Janel Long-Boyle

Low-Dose FK506 (Tacrolimus) in End-Stage Pulmonary Arterial Hypertension

To the Editor: Despite recent advances in therapy, pulmonary arterial hypertension (PAH), characterized by occlusive vasculopathy of the pulmonary arteries, remains a progressive disease without a cure (1–3). Although currently approved PAH medications have not demonstrated antiremodeling properties in humans, novel antiproliferative strategies have shown some benefits and also raised safety concerns (4–6); for example, none target a genetic predisposition of PAH: dysfunctional bone morphogenetic protein receptor 2 (BMPR2) signaling. Loss-of-function mutations in BMPR2 in patients with familial and idiopathic PAH (7–9) are associated with increased pulmonary vasculopathy (10). Furthermore, reduced BMPR2 expression is observed even in patients without a mutation, reinforcing the importance of decreased BMPR2 in PAH (11). In a high-throughput screen of 3,600 drugs approved by the U.S. Food and Drug Administration, we identified low-dose FK506 (tacrolimus) as a potent BMPR2 activator that reversed experimental PAH (12). We therefore hypothesized that low-dose FK506 would be beneficial in patients with PAH by increasing BMPR2 signaling. On the basis of these findings, we initiated a randomized, double-blind, placebo-controlled phase IIa trial (FK506 [Tacrolimus] in Pulmonary Arterial Hypertension [TransformPAH]) to evaluate the safety and tolerability of FK506 in stable patients with PAH. Here, we report our clinical experience with compassionate use of low-dose FK506 in three patients with end-stage PAH who did not qualify for our trial because of the severity of their illness (patient details are provided in the online supplement). We assessed traditional clinical parameters, New York Heart Association (NYHA) functional class, 6-minute-walk distance, serologic biomarkers, and hospital admissions, as well as protocolized cardiac magnetic resonance imaging assessed by blinded readers (13, 14). All patients continued receiving stable doses of PAH medication and diuretics throughout the 12-month period. The TransformPAH clinical trial was registered with www.clinicaltrials.gov (NCT 01647945). Patient 1 Patient 1 is a 36-year-old historically athletic woman, NYHA-IV, with rapidly progressive idiopathic PAH requiring rapid up-titration of epoprostenol and the addition of sildenafil and ambrisentan for recurrent hospitalizations for right ventricular (RV) failure (Figure 1). Despite aggressive treatment, she still reported NYHA-III/IV symptoms, an elevated N-terminal-pro-B type natriuretic peptide, and a Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score of 11, stratifying her as high risk with a potential 1-year mortality risk of 15–30% (3, 15). She was listed for lung transplantation. At that time, she was offered compassionate treatment with FK506 (goal trough blood level, 1.5–2.5 ng/ml). Figure 1. Timeline of symptoms, clinical parameters, events, and therapies for patients 1–3 before and after initiation of FK506. Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score, %1-year survival: score 1–7 = 95–100% ... Within 1 month of FK506 initiation, she reported substantial improvement in symptoms (Figure 2). Within 2 months, she was placed on hold for transplantation by the lung transplant team. After 3 months, her 6-minute-walk distance improved by 100 m, her N-terminal-pro-B type natriuretic peptide decreased more than 50%, and she reported NYHA-I symptoms (Figure 1; see Figure E1A in the online supplement). During the 12-month period, cardiac magnetic resonance imaging showed a stable RV ejection fraction, RV end-diastolic volume index, and cardiac index (Figure E1B). Her REVEAL risk score decreased to 3 (range, 3–6), placing her in the low-risk category (Figure 1). Although the 12 months before FK506 were characterized by three hospitalizations for RV failure, the subsequent 12 months were free of any PAH-associated hospitalizations (Figure 1). At the time of this submission, the patient is 27 months from the initiation of FK506, continues to report NYHA-II symptoms, and has been free from hospitalization or clinical deterioration. Figure 2. Biomarkers for patients 1–3 before and after initiation of FK506. (A) Bone morphogenetic protein receptor 2 (BMPR2) messenger RNA (mRNA)/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in peripheral blood mononuclear cells (PBMCs) of healthy ... Patient 2 Patient 2 is a 50-year-old woman with end-stage systemic sclerosis–associated PAH receiving intravenous treprostinil, sildenafil, and ambrisentan, as well as an intravenous dopamine infusion for end-stage RV failure and hypotension. The patient continued to report NYHA-III/IV symptoms and had an elevated N-terminal-pro-B type natriuretic peptide (range, 4,926–15,161 pg/ml) and four hospitalizations for progressive RV failure and palliative paracenteses (Figure 1). Given the lack of further therapeutic options, she was offered FK506. Cardiac magnetic resonance imaging at baseline and 3 and 6 months showed substantial improvement in RV ejection fraction, stable RV end-diastolic volume index, and improvement in right ventricular stroke volume index and cardiac index (Figure E1B), with a reduction back to baseline at 12 months. Her REVEAL risk score decreased from 12 to 11. At 12-month follow-up, she had stable NYHA-III symptoms, a 94-m increase in 6-minute-walk distance, 30% reduction in her N-terminal-pro-B type natriuretic peptide, and no PAH-related hospitalizations since being on FK506 (Figure 1). The patient is currently 26 months post-FK506 initiation and has not experienced further clinical deterioration. Patient 3 Patient 3 is a 55-year-old woman with severe end-stage drugs-and-toxins–associated PAH, NYHA-III/IV, who is receiving high-dose intravenous treprostinil and sildenafil, has an intolerance to endothelin receptor antagonists, is listed for lung transplantation, and was offered FK506. Despite initial symptomatic improvement (Figure 1), the patient voluntarily discontinued FK506 after 4.5 months. Unfortunately, during the ensuing 7 months, she showed progressive clinical worsening, culminating in an intensive care unit admission for RV failure and large pericardial effusion. On the patient’s wish, she was restarted on FK506 and is currently 12 months after her second FK506 initiation, feeling much better, with compensated NYHA-II symptoms and without any further hospital admission for RV failure. Serologic Biomarkers None of the three patients had mutations in BMPR2, SMAD9, or caveolin-1. We measured BMPR2 expression and specific BMPR2-associated genes and molecules (Id1 [inhibitor of differentiation 1], Smurf-1 [SMAD-specific E3 ubiquitin protein ligase 1], IL-6, LIMK1 [LIM domain kinase 1], Cofilin-1, and microRNAs 21 and 27a) at baseline and 3, 6, and 12 months into FK506 treatment in patients versus healthy controls (n = 12) (see online supplement). Patients had significantly lower BMPR2 messenger RNA expression at baseline (Figure 2), with near normalization of BMPR2 and associated genes after 12 months of FK506 treatment. Strikingly, patient 3, who stopped FK506 after 4.5 months and who worsened clinically during the following 7 months, showed a 12-month BMPR2 profile that was opposite that of patients still receiving FK506 therapy. Discussion Our results suggest a potential clinical benefit of low-dose FK506 in end-stage PAH, judged by the patients’ marked clinical response, stabilization in cardiac function, and freedom from hospitalization for RV failure. Despite the overall positive experience, we caution that these findings are highly preliminary. The efficacy of this therapy must be validated in appropriate, well-designed, prospective clinical trials. Our choice of low-dose FK506 was based on data from preclinical studies (12) and the desire to avoid major immunosuppressive adverse effects in patients with indwelling lines. We did not observe an increase in line sepsis or opportunistic infections. We also did not observe serious adverse effects of posterior reversible encephalopathy syndrome, acute kidney injury or worsening of creatinine, an elevated systemic blood pressure, hyperglycemia, hyperkalemia, anemia, or a change in white blood cell count. The currently underway clinical phase IIa trial will address safety and tolerability in greater detail, as even low-dose immunosuppression over time can lead to complications. This is the first study in patients with PAH that repurposes FK506 to increase BMPR2 signaling. The changes in serologic biomarkers are encouraging and show that we have indeed targeted BMPR2 in patients with reduced levels of BMPR2. It will be of interest to determine whether the same effect can be achieved in patients with documented mutations and whether a subset of patients is particularly sensitive to the beneficial effects of this strategy and could therefore be identified up front as potential “responders” on the basis of BMPR2 levels.

Effect of Weight and Maturation on Busulfan Clearance in Infants and Small Children Undergoing Hematopoietic Cell Transplantation

Little information is currently available regarding the pharmacokinetics (PK) of busulfan in infants and small children to help guide decisions for safe and efficacious drug therapy. The objective of this study was to develop an algorithm for individualized dosing of i.v. busulfan in infants and children weighing ≤12 kg, that would achieve targeted exposure with the first dose of busulfan. Population PK modeling was conducted using intensive time-concentration data collected through the routine therapeutic drug monitoring of busulfan in 149 patients from 8 centers. Busulfan PK was well described by a 1-compartment base model with linear elimination. The important clinical covariates affecting busulfan PK were actual body weight and age. Based on our model, the predicted clearance of busulfan increases approximately 1.7-fold between 6 weeks to 2 years of life. For infants age <5 months, the model-predicted doses (mg/kg) required to achieve a therapeutic concentration at steady state of 600-900 ng/mL (area under the curve range, 900-1350 μM·min) were much lower compared with standard busulfan doses of 1.1 mg/kg. These results could help guide clinicians and inform better dosing decisions for busulfan in young infants and small children undergoing hematopoietic cell transplantation.

Busulfan, Fludarabine, and Alemtuzumab As a Reduced Toxicity Regimen for Children with Malignant and Nonmalignant Diseases Improves Engraftment and Graft-versus-Host Disease without Delaying Immune Reconstitution

For children receiving allogeneic hematopoietic stem cell transplants (HSCTs), the toxicity of the conditioning regimen and graft failure remain challenges. We previously reported that targeted i.v. busulfan, fludarabine, and rabbit anti-thymocyte globulin (rATG) decreased toxicity but had a graft failure rate of 21%. To improve the engraftment rate, we replaced ATG with alemtuzumab, a monoclonal Ab targeting CD52. Thirty-five children with malignant and nonmalignant diseases were enrolled in this phase II prospective study. Twelve children had HLA-matched related donors (MRDs), 16 had 10 of 10, and 7 had 9 of 10 HLA allele-matched unrelated donors (MUDs). Thirty-one of 34 evaluable patients (91%) achieved a durable engraftment. All 3 patients who rejected had a nonmalignant disease and received a MUD transplantation (2 mismatched at 1 antigen). Three patients died of a transplantation-related complication (9% ± 5.2%). Seven patients had disease relapse or progression, 2 of whom died. At a median follow-up of 35 months (range, 15-85 months), the event-free survival (EFS) was 61% ± 9.0% and the overall survival (OS) was 78% ± 7.5%. The median time to neutrophil recovery, B cell, and T cell reconstitution were 16 days, 3 months, and 6 months, respectively. At 1 year, the median donor chimerism in whole blood, CD3, CD14/15, and CD19 subsets were 97%, 87%, 100%, and 99%, respectively. Six patients (17% ± 6.6%) developed acute graft-versus-host disease (aGVHD), only 2 of which were >grade II. Two patients (8% ± 5.4%) progressed to chronic GVHD (cGVHD). These results suggest that replacement of rATG with alemtuzumab may improve engraftment as well as decrease cGVHD rates without resulting in delays in immune reconstitution.

Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis

BACKGROUND Intravenous busulfan combined with therapeutic drug monitoring to guide dosing improves outcomes after allogeneic haemopoietic cell transplantation (HCT). The best method to estimate busulfan exposure and optimum exposure in children or young adults remains unclear. We therefore assessed three approaches to estimate intravenous busulfan exposure (expressed as cumulative area under the curve [AUC]) and associated busulfan AUC with clinical outcomes in children or young adults undergoing allogeneic HCT. METHODS In this retrospective analysis, patients from 15 centres in the Netherlands, USA, Canada, Switzerland, UK, Italy, Germany, and Australia who received a busulfan-based conditioning regimen between March 18, 2001, and Feb 12, 2015, were included. Cumulative AUC was calculated by numerical integration using non-linear mixed effect modelling (AUCNONMEM), non-compartmental analysis (AUC from 0 to infinity [AUC0-∞] and to the next dose [AUC0-τ]), and by individual centres using various approaches (AUCcentre). The main outcome of interest was event-free survival. Other outcomes of interest were graft failure or relapse, or both; transplantation-related mortality; acute toxicity (veno-occlusive disease or acute graft versus-host disease [GvHD]); chronic GvHD; overall survival; and chronic-GvHD-free event-free survival. We used propensity-score-adjusted Cox proportional hazard models, Weibull models, and Fine-Gray competing risk regressions for statistical analyses. FINDINGS 790 patients were enrolled, 674 of whom were included: 274 (41%) with malignant and 400 (59%) with non-malignant disease. Median age was 4·5 years (IQR 1·4-10·7). The median busulfan AUCNONMEM was 74·4 mg × h/L (95% CI 31·1-104·6), which correlated with the standardised method AUC0-∞ (r2=0·74), but the latter correlated poorly with AUCcentre (r2=0·35). Estimated 2-year event-free survival was 69·7% (95% CI 66·2-73·0). Event-free survival at 2 years was 77·0% (95% CI 72·1-82·9) in the 257 patients with an optimum intravenous busulfan AUC of 78-101 mg × h/L compared with 66·1% (60·9-71·4) in the 235 patients at the low historical target of 58-86 mg × h/L and 49·5% (29·2-66·0) in the 44 patients with a high (>101 mg × h/L) busulfan AUC (p=0·011). Compared with the low AUC group, graft failure or relapse occurred less frequently in the optimum AUC group (hazard ratio [HR] 0·57, 95% CI 0·39-0·84; p=0·0041). Acute toxicity (HR 1·69, 1·12-2·57; p=0·013) and transplantation-related mortality (2·99, 1·82-4·92; p<0·0001) were significantly higher in the high AUC group (>101 mg × h/L) than in the low AUC group (<78 mg × h/L), independent of indication; no difference was noted between AUC groups for chronic GvHD (<78 mg × h/L vs ≥78 mg × h/L, HR 1·30, 95% CI 0·73-2·33; p=0·37). INTERPRETATION Improved clinical outcomes are likely to be achieved by targeting the busulfan AUC to 78-101 mg × h/L using a new validated pharmacokinetic model for all indications. FUNDING Research Allocation Program and the UCSF Helen Friller Family Comprehensive Cancer Center and the Mt Zion Health Fund of the University of California, San Francisco.

Population pharmacokinetics of busulfan in pediatric and young adult patients undergoing hematopoietic cell transplant: a model-based dosing algorithm for personalized therapy and implementation into routine clinical use.

Background: Population pharmacokinetic (PK) studies of busulfan in children have shown that individualized model-based algorithms provide improved targeted busulfan therapy when compared with conventional dose guidelines. The adoption of population PK models into routine clinical practice has been hampered by the tendency of pharmacologists to develop complex models too impractical for clinicians to use. The authors aimed to develop a population PK model for busulfan in children that can reliably achieve therapeutic exposure (concentration at steady state) and implement a simple model-based tool for the initial dosing of busulfan in children undergoing hematopoietic cell transplantation. Patients and Methods: Model development was conducted using retrospective data available in 90 pediatric and young adult patients who had undergone hematopoietic cell transplantation with busulfan conditioning. Busulfan drug levels and potential covariates influencing drug exposure were analyzed using the nonlinear mixed effects modeling software, NONMEM. The final population PK model was implemented into a clinician-friendly Microsoft Excel-based tool and used to recommend initial doses of busulfan in a group of 21 pediatric patients prospectively dosed based on the population PK model. Results: Modeling of busulfan time–concentration data indicates that busulfan clearance displays nonlinearity in children, decreasing up to approximately 20% between the concentrations of 250–2000 ng/mL. Important patient-specific covariates found to significantly impact busulfan clearance were actual body weight and age. The percentage of individuals achieving a therapeutic concentration at steady state was significantly higher in subjects receiving initial doses based on the population PK model (81%) than in historical controls dosed on conventional guidelines (52%) (P = 0.02). Conclusions: When compared with the conventional dosing guidelines, the model-based algorithm demonstrates significant improvement for providing targeted busulfan therapy in children and young adults.

High fludarabine exposure and relationship with treatment-related mortality after nonmyeloablative hematopoietic cell transplantation.

Despite its common use in nonmyeloablative preparative regimens, the pharmacokinetics of fludarabine are poorly characterized in hematopoietic cell transplantation (HCT) recipients and exposure-response relationships remain undefined. The objective of this study was to evaluate the association between plasma F-ara-A exposure, the systemically circulating moiety of fludarabine, and engraftment, acute GVHD, TRM and OS after HCT. The preparative regimen consisted of CY 50 mg/kg/day i.v. day –6; plus fludarabine 30–40 mg/m2/day i.v. on days –6 to –2 and TBI 200 cGy on day –1. F-ara-A pharmacokinetics were carried out with the first dose of fludarabine in 87 adult patients. Median (range) F-ara-A area-under-the-curve (AUC(0−∞)) was 5.0 μg h/mL (2.0–11.0), clearance 15.3 L/h (6.2–36.6), Cmin 55 ng/mL (17–166) and concentration on dayzero 16.0 ng/mL (0.1–144.1). Despite dose reductions, patients with renal insufficiency had higher F-ara-A exposures. There was strong association between high plasma concentrations of F-ara-A and increased risk of TRM and reduced OS. Patients with an AUC(0−∞) greater than 6.5 μg h/mL had 4.56 greater risk of TRM and significantly lower OS. These data suggest that clinical strategies are needed to optimize dosing of fludarabine to prevent overexposure and toxicity in HCT.

Comparison of two mycophenolate mofetil dosing regimens after hematopoietic cell transplantation.

Mycophenolic acid (MPA) is the active component of mycophenolate mofetil (MMF). Low MPA exposure is associated with a higher incidence of acute GVHD and possibly worse engraftment. Therapeutic plasma targets have been proposed in hematopoietic cell transplantation (HCT), however, are difficult to achieve in adult patients with MMF doses of 2 g/day. Mycophenolate pharmacokinetics was prospectively studied in adults undergoing nonmyeloablative HCT who received MMF 3 g/day with CYA. The first 15 individuals received 1.5 g every 12 h and the second 15 received 1 g every 8 h. Sampling was performed in each patient with i.v. and oral administration. There were no differences in total or unbound MPA 24-h cumulative area under the curves (AUCs), concentrations at steady state (Css) or troughs between the two dosing regimens (all P>0.01). The previously proposed total MPA Css target of 3 μg/ml and trough ⩾1 μ/ml were achieved in only 13–27% and 20–53% of patients, respectively, on 3 g/day. However, the 3 g/day regimens readily achieved satisfactory unbound 24-h cumulative AUC targets of 0.600 μg*h/ml in 87–100% of subjects. There appears to be no significant difference in daily MPA exposure when MMF of 3 g/day is divided into two or three equal doses.

Population pharmacokinetics of unbound mycophenolic acid in adult allogeneic haematopoietic cell transplantation: effect of pharmacogenetic factors.

To evaluate pharmacogenetic factors as contributors to the variability of unbound mycophenolic acid (MPA) exposure in adult allogeneic haematopoietic cell transplantation (alloHCT) recipients.

Population Pharmacokinetics of Unbound Mycophenolic Acid in Pediatric and Young Adult Patients Undergoing Allogeneic Hematopoietic Cell Transplantation

Mycophenolate mofetil (MMF) is an immunosuppressant routinely used in allogeneic hematopoietic cell transplantation (alloHCT) to promote stem cell engraftment and prevent acute graft vs host disease. Administered as a prodrug, MMF is converted by esterases to the active moiety, mycophenolic acid (MPA). The impact of clinical covariates on unbound MPA exposure was investigated with a population pharmacokinetic approach. Pharmacokinetic data were obtained from routine area under the curve (AUC) monitoring of unbound MPA drug levels in 36 pediatric (n = 31) and young adult (n = 5) patients undergoing alloHCT for a variety of malignant and nonmalignant disorders. Unbound MPA pharmacokinetics were well described by a 2‐compartment model with linear elimination and first‐order absorption. The important clinical covariates affecting unbound MPA pharmacokinetics were weight, estimated creatinine clearance, and total bilirubin. Unbound MPA clearance was reduced, and exposure (AUC0–8) increased in individuals with decreased renal function. In individuals with severe hepatic dysfunction (total bilirubin >10 mg/dL) unbound MPA clearance was approximately 3‐fold lower compared with patients with normal to mild hepatic impairment. In alloHCT recipients with renal dysfunction or severe hepatic injury, dose reductions may be necessary to prevent toxicity and ensure optimal immunosuppression.

Randomised placebo-controlled safety and tolerability trial of FK506 (tacrolimus) for pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease characterised by occlusive pulmonary vasculopathy. Activation of bone morphogenetic protein receptor 2 (BMPR2) signalling by FK506 (tacrolimus) reverses occlusive vasculopathy in rodent PAH models. Here, we determined the safety and tolerability of low-level FK506 therapy in stable PAH patients. We performed a randomised, double-blind, placebo-controlled, 16-week, single-centre, phase IIa trial in PAH patients with New York Heart Association functional class II/III symptoms using three FK506 target levels (<2, 2–3 and 3–5 ng·mL−1). 23 patients were randomised and 20 patients completed the trial. FK506 was generally well tolerated, with nausea/diarrhoea being the most commonly reported adverse event and no observation of line infections in patients on intravenous prostacyclin therapy. PAH patients had significantly lower BMPR2 expression in peripheral blood mononuclear cells versus healthy controls (n=13; p=0.005), which improved after FK506 treatment. While we observed that some patients responded with a pronounced increase in BMPR2 expression as well as improvement in 6-min walk distance, and serological and echocardiographic parameters of heart failure, these changes were not significant. Low-level FK506 is well tolerated and increases BMPR2 in subsets of PAH patients. These results support the study of FK506 in a phase IIb efficacy trial. Low-level FK506 (tacrolimus) is well tolerated in stable PAH and increases BMPR2 in subsets of patients http://ow.ly/t5c530cE1vx