upregulate ACE2 levels irrespective of medications. 20,21Moreover, ACE2 levels have been shown to be higher in men and with increasing age. 22Most of the published data on deaths in COVID-19 disease report that males of increasing age are particularly susceptible to poor outcomes. 12,23r review has also highlighted the variable ACE2 levels in different parts of the body with most of the existing literature focussing on renal and cardiac levels.Responses to drugs may vary depending on cell type and location.Although the lung ACE2 is important to COVID-19, it is unclear if overall COVID-19 mortality might be attenuated by cardiovascular ACE-2 activity levels.We also observed variations in ACE2 levels with drug exposure duration which was relatively short amongst included studies in our review.It is uncertain how dysregulation might continue after starting or stopping these medications.It is also unclear how the observed effects amongst included studies would translate in-vivo in humans and what the net effect on receptor access to the COVID-19 virus is; access to the receptor by the virus may be competitively inhibited by the presence of drugs which also attach to the receptor, so whether upregulation is the key factor in practice is unclear.This is particularly challenging to understand as we found a paucity of data demonstrating the effect of prescribed drugs on ACE2 in the lungs or nasopharynx, where the SARS-CoV-2 virus appears to enact its pathogenic effects.Our results therefore, do not provide convincing evidence on the role of any currently prescribed UK drugs acting through ACE2 regulation that could affect COVID-19 disease.Finally, we found a disproportionate number of studies reporting upregulation or 'positive effects' of drugs on ACE2, compared to studies reporting no effect or downregulation.This may reflect a publication bias that is well-established in the literature, especially amongst animal models. 24,25

Strengths and limitations
We carried out a comprehensive and systematic search of the literature.To our knowledge, this is the first review on the subject.We did not include language restrictions but non-English language studies in the international literature might not have been indexed in the databases we searched.Given the rate of new publications on COVID-19, it is also possible that our search and results may not be up to date.Owing to the limited research on this novel virus, it was necessary to be as inclusive as possible and we therefore considered both animal and human models to look for any drugs acting through ACE2 with potential to affect COVID-19 outcomes.While this inclusive approach may offer insights, the heterogeneity across models makes it hard to interpret findings or translate them directly to patients.Although we were robust in our methodological approach to this review, we were also aware of the urgency to report our findings in the current pandemic.We therefore did not contact authors for more information about their studies beyond what was published.We observed frequent omission of information that would have allowed us to carry out a more detailed quality assessment.Had we pursued this information; the quality assessment of included papers may well have been higher.

Conclusion:
We reviewed the evidence on routinely prescribed drugs in the UK that could up or downregulate ACE2 and thus potentially affect COVID-19 disease.Our review indicates that currently prescribed drugs have been poorly studied in-vivo within the lungs of humans.Until there is better evidence, we cannot recommend starting or stopping prescribed medications during the COVID-19 pandemic.
Acknowledgements: We would like to thank Prof Julia Hippisley-Cox for her early contributions to this project.
Competing Interests: All authors have completed the ICMJE uniform disclosure form and confirm no financial relationships with any organisations that might have an interest in the submitted work in the previous three years, no other relationships or activities that could appear to have influenced the submitted work.Author Contribution: HDM contributed to the design of the study, wrote the analysis plan, conducted the analysis, drafted and revised the paper.AA contributed to the design of the study, led the analysis, drafted and revised the paper.CW and SH contributed to the screening of studies, data extraction and revised the paper.NI revised the paper.SG and PL contributed to the design of the study and revised the paper.HDM is guarantor.

Declaration:
The lead author affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted.The opinions, results, and conclusions reported in this article are those of the authors and are independent from the funding sources.

:
The Southampton, Cambridge and Oxford Primary Care Departments are members of the NIHR School for Primary Care Research and supported by NIHR Research funds.The University of Cambridge has received salary support in respect of SJG from the NHS in the East of England through the Clinical Academic Reserve.SJG is supported by an MRC Epidemiology Unit programme: MC_UU_12015/4.HDM is an NIHR Clinical Lecturer and supported by an NIHR SPCR grant for this work: SPCR2014-10043.The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care

Table 2 : Characteristics and key findings of included studies
Studies reporting on multiple sites or in multiple models have been listed separately and appear more than once in the table *