Efficacy and Pharmacokinetics of Once Daily Prolonged Release Tacrolimus in Calcineurin Inhibitor Dependent Steroid-Sensitive Nephrotic Syndrome: A Pilot Study

Patients

We screened 5-18 year-old patients with idiopathic steroid-sensitive nephrotic syndrome and a difficult-to-treat CNI-dependent course, defined as follows: (i) frequent relapses or steroid dependence; (ii) failure of two or more immunosuppressive agents; and (iii) stable remission (sustained remission or infrequent relapses) during therapy with cyclosporine or tacrolimus. Patients on twice daily therapy with tacrolimus were eligible if cumulative CNI exposure was 6-30 months, disease was in stable remission while on CNI,^3,9 and tacrolimus 12-hour trough (C₀) was 3-7 ng/mL. Patients with initial or late steroid resistance, known secondary cause, estimated glomerular filtration rate (eGFR)¹⁰ <60 mL/min/1.73 m², frequent relapses on tacrolimus therapy, immunosuppression other than prednisolone and tacrolimus in the last 6 months (e.g., cyclosporine, rituximab, mycophenolate mofetil or cyclophosphamide), seizures, recurrent headache, impaired glucose tolerance (fasting glucose >100 mg/dL or glycosylated hemoglobin, HbA1c >5.7%), refractory hypomagnesemia (serum magnesium <1.7 mg/dL, despite therapy with magnesium oxide), concomitant therapy with agents that affect tacrolimus bioavailability [Supplementary Table S2] and chronic infections (tuberculosis, HIV, hepatitis B or C) were excluded.

Intervention

Following written informed parental consent and, in children older than 7 years, written informed assent, eligible patients were enrolled. Details of disease course, therapy, and clinical findings, including anthropometry standard deviation scores (SDS),¹¹ and blood pressure interpretation¹² were documented. Blood levels of creatinine, albumin, glucose, HbA1c, cholesterol, electrolytes, transaminases and tacrolimus 12-hr C₀ were measured. On the day of enrolment, therapy with twice-daily IR-tacrolimus was discontinued and switched to PR-tacrolimus (ODVenta®, Zydus Lifesciences Limited, Ahmedabad, India; available as 0.5 mg and 1 mg capsules) at 1:1 dose equivalence; i.e., one dose of PR-tacrolimus replaced the sum of the two doses of IR tacrolimus that the patient was receiving 12 hours apart. PR-tacrolimus was administered once daily in the morning, 1 hour away from meals, for 6 months. A 24-hour trough C₀ was measured after 2 weeks, and the dose titrated to achieve tacrolimus C₀ of 3-7 ng/mL.

Concomitant medications

If patients were on therapy with prednisolone at enrolment, the dose was tapered to 0.2 mg/kg on alternate days and continued for the 6-month study duration. Therapy with prednisolone was not initiated in patients who were off prednisolone at enrolment until the disease relapsed. Relapse, defined by nephrotic range proteinuria (3+/4+ by dipstick) for 3 consecutive days, was treated with prednisolone at 2 mg/kg/day until remission (negative or trace proteinuria for 3 days), followed by 1.5 mg/kg on alternate days for 4 weeks, 1.0 mg/kg for 2 weeks, 0.5 mg/kg for 2 weeks, and 0.25 mg/kg for 2 weeks and then stopped. All patients received calcium carbonate (250–500 mg) and vitamin D3 (200–400 IU) daily while on prednisolone therapy. Infections were managed as per unit protocols. Hypertension, defined as systolic or diastolic blood pressure ≥95^th percentile for sex, age, and height,¹² was treated with enalapril, administered at a dose of 0.3-0.6 mg/kg/day. Drugs that interfere with the bioavailability or pharmacokinetics of tacrolimus were not used [Supplementary Table S2].

Outcomes

The primary outcome of this prospective pilot study was the proportion of patients with stable remission, defined as sustained remission or occurrence of one relapse during the 6-month follow-up. Secondary outcomes, at 6 months, included the proportions of patients with sustained remission and treatment failure, relapse frequency, C₀/TDD, and change in eGFR,¹⁰ HbA1c, and total cholesterol between baseline to 6 months or the last follow-up. Treatment failure was defined as the occurrence of frequent relapses (≥2 relapses in 6 months), late steroid resistance (non-response to prednisolone at 60 mg/m² daily for 6 weeks), a serious adverse event (SAE) attributed to the intervention, or ≥2 SAE of any nature. Pharmacogenomic studies included the relationship between CYP3A5 polymorphism c.6986A>G and tacrolimus C₀ and C₀/TDD at 2 weeks and 6 months in all patients, and AUC_0-24 at 2 weeks in a subset.

Follow-up and monitoring

Parents were instructed to examine the first morning urine for protein by dipstick, and record details of proteinuria, medications, infections, and adverse events. Visits were scheduled at 2 weeks; and 2, 4, and 6 months; at relapse; at suspicion of SAE; and at diagnosis of treatment failure. At each visit, records were reviewed and physical examination was performed with attention to vital signs, including blood pressure, anthropometry, signs of infections, relapse, and adverse events. Compliance was assessed by history and the pill count of empty blister pack. Investigations included blood levels of albumin, sodium, potassium and creatinine, blood counts, lipid profile, fasting glucose, HbA1c and magnesium, and spot urine protein and creatinine ratio at baseline and 6-month follow-up. All SAE were reported to the Ethics Committee within 48 hours. Study participation was terminated at treatment failure or at 6-months follow up, whichever was earlier. Subsequent management was as per institutional protocols.

Tacrolimus pharmacokinetics

Tacrolimus blood levels were measured using liquid chromatographic mass spectroscopy at the 2-week and 6-month follow-ups. The 24-hr C₀ for PR-tacrolimus was performed after 2 weeks, and the dose was adjusted to achieve a target C₀ of 3-7 ng/mL. The C₀ was measured after every 2-weeks of dose change, if required, to escalate dose to a maximum of 0.2 mg/kg/day. C₀/TDD was calculated to understand inter-individual variations. TDD was corrected for body surface area of 1.73 m² to provide C₀/TDD values comparable to adults (C₀/TDD_BSA-Adult).¹³ In every third patient, AUC_0-24 was estimated at 2 weeks using tacrolimus levels at 8 timepoints (0.5, 1, 2, 4, 6, 8, 12, and 24-hour) in the trapezoid model.¹⁴ AUC_0-24hr was inspected for maximum tacrolimus concentration (C_max) and time taken to reach C_max (t_max).

CYP3A5 polymorphism

All patients underwent testing for the CYP3A5 single-nucleotide polymorphism c.6986A>G using an real time polymerase chain reaction (RT-PCR). Based on the presence of the functional (A, *1 or wild-type) or non-functional (G, *3 or mutant) alleles; patients were categorized as expressor and rapid metabolizer (genotype AA or *1/*1), expressor and intermediate metabolizer (AG or *1/*3), and non-expressor or poor metabolizer (GG or *3/*3).

Statistical analysis

Information from case record forms was entered into Microsoft Excel and analyzed using Stata 14.2 (2015; Statacorp, New College station, Texas). Continuous data was reported as median (IQR) and compared between- and within groups using the Wilcoxon rank sum and sign rank tests, respectively. Proportions are reported with 95% CI. The incidence of relapse is reported as rate per person-year. p value <0.05 was considered significant.

In absence of literature on remission of nephrotic syndrome using PR-tacrolimus, we assumed that PR-tacrolimus will be as effective in maintaining stable remission in children as IR-tacrolimus.¹⁵ Assuming that 95% of patients will show satisfactory remission at 6-month follow-up, at 90% power, allowing for 10% absolute error in the estimate and 10% attrition, 20 patients were required to be enrolled.

Patients

Of 45 patients screened for eligibility, 25 were excluded [Figure 1]; 20 patients were switched to once-daily PR-tacrolimus and followed for 6-months without attrition. Six patients required increase in PR-tacrolimus dose to achieve a target trough between 3-7 ng/mL by 4 weeks. Seven patients underwent AUC_0-24hr estimation.

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Figure 1:

Study flow. CNI: Calcineurin inhibitor.

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Table 1 shows the baseline characteristics of enrolled patients, which included more boys (85%) and patients with steroid dependence (60%). Almost two-thirds of patients had received more than two steroid-sparing therapies during a disease lasting median 8 (IQR 5-10.5) years. CNI therapy had been administered for median 19.5 (IQR 8.3-30) months. Five patients had received rituximab prior to IR-tacrolimus, at median (range) of 30 (12-40) months, before switching to PR-tacrolimus. In the 6-months preceding enrolment, the incidence of relapses was 0.7 (95% CI 0.3-1.4) per person-year; 13 (65%) patients were in sustained remission on IR-tacrolimus.

Outcomes

In the 6 months following the switch to PR-tacrolimus, ten patients were in sustained remission, and eight experienced one relapse each; two patients with two relapses each were termed frequent relapses. Treatment failure, observed in 3 (15%) patients, was attributed to frequent relapses in these two patients and to late steroid resistance, which followed a relapse in one patient. Hence, 17 (85%) patients had stable remission (no or infrequent relapses), including 10 patients with sustained remission [Table 2]. There was no significant association between the disease course with PR-tacrolimus and that during the therapy with IR-tacrolimus (data not shown).

There were no significant changes in eGFR, HbA1c, and total cholesterol from the baseline to the end of study [Table 2]. The incidence (95% CI) of relapses, based on 12 relapses in 117.3 person-months, was 1.23 (0.63, 2.15) per person-year, which was insignificantly higher than the baseline rate (incidence rate ratio 1.76; 95% CI 0.7-4.5; P=0.23). Additional outcomes, not pre-specified, have been shown in Supplementary Table S3.

Adverse events

Ten adverse events observed in eight children, have been listed in Supplementary Table S4. Upper respiratory tract infection was the most common event in seven patients. There were no SAE and/or events requiring therapy discontinuation. No patient experienced tremors, headaches, seizures, hyperglycemia, or hypomagnesemia. PR-tacrolimus was withheld for one week in one patient who developed AKI stage 2 associated with relapse and hypovolemia; eGFR remained stable after PR-tacrolimus was resumed upon AKI resolution.

Therapy compliance and pharmacokinetics

PR-tacrolimus was administered at a dose of 0.09 (0.07, 0.14) mg/kg/day, which was similar to the IR-tacrolimus dose at baseline [Table 3]. The compliance to doses was 98.8% (98.3%, 100%). While the C₀ and tacrolimus daily dose indexed to body weight were similar between baseline and after 6 months, the C₀/TDD declined over 6-months’ follow-up [P=0.032; Supplementary Table S5]; similarly, there was an insignificant decline in the C0/TDD values adjusted for body surface area [C0/TDD_BSA-Adult; P=0.089; Supplementary Table S5]. Individual trends for these parameters have been shown in Supplementary Figure S1. In 7 patients, AUC_0-24hr was 130.3 (81.6, 158.6) ng*hr/mL, with a C_max of 10.40 (5.80, 18.30) ng/mL and t_max of 2 hours [Supplementary Table S6; Figure 2]. Both the C₀ and C_max correlated modestly with the AUC_0-24hr [Supplementary Figure S2].

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Figure 2:

Area under the curve (0-24 hours) for 7 children receiving prolonged release tacrolimus. Solid and dashed lines indicate patients with CYP3A5 genotypes *1/*3 (expressors) and *3/*3 (non-expressors), respectively. The shaded area indicates the conventional target range (3-7 ng/mL) for tacrolimus trough in children with nephrotic syndrome.

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CYP3A5 polymorphism

Twelve patients were intermediate metabolizers (*01/*03) and eight were poor metabolizers (*03/*03); none had rapid metabolism (*01/*01). Patients with intermediate metabolism had significantly lower C₀ at enrolment and 2 weeks and 6 months, as compared to the poor metabolizers [Table 3]. While the C₀/TDD_BSA-Adult were significantly lower for intermediate metabolizers at enrolment and at 2 weeks, these values were comparable between the groups at 6-months [Table 3].