Vascular calcification is associated with increased morbidity and mortality in dialysis patients (1,2) and new therapeutic strategies are needed to tackle this challenge. Sodium thiosulfate (STS, Na2S2O3·5H2O, MW 248 g) is an old drug used for decades as an antidote against cyanide poisoning (3), because STS supplements the cyanide-detoxifying enzyme rhodanese (4,5), which is part of the endogenous thiosulfate (TS) synthesis pathway (6). Recently, STS has been prescribed for the treatment of soft-tissue calcifications in patients with calciphylaxis (7–12). Furthermore, systematic studies in rats (13) and humans (14) have investigated the effect of STS on aortic and coronary artery calcifications. These studies support the notion of vascular calcification, slowing or even preventing properties of STS. The underlying mechanism for the efficacy of STS is not clear. Apparently, the beneficial effects of STS are mediated by a combination of calcium-solubilizing, antioxidative, acidosis-inducing, and possibly other not-yet-identified mechanisms (13,15–17). Given the favorable tolerability even in long-term use along with its low cost, STS has the potential to become a more widely applied therapeutic agent for patients predisposed to vascular calcifications.
Before new xenobiotics can be applied, their pharmacokinetics have to be established in a restricted number of patients from the target population. Surprisingly, this was not done in the case for STS. One can only speculate why no formal pharmacokinetic studies have been previously performed. First, decades ago when STS was first used (3,18) the legal prerequisites for new drugs were not as stringent as those today. Second, when STS is given as a cyanide antidote, the application is of short duration and occurs in patients with normal renal function.
With the new indications and the prolonged use of STS, the pharmacokinetics have to be investigated for rational dosing, potential pharmacokinetic-pharmacodynamic modeling, and safety reasons. Therefore, we investigated the kinetics of STS in dialysis patients on- and off-hemodialysis and in healthy volunteers (HV) and present the numbers derived from classic pharmacokinetic calculations and from a newly developed STS population pharmacokinetic model.
Materials and Methods
Patients, HV, and Study Protocol
All patients and HV (Table 1, Table 2) were studied at the Department of Nephrology/Hypertension of the University-Hospital Bern, Switzerland. The study was approved by the ethics committee and registered (NCT01008631). Ten patients and nine HV were enrolled after giving written consent. Inclusion criteria for patients were as follows: Age 18 to 80 years and dialysis vintage >3 months. Hemodialysis was performed 3 times per week through an arteriovenous fistula with high-flux membranes using the Fresenius-5008 device. Dialysate flow rate was set at 600 ml/min.
The protocol for patients involved two single intravenous doses of 8 g (32,250 μmol) of STS (Köhler-Chemie, Bensheim, Germany), diluted in 50 ml of 0.9% NaCl and infused over 8 minutes. A detailed protocol overview is given in Figure 1.
Similarly, HV between 18 and 80 years without regular medications and a normal renal function according to the Modification of Diet in Renal Disease (MDRD) formula (GFR >70 ml/min per 1.72 m2) were included. During the study, individual creatinine clearances were calculated from the 24-hour urine collection. Intravenous and oral pharmacokinetics were investigated with a washout period of 7 days between the two applications. Intravenous STS (8 g) was infused as described above for dialysis patients. Blood samples were collected at baseline and 15, 30, 60, and 180 minutes after the end of the STS infusion. Urine was collected for 24 hours.
For oral application, 5 g (20,000 μmol) of the intravenous solution was diluted in 100 ml of water and rapidly ingested. Sampling was performed as described for the intravenous application. All samples were processed immediately and frozen at −80°C.
Determination of Thiosulfate Concentrations in Blood, Urine, and Dialysate
Thiosulfate (TS) was determined by a specific HPLC method as described previously (19,20). To prevent the premature clotting of the HPLC column by lipids, we introduced a delipidation step. Thereby, the life span of the column was enhanced for several hundred samples.
Briefly, serum samples (150 μl) were delipidized with dichloromethane by centrifugation. Twenty-five microliters of the supernatant was derivatized with 5 μl of 46 mM monobromobimane, 25 μl of acetonitrile, and 25 μl of 160 mM HEPES/16 mM EDTA pH 8 buffer (Invitrogen, Carlsbad, CA) for 30 minutes in the dark. Derivatization of thiol groups was stopped by 50 μl of 65 mM methanosulfonic acid (Fluka, Buchs, Switzerland) and proteins were removed by recentrifugation. Urine and dialysate samples were processed identically but without the delipidation step.
HPLC was performed using a Waters-2695 module, coupled to an RF10-AXL (Shimadzu, Kyoto, Japan) fluorescence detector (excitation 380 nm, emission 480 nm) and a Merck-LiChro-CART-125-4, LiChrospher-60, RP-select-B (5 μm) reverse-phase column. PIPES (10 mM, pH 6.6) and methanol (gradient) were used as eluant at a flow rate of 1 ml/min. Area under the curves (AUCs) of the TS peaks were integrated by the software of the Waters-2695 device.
Using this method, we obtained linearity from 2 to 100 μM and a maximal intraday and interday variability of 8.6% and 9.3%, respectively. The lower detection limit was approximately 2 μM.
Pharmacokinetic and Statistical Analyses
Individual estimates of pharmacokinetic parameters were calculated by a noncompartmental approach (21,22). The total body clearance was calculated by dividing the intravenous dose by the corresponding AUC, the renal clearance by dividing the amount of TS recovered in urine by the AUC, and the nonrenal clearance by subtracting the renal from the total body clearance (23). For population pharmacokinetic modeling, the serum concentration time data and TS amounts recovered in urine and dialysate of dialysis patients and from HV after intravenous STS application were pooled and analyzed by a nonlinear mixed-effects modeling approach using NONMEM-6.2 (GloboMax LLC, Hannover, Germany), double precision, with the g95-FORTRAN compiler on a Windows-NT-6.0 platform. The NONMEM runs were executed using PsN-2.3.0 (24). Serum TS concentrations were fitted to a one-compartment model using user self-supplied differential equations (ADVAN6). Interindividual variability of estimated parameters was best described by an exponential model whereas the residual error was implemented with an intercept-slope residual variability model. Data were analyzed using the first-order conditional estimation with INTERACTION. Statistical comparison of nested models was based on a χ2-test of the difference in the objective function (OFV). A decrease in OFV of 3.84 units (P < 0.05) per 1 additional degree of freedom (df) was considered significant. Goodness of fit and visual predictive checks were performed using PsN-2.3.0 and XPose-4 (24,25). Posterior empirical Bayesian estimates were further used for statistical inference if calculated shrinkage was lower than 20%.
The R.2.10.1 (R-Development Core-Team ) was used to compute AUCs based on the linear trapezoidal rule. GraphPad5 (GraphPad, La Jolla, CA) was used for statistics and figures.
STS Pharmacokinetics in Hemodialysis Patients
STS was given intravenously immediately after the first dialysis session of the week. Endogenous baseline TS serum concentrations before and after this dialysis session were comparable (6.6 ± 2.4 and 6.2 ± 2.8 μmol/L). Serum TS concentrations after the single dose given off-dialysis are shown in Figure 2A and individual estimates of pharmacokinetic parameters obtained by noncompartmental analyses in Table 3. Mean total body clearance was 2.04 ± 0.72 ml/min per kg. This value largely reflects the nonrenal clearance because minimal residual renal function (GFR <6 ml/min) was present in only two patients.
Assuming a constant endogenous production (G) of TS, G can be estimated from the steady-state concentration and the total body clearance. G was estimated to be 1.05 μmol/min (14.6 ± 6.1 nmol/min per kg), a value used for further calculations.
With use of the population pharmacokinetics approach described above, simulated time-serum concentration profiles of a single 25-g STS dose (which represents the usually applied dose in the literature [7–10]) in dialysis patients are shown in Figure 3A.
Mean endogenous baseline TS serum concentrations before the mid-week session were 7.6 ± 3.0 μmol/L and therefore comparable with the values obtained before and after the first dialysis session of the week, indicating that the STS applied after the first dialysis had been cleared. When all endogenous TS concentrations were summed up, the mean value was 7.1 ± 2.7 μmol/L. TS serum concentrations during dialysis are shown in Figure 2B. Thirty minutes after starting dialysis, simultaneous arterial (TSa) and venous (TSv) samples from a pre- and postdialyzer tubing port as well as dialysate (TSd) samples were obtained from the dialysis circuit. TSa, TSv, and TSd were 748 ± 191, 309 ± 90, and 236 ± 77 μmol/L, respectively. For the calculation of the amount of TS lost from serum and recovered in the dialysate the blood flow (Qb), ultrafiltration (UF), dialysate flow (Qd), and partitioning into the red blood cells have to be considered. Because no rebound of STS at the end of dialysis was observed, we used a one-compartment model without unequal partitioning and derived the amount of STS recovered from the following equation: TSd*Qd = TSa*Qb − TSv*(Qb − UF*t). The ratio between STS recovered in the dialysate and STS filtered from the blood was 1.00 ± 0.41 (median 0.93, range 0.28 to 1.7).
The serum concentrations at the end of the dialysis treatment and 30 minutes later were comparable (24.5 ± 8.7 versus 20.5 ± 8.4 μmol/L, NS), suggesting absence of a pronounced TS rebound and possibly ongoing nonrenal clearance.
STS Pharmacokinetics in HV
STS Kinetics after Intravenous Application.
STS (8 g) was given intravenously to nine HV. Mean endogenous baseline TS serum concentrations were 5.5 ± 1.8 μmol/L and not significantly different from those of dialysis patients. The AUC0→∝ was significantly smaller in HV than in patients off-dialysis (P < 0.001; Table 2, Table 3, Figure 2C). The renal clearance (1.86 ± 0.45 ml/min per kg) accounted for about 50% of the total body clearance (4.11 ± 0.77 ml/min per kg; Table 2). Urinary recovery of the intravenous TS dose of 32,250 μmol was 43% (13,869 ± 1847 μmol).
The simulated expected time-concentration profile after an assumed single dose of 25 g in HV is displayed in Figure 3B.
STS Kinetics after Oral Application.
Each HV ingested a single STS dose of 5 g. The time-serum concentration profile is shown in Figure 2D. The peak TS concentrations were variable and only slightly higher than the usually measured steady-state endogenous serum concentrations. Within 180 minutes after STS intake, peak TS serum concentrations (Cmax) were observed in only 5 of 9 HV. Thus, the calculation of the bioavailability by dividing the oral AUCs by the intravenous AUCs was prone to yield inaccurate results. Therefore, bioavailability was estimated by two alternative methods, both taking into account the different doses of STS given orally and intravenously. First, the 24-hour urinary TS excretion after oral dosing was divided by the excretion after intravenous dosing. This method yielded a low bioavailability ranging from 0.8% to 26% (median 7.6%; Table 4). Second, with use of the population pharmacokinetic approach (see below), a similar bioavailability ranging from 2.3% to 11.2% (median 6.6%; Table 5) was found. The mean urinary recovery of oral STS was 4%. A summary of all pharmacokinetic estimates obtained from noncompartmental analysis (21,22) is given in Table 4.
Population Pharmacokinetic Model
All intravenous data from dialysis patients during and between dialyzes as well as those from HV were combined into one population pharmacokinetics model, reflecting the mean of all participants. Because no rebound was observed 30 minutes after dialysis sessions, a one-compartment distribution of STS was assumed. G was set at 1.05 μmol/min. Mean population estimates of the final model are given in Table 5. The validity of the model is depicted in Figure 4. In a final step, all model parameters were fixed at their estimates, and as indicated above, a median bioavailability of 6.6% was estimated by adding the oral data from HV.
The present investigation revealed a low and highly variable oral bioavailability of STS in HV (0.8 to 26%). We therefore decided not to expose dialysis patients to oral STS until a suitable galenic formulation of STS with an enhanced bioavailability is available. The mechanism for the low bioavailability is open to speculation. It might be at least partially explained by the STS degradation in the acidic environment of the stomach (Na2S2O3 + 2HCl → 2NaCl + H2O + S + SO2). Alternatively, the low and variable bioavailability may be due to STS degradation by intestinal bacteria (26) and/or different expression levels of a putative TS transporter in the gut mucosa, which might exist in analogy to a sulfate-inhibitable TS transporter in the dog renal tubule (27). Interestingly, despite the very low oral bioavailability of STS, the successful prevention of renal stones in humans (28), and rats (29), and of the progression of calciphylaxis (30) and nephrocalcinosis (31) has been reported by comparable oral STS doses as those used in our investigation.
More than 50 years ago, a good correlation between renal TS and inulin or creatinine clearance was shown (32,33). This was explained by the assumption that TS mainly undergoes glomerular filtration without quantitative tubular secretion or reabsorption. In our HV the mean creatinine clearance was in the same range (101 ± 28 ml/min) as the renal clearance of TS. Despite the excellent correlation between renal clearance of TS and the values of the GFR assessed by using creatinine, endogenous TS cannot be used as an estimate of the GFR for two reasons. First, the endogenous TS generation is variable (Table 4) and depends among other factors on the diet; and second, renal and nonrenal mechanisms contribute about equally to the elimination of STS in normal HV (Table 2). The nonrenal clearance varies even between HV (Table 2, Table 4). Therefore, the GFR determined by Newman and Gilman (32,33) required the exogenous administration of STS and the simultaneous measurement of TS in serum and urine.
The nonrenal elimination of TS accounts for about 50% in HV, an observation consistent with previous investigations in humans and dogs (4,34). When the nonrenal clearance of other xenobiotics was compared between healthy subjects and dialysis patients, the values observed in dialysis patients were either higher, lower, or identical depending on the agent and the metabolic pathways analyzed (35–37). Here, we showed for the first time that the nonrenal clearance of TS is similar in hemodialysis patients and in HV (Table 4). The vast proportion of TS cleared by nonrenal mechanisms appears to be metabolized to sulfate (4,29,38) possibly predominantly in the liver but also in other tissues (39). TS is considered to be the principal, rapidly disappearing precursor of sulfate in mammalians (40). Thus, our study suggests that the TS metabolism is unaltered in patients with chronic renal failure. Surprisingly, however, when we measured endogenous serum concentrations of TS during dialysis treatment without administration of exogenous STS, the TS concentrations remained stable and did not decline as expected during dialysis treatment, although TS is well dialyzable (Table 3). Patients did not consume TS-containing food during dialysis treatment. Therefore, the stable TS concentrations during dialysis are difficult to interpret. One can only speculate whether the TS synthesis is upregulated during dialysis or the removal of TS by the dialysis is negligible at low concentrations.
On the basis of our population kinetic modeling, we can predict the concentration versus time curves in patients and HV and the model accurately reflects the measured values (Figure 4). The concentrations predicted from a given dose are potentially useful for targeting a final or intermediate therapeutic endpoint (41). The therapeutic endpoint of STS with respect to calciphylaxis or vascular calcification is the disappearance of calcium deposited in soft tissues and the vasculature. TS serum concentrations between 5 and 10 mmol/L have been shown to transiently lower ionized serum calcium in vitro and in vivo (13). Such high concentrations are achieved only for <30 minutes when the usual dose of 25 g of STS is given to patients off-dialysis (Figure 3A). Thus, it remains unknown whether the presumed mechanism of calcium solubilization by chelation is therapeutically relevant (13). As a second, alternative mechanism the induction of a high anion-gap acidosis (8,13) by STS, which directly inhibits the precipitation of calcium and phosphate, has been proposed (16). However, this mechanism has been questioned by the observation that vascular calcifications can be prevented by STS even in the presence of alkaline serum values in rats (13). As a third mechanism for the efficacy of STS, an anti-oxidative effect improving endothelial dysfunction and promoting vasodilatation has been discussed (42). The biologically relevant concentrations of TS for this effect are unknown at the present time. A fourth mechanism may be related to STS-induced changes in serum inhibitors of vascular calcification (43,44), as described for the matrix-Gla-protein in previous studies with STS-treated rats (13). However, the concentrations required for this effect are unknown, too. Thus, for future rational dosing of STS, concentration-effect studies are mandatory.
In conclusion, the present investigation established a kinetic model for predicting TS concentrations after intravenous dosing of STS. Oral application cannot be recommended at the present time, given the low and variable bioavailability. The mechanisms of this low bioavailability have to be clarified for the development of strategies to improve the bioavailability of oral STS. The hemodialysis clearance of STS is high. As a corollary, if the main mechanism of action of STS is the removal of calcium, then STS should be given just before dialysis treatment is started. If the putative mechanism requires prolonged presence of STS in the tissues, then dosing off-dialysis might rather be warranted.
This study was supported by a Baxter Company Clinical Evidence Council (CEC) grant (#08CEC2EU001) to A Pasch, S Farese and D Uehlinger and by the Alfred and Erika Br-Spycher Foundation to S. Farese and A Pasch.
Published online ahead of print. Publication date available at www.cjasn.org.
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