Changes in Urinary Uric Acid Concentration after Dotinurad Administration to Patients with Hyperuricemia: A Post Hoc Analysis of Two Clinical Trials in Japan

Dotinurad has been approved in Japan as a selective urate reabsorption inhibitor for the treatment of gout and hyperuricemia. The relationship between uric acid crystallization and the use of uricosuric drugs is widely acknowledged; however, the relationship between changes in urinary uric acid concentration and urine pH or volume has not been sufficiently analyzed. Therefore, we investigated the changes in urinary uric acid concentration following dotinurad administration as well as the relationship between urine pH or volume and urinary uric acid concentration. This post hoc analysis used data from 2 clinical trials that included 12 and 26 patients with hyperuricemia who received dotinurad treatment (for 7 days on an inpatient basis and 14 weeks on an outpatient basis, respectively). The urinary uric acid concentration transiently increased in the early stages of dotinurad use and when its dose was increased, but decreased over time. No uric acid concentrations exceeded the soluble limit at any urine pH. An inverse correlation was observed between urine volume and urinary uric acid concentration. This study highlights the significance of adequately managing urinary uric acid concentrations by increasing urine volume and alkalinizing urine to prevent uric acid crystallization during dotinurad administration.

Two types of medications are used for the treatment of gout and hyperuricemia: xanthine oxidase inhibitors, which target uric acid production and prevent its overproduction; and uricosuric agents (UAs), which increase its renal excretion.However, safer and more effective UAs are required due to concerns regarding the effectiveness and safety of these medicines. 1otinurad is a novel selective urate reabsorption inhibitor for the treatment of hyperuricemia that was developed to address the safety concerns associated with conventional UAs, while maintaining strong serum uric acid-lowering effects. 2 Although dotinurad potently inhibits the uptake of urate (halfmaximal inhibitory concentration, 0.0372 μM) by urate transporter 1-overexpressing Madin-Darby Canine kidney II cells, 1,3 it minimally inhibits adenosine triphosphate-binding cassette subfamily G member 2 and organic anion transporters 1, and 3. 4 Additionally, the noninferiority of dotinurad to benzbromarone 5 and febuxostat 6 has been confirmed in clinical trials.
In the late Phase II and long-term administration studies, most patients who were administered a maintenance dose of dotinurad (2 or 4 mg) achieved the target serum urate level of 6 mg/dL or less. 4Furthermore, the estimated glomerular filtration rate was not affected by dotinurad in a long-term Phase III study, and adverse drug reactions of renal calculi, found in 1.5% of the patients, were not deemed serious or did not require treatment. 7These results indicate that there are no safety concerns associated with the long-term use of dotinurad. 4,7yperuricemia, a risk factor for gout, is caused by increased production or reduced renal excretion of uric acid. 8Recent studies have shown that hyperuricemia is closely associated with lifestyle-associated diseases, such as chronic kidney disease, hypertension, and diabetes mellitus. 2Therefore, the treatment of hyperuricemia is important for gout and various other diseases.Hyperuricemia increases the urinary uric acid concentration and is frequently associated with urolithiasis, which is worsened by low urinary pH. 9 Urolithiasis causes severe pain and symptoms associated with urinary tract obstruction due to urinary stones.A study conducted in 2015 reported that among patients with urolithiasis, calcium oxalate/phosphate stones were found in 82.8% of males and 77.1% of women, while uric acid stones were found in only 3.4% of men and 1.2% of women. 10The occurrence of urinary stones has been linked to one's lifestyle such as diet. 11ric acid stones tend to form more easily in acidic urine 12 ; therefore, alkalinization of urine is essential to prevent uric acid stone formation in patients with hyperuricemia or gout.Furthermore, increasing urine volume through fluid intake is considered effective for preventing uric acid stone formation. 13n analysis of clinical trials of dotinurad revealed that the amount of urinary uric acid excretion transiently increased after dotinurad administration. 14,15owever, it is unclear whether the use of dotinurad causes an increase in uric acid stones.Therefore, the potential risk of uric acid stone formation should be carefully monitored. 16hanges in urinary uric acid concentration, urine pH, and urine volume after dotinurad administration have not been analyzed in clinical trials. 14,15Additionally, no clinical reports are available on the preventive control of uric acid stone formation in patients treated with dotinurad.
Therefore, we conducted a post hoc analysis of 2 clinical trials 14,15 to evaluate the following after administration of dotinurad: (1) the change in urinary uric acid concentration, (2) the distribution of urinary uric acid concentration according to urine pH, and (3) the relationship between urinary uric acid con-centration and urine volume.Clarifying these data enables the evaluation of the possibility of uric acid crystallization in urine and the assessment of the changes in uric acid kinetics caused by dotinurad administration.

Study Design and Treatment
This is a post hoc analysis based on data on urinary uric acid concentrations from an open-label Phase II study (FYU-981-008: short-term study) 14 and a multicenter, open-label, forced-titration Phase I study (FYU-981-013: long-term study) 15 conducted in Japan from 2016 to 2018 (Table S1).
The short-term (FYU-981-008) and long-term (FYU-981-013) studies were conducted in accordance with the ethical principles of the Declaration of Helsinki and in compliance with Good Clinical Practice.The protocols were approved by the institutional review board of each medical site.Two clinical trials were registered in the ClinicalTrials.govdatabase (ClinicalTrials.govIdentifier: NCT02837198 and NCT03375632).Written informed consent was obtained from all patients to publish this study.
In the short-term study, patients received dotinurad 1 mg once daily for 7 days on an inpatient basis.In the long-term study, patients received dotinurad for 14 weeks on an outpatient basis in escalating doses: a starting dose of 0.5 mg/day once daily for 2 weeks followed by 1, 2, and 4 mg/day every 4 weeks.

Patients
The main inclusion criteria of the short-term study were patients with serum uric acid levels of 7.0 mg/dL or greater on screening and on Day −1, while those of the long-term study were patients with gout who had a serum uric acid levels of 7.0 mg/dL or greater (patients with a history of gout or with gouty tophi), or patients with hyperuricemia who had a serum uric acid levels of 8.0 mg/dL or greater (patients with hypertension, diabetes mellitus, or metabolic syndrome under treatment or follow-up) or 9.0 mg/dL or greater (patients without the aforementioned complications); men aged 20 years or older when informed consent was given and patients who were classified as the uric acid overproduction or underexcretion on screening and on Day −1 or on the first day of the run-in period were included in both clinical trials.
Blood and 60-minute urine samples were collected on the first day of the run-in period.Afterward, urinary extraction of uric acid (E UA [mg/kg/h]) and uric acid clearance (C UA [mL/min/1.73m 2 ]) were determined to identify the patients' disease type according to the following criteria: (1) overproduction type, E UA greater than 0.51 and C UA 7.3 or greater; (2) underexcretion type, E UA less than 0.48 or C UA less than 7.3; and (3) combined type, E UA greater than 0.51 and C UA less than 7.3. 15he main exclusion criteria of the short-term study were patients with a history of gouty arthritis or gouty tophi within 1 year before the day informed consent was given or patients who had gouty arthritis during the period from the day informed consent was given to immediately before the initial dotinurad administration, while that in the long-term study were patients with unresolved gouty arthritis by 14 days before the end of the run-in period.Additionally, in both clinical trials, patients with an HbA 1c of 8.4% or greater, serum aspartate aminotransferase and/or alanine aminotransferase of 100 IU/L or greater, estimated glomerular filtration rate less than 30 mL/min/1.73m 2 , and systolic blood pressure of 180 mm Hg or greater or diastolic blood pressure of 110 mm Hg or greater on screening (and on Day −1) or on the first day of the run-in period were excluded.

Study End Points
Based on the urinary data obtained from each clinical trial, change in urinary uric acid concentration, distribution of urinary uric acid concentration according to urine pH, and relationship between urinary uric acid concentration and urine volume were analyzed and graphically shown.The 24-hour urine collection schedules are shown in Figures S1 and S2.

Statistical Analyses
Statistical testing and interval estimation were not performed.The missing values were not imputed, and outliers were included.SAS Version 9.4 (SAS Institute Inc.) was used for the analyses.
The change in urinary uric acid concentration was compared with the soluble limit at pH 6.0, with 95 mg/dL as a reference value.The distribution of urinary uric acid concentration and urine pH was quoted from the solubility curve (Figure S3), and the relationship between urinary uric acid concentration and urine pH was plotted.
The saturated solubility of uric acid in urine is higher than that in serum. 17,18Theoretically, the solubility of urinary uric acid increases with an increase in pH.However, the solubility varies due to urine pH and formation of various types of salts with coexisting substances in urine. 18Consequently, the soluble limit for urinary uric acid was defined as the upper limit of metastable supersaturation (approximately 55, 95, 145, and 135 mg/dL at urine pH 5.0, 6.0, 7.0, and 8.0, respectively; Figure S3).A wide range of saturation zones was attributed to the different solubilities of uric acids in the individual samples.

Patient Characteristics
The patient flowchart is shown in Figures S4 and S5.Overall, 12 of 24 enrolled patients from the short-term study (6 patients each with uric acid overproduction and underexcretion) and all 26 enrolled patients from the long-term study (13 patients each with uric acid overproduction and underexcretion) were included in the analyses.In the short-term study, 12 patients were excluded; 6 were excluded because they had been enrolled before the protocol amendment (the length of hospital stay was shortened from 4 days to 1 day) and had not undergone the same study procedures as those for the patients enrolled after the amendment, and 6 were excluded due to the concomitant use of topiroxostat, which may have affected the production of uric acid.
All patients included in the analysis received at least 1 dose of dotinurad.All of the 12 patients (6 patients each with uric acid overproduction and underexcretion) in the short-term study and 13 (8 with overproduction and 5 with underexcretion of uric acid) in the long-term study completed the trials.The remaining 13 patients in the long-term study were withdrawn because urinary uric acid excretion 1 day after the initial dose or dose escalation of dotinurad increased by 15% or more than that on the day of starting treatment at each dose.
The baseline characteristics of patients are shown in Tables 1 and 2.

Change in Urinary Uric Acid Concentration
Changes in urinary uric acid concentrations are shown in Figure 1.In the short-term study, the median urinary uric acid concentrations of patients with uric acid overproduction and underexcretion at baseline In the long-term study, the median urinary uric acid concentrations at baseline were 43.3 and 34.2 mg/dL in patients with uric acid overproduction and underexcretion, respectively.After starting treatment, the median uric acid levels ranged from 39.7 to 70.3 mg/dL and from 38.5 to 50.8 mg/dL in patients with uric acid overproduction and underexcretion, respectively.Peak uric acid levels were achieved on the first day of dotinurad administration at each dose and decreased on the second day.Individually, uric acid levels increased to 95 mg/dL or more in 3 patients (1 time point in each patient) (Figure S6); all 3 patients had uric acid overproduction.The uric acid levels were 99.0 mg/dL at the end of treatment in the first patient (A1), 95.7 mg/dL at 0.5 mg/day on Day 2 in the second patient (A2), and 95.0 mg/dL at 1 mg/day on the day before administration in the third patient (A3).

Urinary Uric Acid Concentration According to Urine pH
Shimizu et al estimated the solubility of uric acid in human urine by dissolving anhydrous uric acid or monosodium urate in human urine samples. 19igure S3 is a superimposed schematic diagram of the solubility of anhydrous uric acid and monosodium urate in urine from their study.In this diagram, the "oversaturation" zone was defined as the concentration in which neither anhydrous uric acid nor monosodium urate was dissolved.The "saturation" zone was defined as the concentration in which both anhydrous uric acid and monosodium urate was dissolved.The "metastable supersaturation" zone was defined as the concentration in which either anhydrous uric acid or monosodium urate was dissolved.It is presumed that crystallization occurred more easily in the metastable supersaturation zone than in the supersaturation zone.Data from their in vitro study were compared with the urine uric acid concentrations after dotinurad administration (Figure 2).In Figure 2, the upper limit of the uric acid solubility in urine, estimated based on the in vitro study, is indicated by the red line and the lower limit by the black line.None of patients had urinary uric acid concentration that exceeded the relevant soluble limit.In the short-term study, no concentration reached the level of metastable supersaturation at any urine pH.However, in the long-term study, uric acid concentrations reached the level of metastable supersaturation at 7 of 48 (14.6%) time points in 5 of 13 (38.5%)patients with uric acid overproduction and at 2 of 42 (4.8%)time points in 2 of 13 (15.4%)patients with uric acid underexcretion; the percentage was higher in patients with uric acid overproduction.Meanwhile, based on the urine pH, urinary uric acid concentrations reached the level of metastable supersaturation at 5 of 46 (10.9%) time points in 5 of 19 (26.3%) patients with a pH of less than 6.5 and at 4 of 44 (9.1%) time points in 3 of 18 (16.7%)patients with a pH of 6.5 or greater; there was no notable difference between the groups.No adverse events associated with urinary stones were reported.

Relationship Between Urinary Uric Acid Concentration and Urine Volume
The relationship between the urinary uric acid concentration and urine volume is shown in Figure 3.Among inpatients in the short-term study, there were no differences in urine volume between hyperuricemia types (uric acid overproduction: ranged from 1528 to 3964 mL [median, 2672 mL] vs uric acid underexcretion: ranged from 1422 to 3836 mL [median, 2685 mL]).On the other hand, among outpatients in the long-term study, urine volume was lower in patients with uric acid overproduction (ranged from 650 to 3350 mL [median, 1500 mL]) than in those with uric acid underexcretion (ranged from 550 to 6450 mL [median, 1750 mL]).Moreover, patients with uric acid overproduction tended to have higher urinary uric acid concentrations compared with those with uric acid underexcretion.
Figure 2. Individual data on urinary uric acid concentration versus urine pH in the short-and long-term studies.The upper limit of the uric acid solubility in urine, estimated on the basis of the in vitro study, is indicated by the red line and the lower limit by the black line.The "metastable supersaturation" zone was defined as the concentration at which either anhydrous uric acid or monosodium urate was dissolved.It is presumed that crystallization occurred more easily in this zone than in the supersaturation zone.The soluble limits between pH 7.0 and 8.0 are estimated values and are shown in dashed lines.

Discussion
The treatment of hyperuricemia is important for gout and various other diseases.Generally, hyperuricemia is classified into the overproduction and underexcretion type based on the amount of renal uric acid excretion.Because the prevalence of the underexcretion type is evidently higher (approximately 60% or higher) than that of the overproduction type in patients with Japanese ethnic extraction, the importance of choosing drugs with selective urate reabsorption inhibitor property will be inevitably and naturally expanding in the future.However, one must be alert to the potential fear of enhancing uric acid stone formation induced by UAs. 16herefore, we conducted a post hoc analysis to evaluate the change in urinary uric acid concentration, the distribution of urinary uric acid concentration according to urine pH with in vitro data, 19 and the relationship between urinary uric acid concentration and urine volume.
In both patients with uric acid overproduction and underexcretion, peak urinary uric acid concentrations were achieved on the first day of dotinurad administration at each dose and decreased on the second day.In the short-term study, the urinary uric acid concentration in all patients increased on Day 1, gradually decreased to near-baseline levels, and were maintained until Day 7. In the long-term study, the urinary uric acid concentration at baseline was higher and had a larger variation than those in the short-term study.This difference may be partly due to the long-term study being conducted on an outpatient basis wherein data may have been influenced by lifestyle habits such as diet and fluid intake.
In patients with a urine pH of 5.5 or 6.0, the urinary uric acid concentration did not exceed the soluble limit (Figure 2).However, considering the circadian and daily variations in urine pH, the concentration might have exceeded the soluble limit if the urine pH was acidic.We assume a solubility of total uric acid curve like Figure S3, based on in vitro experimental results. 19olubility of uric acid is pH dependent, and the superiority of uric acid and urate switches around the acid dissociation constant (pK a1 ).In human body fluids at pH 7.4, almost all of the uric acid is present in the form of monosodium urate.In urine, the solubility of total uric acid increases following the solubility curve of uric acid up to approximately pH 6.0, and slowly decreases following the curve of monosodium urate in most cases above pK a1 .However, monosodium urate tends to remain in the metastable supersaturated state, and it does not crystallize easily.Therefore, a wide metastable supersaturation zone may be formed above the saturation line.Accordingly, clinical attention needs only in acidic urine with pK a1 or less.
Based on these results, the risk of uric acid crystallization was high in patients with uric acid overproduction whose urinary uric acid concentration was elevated and in patients with a urine pH of 6.0 or less.Additionally, a diet high in animal proteins leads to higher urinary uric acid excretion and lower urine pH than fruit-or vegetable-based diets. 20To reduce the risk of uric acid crystallization in dotinurad-treated patients with the above-mentioned risk factors, it is necessary to improve their diet, increase their water intake, and prescribe urinary alkalinizers to increase the urine pH.Generally, the pH should be maintained in the range of 6.5-7.0 for dissolving uric acid calculi. 21It is also important to establish measures to prevent uric acid crystallization, especially in the early stages of dotinurad treatment or when increasing the dose of dotinurad wherein urinary uric acid concentrations transiently increase.
In theory, fluid intake reduces the risk of urinary stone formation in patients with gout or hyperuricemia.However, few studies have discussed the relationship between urine volume and uric acid concentration, and there is insufficient evidence to support the effectiveness of fluid intake in preventing uric acid crystallization.
In this study, an inverse correlation between urine volume and uric acid concentration was observed.Urine volume tended to be lower, and urinary uric acid concentration tended to be higher, in outpatients with uric acid overproduction than in those with uric acid underexcretion.We considered that low fluid intake was one reason for the smaller urine volume.Although stronger evidence from clinical trials is required, increased fluid intake can be used as a strategy to prevent primary urolithiasis.A target urine volume of 2000 mL/day or greater, achieved by increasing fluid intake, can be recommended to prevent the recurrence of urolithiasis. 22When this target urine volume is taken into consideration, the urinary uric acid concentration ranged from 28.1 to 99.0 mg/dL (median, 50.7 mg/dL) in patients with a urine volume of less than 2000 mL and from 9.0 to 60.0 mL/dL (median, 31.4 mg/dL) in patients with a urine volume of 2000 mL or greater.The urinary uric acid concentration greatly decreased with an increase in urine volume even after the administration of dotinurad, suggesting that fluid intake is important for lowering the risk of uric acid crystallization.
This analysis has the following limitations: (1) These results may be different from those in real-world clinical practice because data were obtained from patients who were hospitalized prior to dotinurad administration and those who were treated with dotinurad in a manner different from the approved regimen; (2) statistical testing was not possible because of the small sample size; and (3) the accurate solubility of urinary uric acid could not be estimated because solubility of uric acid in renal tubule is currently under discussion, 17,23 there is no established standard of solubility, and necessary parameters were not collected sufficiently in the clinical trials.There are various cations such as Na + and K + in urine, which form salts with urate, and the solubility varies depending on the cation.In addition, macromolecules that affect the dissolution of uric acid are also known, 24 and it is thought that interindividual differences in these concentrations affect the stability of uric acid in the metastable supersaturation and supersaturated zone.(4) Insufficient concentration data of substances that affect the solubility of uric acid in urine.Ogawa reported circadian variations in the relative supersaturation of uric acid, sodium urate, and ammonium urate. 25As the trials did not provide sufficient data, further studies are needed to analyze the relative supersaturation, which may be useful for examining the pharmacokinetics of urinary uric acid in patients treated with dotinurad.

Conclusions
This study indicates the importance of adequate control of urinary uric acid concentrations by increasing urine volume and alkalinizing urine to prevent uric acid crystallization during treatment with dotinurad.We hope that real-world data on the urine of dotinurad-treated patients will be accumulated, which will facilitate further discussions on the standards and methods for the preventive control of uric acid crystallization that reflect actual clinical situations.

Figure 1 .
Figure 1.Individual data on urinary uric acid concentration in inpatients from the short-term study (A) and in outpatients from the long-term study (B).Bars indicate median values.

Figure 3 .
Figure 3. Scatter plot of urinary uric acid concentration versus urine volume in the short-and long-term studies.

Table 1 .
Baseline Characteristics of the Patients in the Short-= 194 × serum creatinine −1.094 × age −0.287 .SAS Version 9.2 was used for statistical analysis of the short-term study.This table was modified from the table in Okui et al. 14 with permission.

Table 2 .
Baseline Characteristics of the Patients in the Long-Term Study , body mass index; eGFR, estimated glomerular filtration rate; SD, standard deviation.SAS Version 9.3 was used for statistical analysis of the long-term study.This table was modified from the table in Hosoya et al. 15 with permission.
BMIa eGFR (mL/min/1.73m 2 ) = 194 × serum creatinine −1.094 × age −0.287 .b Definition of drinking habit: consumption of alcohol for >3 days/week and consumption of >500 mL of beer or >60 mL of whiskey/day.were 24.7 and 19.2 mg/dL, which increased to 45.7 and 49.0 mg/dL on Day 1, respectively.Subsequently, uric acid concentrations gradually decreased to nearbaseline levels starting on Day 4 and were maintained until Day 7.Moreover, the concentration was lower than 95 mg/dL in all patients.