Association between methotrexate-induced Stevens-Johnson syndrome/toxic epidermal necrolysis and furosemide: a real-world disproportionality analysis

ABSTRACT Background Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare and life-threatening skin adverse reactions that are usually induced by drugs. This study aimed to assess the association between methotrexate and SJS/TEN when combined with furosemide. Research design and methods Data on suspicious, interactions (PS, SS, I) from the FDA Adverse Event Reporting System database for 2016–2021 were analyzed using the reporting odds ratio (ROR), information component (IC), proportional reporting ratio (PRR) and the Medications and Health Care Products Regulatory Agency (MHRA). Results We identified 28 case reports of TEN associated with the combination of furosemide and methotrexate and 10 reports of SJS associated with furosemide and methotrexate. The association of methotrexate with SJS/TEN was more significant in the entire data set when combined with furosemide than when methotrexate was not combined with furosemide. The association of methotrexate with SJS/TEN remained significant when furosemide was combined with methotrexate in a tumor-based disease context. After sensitivity analysis of the entire dataset as well as all antineoplastic drug datasets, consistent results were observed for TEN. Conclusions Our study confirmed a significant association between methotrexate and SJS/TEN when combined with furosemide, with an increased risk of SJS/TEN.


Introduction
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare and life-threatening skin reactions with an incidence of about 1-2 cases per million people per year, characterized by the formation of flaccid blisters, rapid development and extensive necrosis with epidermal detachment, and in severe cases, the acute phase may be associated with various systemic complications, including multi-organ failure.SJS involves<10% of the total body surface area (TBSA), and TEN involves more than 30% of the TBSA, TBSA impairment between 10% and 30% was defined as SJS-TEN overlap [1,2].Mortality from SJS/TEN is approximately 10%-40% [3,4], and the high mortality rate and complications seriously affect the health of patients.It has been reported that more than 95% of patients with TEN and about 50% of patients with SJS are associated with the use of specific drugs [5,6].
SJS and TEN are listed in both methotrexate and furosemide drug labels as one of the possible dermal adverse reactions that can occur.Methotrexate continues to play a key role as one of the most widely used antimetabolites in cancer chemotherapy and is recommended as an important component of chemotherapy for acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and osteosarcoma [7,8].Methotrexate is also widely used as a treatment for rheumatism, psoriasis, psoriatic arthritis and systemic lupus erythematosus because of its antiinflammatory and immunomodulatory effects [9,10].However, studies have shown that methotrexate metabolites are more difficult to dissolve under acidic conditions, deposition of methotrexate metabolite can lead to crystal formation in intratubular cells during the excretion process, which can cause kidney damage.Therefore, patients are advised to drink a lot of water every day when taking methotrexate to promote excretion and reduce the accumulation of metabolites, and to take diuretics when the urine volume is low [11].Furosemide is one of the most commonly used highly effective diuretics, and is mainly used in the treatment of congestive heart failure, hypertension, acute pulmonary edema, cerebral edema, and in the treatment of edema due to cirrhosis and renal disease [12], so there are clinical cases where methotrexate is combined with furosemide.We searched PubMed and Web of Science for SJS/TEN case reports related to methotrexate and furosemide for an unlimited period of time, excluding duplicate reports and including only publicly published literature, and found a total of 19 SJS/TEN cases related to methotrexate and 2 SJS/TEN cases related to furosemide, and in the literature with clear information on the combination we have no cases of methotrexate and furosemide combination were identified (see Appendix S1).Therefore, whether methotrexate causes a potential increased risk of SJS/TEN when combined with furosemide has not been reported in studies conducted in the literature.We also found that the drug label for furosemide clearly states that methotrexate may cause drug interactions when used in combination with furosemide, but the relationship between furosemide and methotrexate is not clearly stated in the methotrexate drug label.Methotrexate and furosemide are both older and more commonly used drugs, and SJS/TEN mortality is relatively high, so it is necessary to evaluate the association of methotrexate and furosemide combination with SJS/TEN.
Generally, spontaneous reporting system can be used to detect new, rare and serious adverse events.Both SJS/TEN with methotrexate and SJS/TEN with furosemide in the published literature are limited to a small number of case reports, and no studies in the literature on the combination of methotrexate and furosemide were identified, including a pharmacovigilance analysis of the FAERS database, so this warrants a study of the risk of SJS/TEN with the combination.Therefore, the aim of this study was to assess the association with SJS/TEN when methotrexate and furosemide are combined by adverse events reported in the US FDA Adverse Event Reporting System (FAERS).We counted relevant information from reports of methotrexate in combination with furosemide, including demographic and administrative information, adverse event information, and patient outcomes; and performed disproportionality analysis including secondary sensitivity analysis for different data contexts to assess the association with SJS/TEN when methotrexate is combined with furosemide.

Data source
Data were obtained from the FAERS quarterly database of drug data classified as suspicious, interacting (PS, SS, I) drugs reported from the first quarter of 2016 to the fourth quarter of 2021, with a total of 4,713,954 reports included for analysis as the entire dataset.To reduce confounding by relevant disease background, all reports containing antineoplastic drugs were extracted as a subset of data for controlled analysis based on all suspected, interacting (PS, SS, I) drug data from 2016-2021, with a total of 734,746 included in this study.The list of antineoplastic drugs is defined according to the secondary active substance corresponding to the anatomical therapeutic chemical (ATC) classification (code: L01), see Appendix S2.
In order to better compare the association between methotrexate and furosemide in combination and methotrexate without furosemide with SJS/TEN, the names of the entire data set and data subsets were standardized.The 'methotrexate' without furosemide in the report is changed to 'not combined with furosemide-methotrexate,' and the 'furosemide' without methotrexate in the report is changed to 'not combined with methotrexate-furosemide;' For reports containing furosemide and methotrexate at the same time, 'methotrexate' was changed to 'combination with furosemidemethotrexate.' All the name changes were made in the column 'prod_ai' of the pharmaceutical active ingredient in the 'DRUG' table.

Definition of adverse events
Medical Dictionary for Regulatory Activities (MedDRA) is a terminology set developed under the auspices of the International Technical Coordination Committee for Registration of Pharmaceuticals for Human Use.Adverse events in the FAERS were coded using the MedDRA preferred terms (PTs), which are grouped by defined medical condition or area of interest.The following two preferred terms were used in this study to precisely match SJS and TEN: SJS/ PT10042033, TEN/PT10044223.

Disproportionality analysis
In this study, disproportionality analysis is used to detect adverse drug reaction (ADR) signals, including the proportional reporting ratio (PRR), the reporting odds ratio (ROR), the Medications and Health Care Products Regulatory Agency (MHRA), and the information component (IC).The data such as the target drug-ADR, and non-target drug-ADR are obtained by constructing a two-by-two contingency table, and each signal value is calculated according to the formulas, see Appendix S3 and Appendix S4 [13,14].The conditions for each method to detect the positive signal are as follows: for ROR [15] and PRR [16], the number of reports≥3 and the lower limit of the 95% confidence interval (CI)＞1; for MHRA [17], the number of reports≥3, PRR≥2 and χ 2 ≥4; for IC [18], the number of reports≥3 and the lower limit of the 95%CI＞0.The positive signal was considered to be statistically significant between the target drug and the target ADR.To make the results more intuitive and considering that ROR and IC are two commonly used and different signal detection methods, ROR belongs to frequency count method rather than Bayesian frequency method and IC belongs to Bayesian method, the ROR and IC methods are chosen for comparison and presented as forest plots.Use R 4.0.1 for signal detection as well as data processing and analysis.
Analyzing specific patients with common risk factors can mitigate the effects of confounding factors, so this study first performed a disproportionate analysis based on the entire dataset, and then performed a subset analysis of the data based on the antineoplastic drug context.To assess the robustness of the results of the analysis, we performed a sensitivity analysis: the most commonly reported drugs listed in the literature known to cause SJS and TEN were excluded, including antiepileptic drugs such as carbamazepine, sulfonamide antibiotics, NSAIDs, and allopurinol [19][20][21].Use ATC codes to determine the drug list for antiepileptic drugs (N03), sulfonamide antibiotics (J01E), nonsteroidal antiinflammatory drugs (M01A), and allopurinol (M04AA01), as shown in Appendix S5.

Descriptive analysis
A total of 322 adverse events were reported for the combination of methotrexate and furosemide, including 10 cases of SJS and 28 cases of TEN.We analyzed the adverse event reporting characteristics of methotrexate in combination with furosemide, including data on induced SJS and TEN, and the patient characteristics are summarized in Table 1.Among the cases of methotrexate in combination with furosemide, the indications for methotrexate were mainly rheumatoid arthritis (21.12%), lymphoma (9.01%), psoriasis (7.14%), and leukemia (5.90%), among the SJS and TEN case reports, the indications for methotrexate were mainly focused on leukemia and lymphoma.It is noteworthy that among the SJS and TEN adverse event reports, the highest percentage of case events was death, 90% and 67.86%, respectively.

Signal analysis
Based on the signal detection of the entire data set, there were 131 and 62 case reports of adverse reactions as TEN and SJS for methotrexate alone; 301 case reports of TEN and 113 case reports of SJS for furosemide alone; and 28 and 10 cases of TEN and SJS, for the combination(See Table 2 and Figure 1).The results of the four signal detection methods, ROR, PRR, MHRA, and IC, were consistent, with no signal detected for either TEN or SJS for methotrexate alone, and although a signal was detected for furosemide alone, the signal values were much lower than for the two-drug combination, as shown in Table 2.The combination of methotrexate and furosemide detected ROR and PRR signal values above 10 for TEN and above 4 for SJS, this is statistically significant.There was a significant association between methotrexate and SJS/ EXPERT OPINION ON DRUG SAFETY TEN when furosemide was combined with methotrexate; the risk of TEN and SJS was greatly increased in patients taking both methotrexate and furosemide compared to methotrexate alone.It can also be visualized in Figure 1 that the signal is very significant when the two drugs are combined compared to the single drug(SJS: ROR95% CI: 7.18-24.90;IC95% CI: 1.99-4.14;TEN: ROR95% CI: 18.58-39.23;IC95% CI: 3.57-4.83).
Based on data from a subset of case reports containing antineoplastic drugs, there were 131 and 62 case reports of adverse reactions as TEN and SJS for methotrexate alone; 6 case reports of TEN and 4 case reports of SJS for furosemide alone; and 28 and 10 cases of TEN and SJS, respectively, for the combination(See Table 3 and Figure 2).The results of the four signal assays, ROR, PRR, MHRA, and IC, remained consistent, with no positive signals detected for either methotrexate alone or furosemide alone(Table 3).Notably, when furosemide was combined with methotrexate, the association between methotrexate and SJS/TEN remained highly significant and was higher than the signal values detected in the context of the entire data.Statistically significant ROR and PRR signal   values of upwards of 18 were detected for TEN and upwards of 7 for SJS when the two drugs were combined.From Figure 2, for the ROR and IC method, the signal values were much higher with the combination than with the single drug (SJS: ROR95% CI: 7.18-24.90;IC95% CI: 1.99-4.14;TEN: ROR95% CI: 18.58-39.23;IC95% CI: 3.57-4.83).

Sensitivity analysis
Sensitivity analyses were performed on the entire dataset and a subset of all antitumor drug data according to drugs known to cause TEN, SJS, and the results are summarized in Table 4.After sensitivity analysis, there were 18 case reports of TEN with the combination of methotrexate and furosemide.SJS was not discussed in this section because there were no case reports of SJS with the combination after sensitivity analysis due to the low number of reports.The results of the sensitivity analysis were almost identical to those found in the results of the signal analysis above.All four methods, ROR, PRR, MHRA, and IC, detected positive TEN signals when the two drugs were combined, and the signal values were much higher than those of the single drug.In the sensitivity analysis of the entire data set, the ROR and PRR signal values for the combination were upwards of 16; in the sensitivity analysis of the subset of data, the ROR and PRR signal values for the combination were greater than 25, which was statistically significant.In conclusion, the association between methotrexate and TEN when furosemide was combined with methotrexate remained highly significant in the entire dataset as well as in the subset based on the antineoplastic drug context with a much higher risk of TEN than when methotrexate was not combined with furosemide, especially in the antineoplastic drug context.

Discussion
The association between methotrexate and SJS/TEN was confirmed by our signal mining analysis of the FAERS database.In  this study, we found that although the number of reported cases of SJS and TEN when methotrexate was combined with furosemide was not high, which may be related to the fact that SJS and TEN themselves are relatively rare adverse reactions, the mortality rate of SJS and TEN in the reports of furosemide in combination with methotrexate has exceeded 60% by the statistics of this study.We performed an additional count of 683 reports of methotrexate in combination with all diuretics among those classified as suspected, interacting adverse drug reactions, with 652 adverse reactions and 10 and 28 cases of SJS and TEN, respectively, identifying the list of diuretics according to ATC code (C03) in Appendix S6.We found that in all methotrexate in combination with furosemide was used in all the reports where SJS and TEN occurred, and none of the reports produced SJS/TEN when methotrexate was combined with other diuretics, so this warrants a study of the risk of SJS/TEN.In our descriptive statistical analysis, the cases of TEN were concentrated in the elderly, where impaired cognitive function and reduced urinary clearance can lead to increased drug toxicity [22].In addition, the results of disproportionate analysis showed that methotrexate without furosemide did not produce signals related to SJS and TEN, no matter in the background of FAERS' drug data or tumor diseases.Whereas when combined with furosemide, methotrexate found significant signals with SJS and TEN and also significantly higher signals than furosemide without methotrexate.Therefore, from the signal results, the combination of furosemide and methotrexate increases the risk of SJS and TEN, and this result will improve the understanding of the potential risk of SJS/TEN with the combination of furosemide and methotrexate.
The etiological mechanism of SJS/TEN is unknown, CD8 T-cells as well as the cytolytic molecules FasL and granulysin are key substances in the pathogenesis of SJS/TEN, and studies suggest that it may be a drug-induced immune mechanism [23,24].Due to the rare incidence of SJS/TEN, there is no risk signal mining for methotrexate or furosemide associated with SJS/TEN, and only a few case reports of methotrexate or furosemide have been reported in the literature (Appendix S1).Akinci B [25]reports a case of SJS associated with methotrexate treatment for acute lymphoid leukemia in a patient who was successfully treated and again continued on methotrexate at the same dose.Cases of SJS/TEN induced by high doses of methotrexate have also been reported in the literature, Primka EJ [26]described a patient with psoriasis who responded well to a course of methotrexate and then received intramuscular methotrexate 25 mg for septic psoriasis and bumetanide as a diuretic, shortly thereafter began to experience increased skin pain and burning, with extensive exfoliation and blistering of the patient's trunk and proximal extremities, and was diagnosed with TEN-like disease, the mechanism of induction of which is unknown.It may be secondary to chronic high circulating levels of methotrexate toxicity.However, it has been reported in the literature that SJS/TEN etiology may be caused by cutaneous malignancies and not methotrexate-induced [27].Not only that, case reports of successful treatment of SJS with methotrexate have been reported in the literature, showing significant regression of SJS skin lesions after 4 weeks of methotrexate administration [28].Interestingly, in our present pharmacovigilance study, methotrexate when not combined with furosemide did not find a significant signal with SJS/TEN.In summary, whether methotrexate induces SJS/TEN or whether SJS/TEN is an allergic or dose-related toxicity of methotrexate remains controversial and needs to be studied in further clinical trials.
In addition, we also found that furosemide was unstable in signal detection in both dataset contexts, being associated with SJS/TEN in signal detection across the dataset, while no signal was detected in the tumor-based disease context.Paret N [29] performed a descriptive analysis of 32 case reports of TEN and 14 cases of SJS in patients treated with furosemide in the French database and scored, but without signal detection and without analysis of the induction of SJS/TEN with the combination of furosemide and methotrexate, but the data emphasize the possibility that furosemide and SJS/TEN have morbidity in very few cases, and furosemide should also be considered before other suspected drugs are identified.Mebazaa A [30]and Kennedy AC [31]also reported furosemide-induced SJS/TENlike cases.The mechanism of SJS/TEN induction by furosemide has not been investigated, and among the most common drugs causing SJS/TEN are sulfamethoxazole, sulfadiazine, and sulfapyridine [32], and we speculate that the induction of SJS/TEN by furosemide may be related to its sulfonamide structure.
Pierard-franchimont C [33] elaborated that in some patients, high dosages of antifolate drugs (eg: methotrexate) used in cancer chemotherapy alter the keratinocytes, endothelial cells and Factor XIIIa+ dermal dendrocytes in a range of various severities.Occasionally, TEN/SJS-like conditions occur in anti-folate cytotoxic skin reactions (ACSR), but whether or not the TEN/SJS presentation of ACSR is a regular condition similar to that induced by other drugs or a variant condition supported by a unique pathomechanism is unsettled.Cases of SJS/TEN induced when methotrexate was combined with other drugs have been reported in the literature, including the combination of methotrexate and etoricoxib resulting in overlapping SJS/TEN, which highlights a possible pharmacokinetic interaction between methotrexate and etoricoxib [23].Methotrexate in combination with acetylsalicylic acid (ASA) induced SJS, and the authors concluded that the increased toxic effect of methotrexate on keratinocytes of patients was likely caused by the reduced plasma binding capacity and decreased renal excretion of methotrexate due to concomitant administration of ASA [34].SJS/TEN also appeared after the combination of methotrexate and trimethoprim-sulfamethoxazole [35].King LD [36]suggests that current Australian guidelines recommend avoiding concomitant administration of highdose methotrexate and trimethoprim-sulfamethoxazole, but the evidence to support this recommendation is unclear.Several mechanisms in the literature may be associated with this interaction; firstly, trimethoprim-sulfamethoxazole may decrease the renal clearance of methotrexate; secondly, trimethoprim-sulfamethoxazole may lead to substitution of methotrexate from plasma proteins, resulting in increased free methotrexate concentrations; and finally, since both methotrexate and trimethoprim-sulfamethoxazole inhibit dihydrofolate reductase, the combined use may produce additive effects, leading to excessive folate inhibition [37][38][39].Our results show that furosemide can increase the risk of SJS/TEN of methotrexate, and the results seem to be more inclined to the fact that SJS/TEN of methotrexate is induced by other drugs.Although few case reports of fur-osemide2020a, in combination with methotrexate have been reported in the literature, the labeling of furosemide mentions that furosemide decreases the renal elimination of methotrexate, leading to increased serum levels of the drug and thus increased toxicity of methotrexate; furosemide may also compete with methotrexate for plasma binding proteins, and this interaction of furosemide and methotrexate is similar to that of trimethoprim-sulfamethoxazole, explaining the results of this study from a pharmacokinetic perspective.
There are several limitations to our study.First, because this study was based on a spontaneous reporting system, a known limitation of spontaneous reporting is that a causal relationship between an adverse event and a drug does not have to be established to submit a report, which may be influenced by concomitant medications and patient comorbidities [40].Submission of reports is voluntary and subject to underreporting and underreporting.Second, because SJS/TEN is relatively rare, we did not collect a large number of reports, so we failed to consider the limitations of age, gender, and race.Finally, the number of SJS cases in which furosemide was combined with methotrexate was small, and there were differences in SJS in sensitivity analysis, and we failed to collect reports of SJS in which furosemide was combined with methotrexate.

Conclusion
This study is the first to evaluate the association between furosemide and methotrexate co-administration and SJS/TEN using the FAERS database.Signal detection results from realworld pharmacovigilance data suggest an increased risk of SJS/TEN with methotrexate when furosemide is combined with methotrexate.Clinicians and healthcare professionals, as well as patients, should be more aware of this risk and monitor for its presence when using methotrexate along with furosemide, including symptoms such as fever, eye inflammation, mouth ulcers, and erythema and blistering of the skin, and prompt treatment may help reduce the number as well as the severity of adverse events.Finally, it is recommended that the labeling of methotrexate should clearly state the drug interactions between methotrexate and furosemide, consistent with the labeling of furosemide.

Table 1 .
Characteristics of adverse events reported with the combination of methotrexate and furosemide.
a Indications for methotrexate when used in combination with furosemide; SJS, stevens-Johnson syndrome; TEN, toxic epidermal necrolysis.

Table 2 .
Signal detection results of 4 methods based on the whole dataset.

Table 3 .
Signal detection results of the four methods based on data subsets.

Table 4 .
Sensitivity analysis of the combination of methotrexate and furosemide based on different backgrounds.
RORPRR MHRA IC ADR N The lower limit of the 95% CI The lower limit of the 95% CI PRR; χ2The lower limit of the 95% CI Sensitivity analysis of the entire dataset Not combined with FUR-MTX *signal detected; ADR, adverse drug reaction; TEN, toxic epidermal necrolysis; MTX, methotrexate; FUR, furosemide; CI, confidence interval; ROR, reporting odds ratio; PRR, proportional reporting ratio; MHRA, the Medications and Health Care Products Regulatory Agency; IC, information component.