Barr, Frederick E.

Campbell, Andrew

Cunningham, Gary

Darbari, Deepika

Desai, Jagdish

Majumdar, Suvankar

Mashegu, Hafsat

Nickel, Robert

Shannon, Neil T.

Summar, Marshall

Tirona, Rommel

English

Scholarship@Western

Western University Scholarship@Western Paediatrics Publications Paediatrics Department 2-1-2019 A phase 1 dose-finding study of intravenous L-citrulline in sickle cell disease: a potential novel therapy for sickle cell pain crisis Suvankar Majumdar Childrens National Health System Rommel Tirona Schulich School of Medicine & Dentistry, rommel.tirona@schulich.uwo.ca Hafsat Mashegu The Children's Hospital of Philadelphia Jagdish Desai University of Mississippi Medical Center Neil T. Shannon University of Mississippi Medical Center See next page for additional authors Follow this and additional works at: https://ir.lib.uwo.ca/paedpub Citation of this paper: Majumdar, Suvankar; Tirona, Rommel; Mashegu, Hafsat; Desai, Jagdish; Shannon, Neil T.; Summar, Marshall; Cunningham, Gary; Darbari, Deepika; Nickel, Robert; Campbell, Andrew; and Barr, Frederick E., "A phase 1 dose-finding study of intravenous L-citrulline in sickle cell disease: a potential novel therapy for sickle cell pain crisis" (2019). Paediatrics Publications. 1484. https://ir.lib.uwo.ca/paedpub/1484 Authors Suvankar Majumdar, Rommel Tirona, Hafsat Mashegu, Jagdish Desai, Neil T. Shannon, Marshall Summar, Gary Cunningham, Deepika Darbari, Robert Nickel, Andrew Campbell, and Frederick E. Barr This letter to the editor is available at Scholarship@Western: https://ir.lib.uwo.ca/paedpub/1484 A phase 1 dose-finding study of intravenous L-citrulline insickle cell disease: a potential novel therapy for sickle cell paincrisisAcute pain from vaso-occlusion (VOC) in sickle cell disease(SCD) is the most frequent cause of emergency room visitsand hospital admissions, contributing to the high burden ofhealth care costs (Lanzkron et al, 2010). While majoradvances in the care of patients with SCD have occurred overthe last 30 years, very little progress has been made in theactual treatment of VOC.Nitric oxide (NO) is a powerful vasodilator that plays afundamental role in VOC (Morris, 2008). NO is producedfrom L-citrulline and L-arginine, amino acids generatedthrough the urea cycle from the NO synthase (NOS) familyof enzymes (Moncada & Higgs, 1993). There are three NOSisoforms: neuronal (nNOS) found in neuronal tissue, induci-ble NOS (iNOS) found in cells and tissues, and endothelialNOS (eNOS) found in vascular endothelial cells. Shen et al(2005) showed that citrulline was the major supply for intra-cellular L-arginine and endothelial NO production in murineendothelial cells. Furthermore, Wijnands et al (2012) showedthat L-citrulline supplementation restored intracellular NOproduction, which was related to the degree of eNOS phos-phorylation. Moreover, enhanced arginase-induced arginineconsumption is believed to play an integral role in the patho-genesis of sickle cell complications. In a more recent study,L-citrulline supplementation increased NO production andimproved microcirculatory flow during conditions with argi-nase-induced arginine deficiency (Wijnands et al, 2015).However, intravenous citrulline has never been evaluated inhuman SCD. Hence, this study aimed to characterize thepharmacokinetic (PK) and safety profile of intravenous (IV)citrulline in this unique patient population.A single centre open label phase 1 trial of IV citrulline wasperformed in participants with SCD following approval fromthe University of Mississippi Medical Center (UMMC) insti-tutional review board. The study was registered at Clini-calTrials.gov (NCT02314689; NCT02697240), where theinclusion and exclusion criteria are described. Th phase 1study was performed in two steps. Step 1 included a doseescalation bolus infusion of IV citrulline in steady-state SCDto determine the PK and safety profile with a peak goalplasma citrulline concentration of 80–100 lmol/l (Barr et al,2007). Step 2 of the study was performed to evaluate safetyand PK during a vaso-occlusive crisis.The study drug, L-citrulline, was administered as openlabel vials of 50 mg/ml (5%) isotonic solution. Plasmasampling for PK studies were collected at specific timepoints. Briefly, for amino acid analysis, deproteinated plasmasamples were subjected to cation exchange chromatographyusing a 4-component pH and ionic strength graded lithiumcitrate buffer system on a Beckmann 7300 amino acid analy-ser (Beckmann, Palo Alto, CA). Data obtained for eachpatient was fitted to a single-compartment PK model. Theappearance of citrulline in plasma was described by a zero-order process (rate of citrulline appearance, Rapp) to accountfor endogenous production, whereas the removal of citrullinewas determined by a first-order process (constant of citrul-line removal, krem). It was assumed that the values of allparameters remained constant for each patient during thecourse of plasma sampling. Scientist v2.0 (Micromath Scien-tific Software, St. Louis, MO) was used to fit the plasmacitrulline concentration to the PK model by a weighted, leastsquares procedure to obtain values for Rapp, krem, and thevolume of distribution (Vd). Clearance was calculated as theproduct of krem and Vd.For safety assessments, the Investigator determined theintensity of any adverse event (AE) according to the NationalCancer Institute Common Terminology Criteria for AdverseEvents Version 4.03 (https://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf) and theircausal relationship. A Data Safety Monitoring Board, com-prising 3 physicians who were not related to the study,reviewed every third subject.A total of 8 subjects with SCD were enrolled in this phase1 study of IV citrulline: four participants were enrolled inStep 1 and another four participants in Step 2. Patient demo-graphics, genotype and baseline blood counts are shown inTable SI. In the first cohort of four participants, the IV bolusinfusion of 20 mg/kg of L-citrulline yielded a mean peaklevel of 259 lmol/l and trough level in the range of20–40 lmol/l at 4 h after infusion (Fig 1). Citrulline PKparameters for Step 1 with bolus infusion are shown inTable SII. Pharmacokinetic model simulations indicated a20 mg/kg bolus dose of IV citrulline followed by 7 mg/kgper hour continuous infusion was needed to maintain thetarget citrulline plasma concentration of 100 lmol/l. Subse-quently, four subjects with VOC were enrolled to receive IVcitrulline bolus and continuous infusion. Individuals withVOC showed significantly lower baseline citrulline levelscompared to steady-state (mean  SD: 937  143 vs.correspondencedoi: 10.1111/bjh.15124ª 2018 British Society for Haematology and John Wiley & Sons LtdBritish Journal of Haematology, 2019, 184, 634–6962228  68, P = 001) (Figure S1). After the IV bolus, meanpeak plasma concentrations was 257 lmol/l and citrullineplasma concentrations of approximately 100 lmol/l wereachieved during a 7 mg/kg per hour continuous infusion.Furthermore, there was a robust and durable rise in bothcitrulline and arginine levels (Fig 2). The citrulline PKparameters for Step 2 with the bolus and IV continuousinfusion are shown in Table SIII. The endogenous citrullineappearance rate (Rapp) was significantly lower in the VOCcohort compared to steady-state (50 lmol/h/kg vs111 lmol/h/kg, P = 0014).Overall, intravenous citrulline in SCD was well toleratedand safe. There were no AEs ≥ grade 2 level of toxicity.Drowsiness was noted in 6 participants, but not severeenough to discontinue study medication. One patientreported feeling cold and one patient had nausea (no vomit-ing) associated with the drowsiness. Due to the theoreticalrisk of vasodilation from the NO boost, vital signs were fol-lowed closely (Figure S2). In one subject, the diastolic bloodpressure transiently dropped >20% from baseline during thefirst 30 min of drug administration but normalized within1 h without any intervention. There were no significantchanges in the complete blood count and renal function tests(Table SIV). However, one subject was readmitted about2 weeks later with right upper quadrant painelevated alaninetransaminase and aspartate transaminase, peaking at 367 and335 u/l respectively, which resolved by day 31. The subjectdeveloped fever during hospitalization; tests revealed cytome-galovirus (CMV) IgM positivity and CMV polymerase chainreaction of 6300 copies, suggesting acute CMV infection asan aetiology. This was reported to the US Food and DrugAdministration as a serious AE.This is the first report on the use of intravenous citrullinein SCD. While this intervention was well tolerated, drowsi-ness was a potential side effect of unclear aetiology thatcould be related to vasodilation of the cerebral vasculatureor, perhaps, improvement in pain. Given the potential bene-fit of boosting endothelial NO and contributing to microcir-culatory vasodilation, studies are needed to evaluate theefficacy of intravenous citrulline during a vaso-occlusivecrisis.AuthorshipSM designed the study and wrote the manuscript. TRG per-formed the PK analysis, MH and NS conducted the study.JD analysed the data. MS designed the study. GC performedthe amino acid testing. DD, RN and AC critically revised themanuscript. FB designed the study.AcknowledgementsThis work was supported by a University of MississippiMedical Center (UMMC) intramural grant (#68599340412)Fig 1. Graphs showing plasma citrulline andarginine levels over time after an intravenousbolus infusion of 20 mg/kg L-citrulline.Fig 2. Graphs showing plasma citrulline andarginine levels over time after an intravenousbolus of 20 mg/kg followed by a continuousinfusion of 7 mg/kg per hour L-citrulline.Correspondenceª 2018 British Society for Haematology and John Wiley & Sons Ltd 635British Journal of Haematology, 2019, 184, 634–696(S.M.). We are grateful to the patients and families for agree-ing to participate in this phase 1 study. We thank theresearch nurse, Heather Atterberry, RN, and the studypharmacist Richard Ogletree Jr, Pharm D. We also thankAsklepion pharmaceuticals for supplying the study drug atno cost.DisclosuresU.S. Provisional Patent Application No. 62/463,931 (Intra-venous citrulline for treatment of sickle cell crisis).Suvankar Majumdar1,2Rommel Tirona3Hafsat Mashegu4Jagdish Desai2Neil T. Shannon2Marshall Summar5Gary Cunningham5Deepika Darbari1Robert Nickel1Andrew Campbell1Frederick E. Barr21Division of Hematology, Children’s National Medical Center, GeorgeWashington School of Medicine and Health Sciences, Washington, DC,2Department of Pediatrics, University of Mississippi Medical Center,Jackson, Mississippi, 3Departments of Physiology & Pharmacology andMedicine, Schulich School of Medicine & Dentistry, The University ofWestern Ontario, London, Canada, 4Children’s Hospital of Philadelphia,Philadelphia, PA, and 5Division of Genetics, Children’s National MedicalCenter, Washington, DC, USA.E-mail: smajumdar@childrensnational.orgKeywords: sickle, citrulline, pharmacokineticFirst published online 07 February 2018doi: 10.1111/bjh.15124Supporting InformationAdditional Supporting Information may be found in theonline version of this article:Figure S1. Box-plot showing baseline plasma citrullinelevels in steady-state sickle cell subjects (n = 4) and during asickle cell pain crisis (n = 4); P = 001.Figure S2. Vitals signs (mean  standard error) in thefirst 4 subjects after a bolus infusion of L-citrulline.Table SI. Showing the demographic, genotype and base-line complete blood count of participantsTable SII. Citrulline pharmacokinetic model parameterestimates for I.V. bolus cohort.Table SIII. Citrulline pharmacokinetic model parameterestimates for intravenous bolus plus continuous infusioncohort. Rapp, rate of citrulline appearance; krem, constant ofcitrulline removal.Table SIV. Showing the laboratory profile after receivingthe bolus and continuous intravenous L-citrulline at baselineand at 24 h (end of infusion).ReferencesBarr, F.E., Tirona, R.G., Taylor, M.B., Rice, G.,Arnold, J., Cunningham, G., Smith, H.A., Camp-bell, A., Canter, J.A., Christian, K.G., Drinkwater,D.C., Scholl, F., Kavanaugh-McHugh, A. & Sum-mar, M.L. (2007) Pharmacokinetics and safety ofintravenously administered citrulline in childrenundergoing congenital heart surgery: potentialtherapy for postoperative pulmonary hyperten-sion. Journal of Thoracic and Cardiovascular Sur-gery, 134, 319–326.Lanzkron, S., Carroll, C.P. & Haywood, C. Jr(2010) The burden of emergency departmentuse for sickle-cell disease: an analysis of thenational emergency department sample data-base. American Journal of Hematology, 85, 797–799.Moncada, S. & Higgs, A. (1993) The L-arginine-nitric oxide pathway. New England Journal ofMedicine, 329, 2002–2012.Morris, C.R. (2008) Mechanisms of vasculopathyin sickle cell disease and thalassemia. Hematol-ogy/the Education Program of the American Soci-ety of Hematology, 2008, 177–185.Shen, L.J., Beloussow, K. & Shen, W.C. (2005)Accessibility of endothelial and inducible nitricoxide synthase to the intracellular citrulline-argi-nine regeneration pathway. Biochemical Pharma-cology, 69, 97–104.Wijnands, K.A., Vink, H., Briede, J.J., van Faassen,E.E., Lamers, W.H., Buurman, W.A. & Poeze,M. (2012) Citrulline a more suitable substratethan arginine to restore NO production and themicrocirculation during endotoxemia. PLoSOne, 7, e37439.Wijnands, K.A., Meesters, D.M., van Barneveld,K.W., Visschers, R.G., Briede, J.J., Vandendriess-che, B., van Eijk, H.M., Bessems, B.A., van denHoven, N., von Wintersdorff, C.J., Brouckaert,P., Bouvy, N.D., Lamers, W.H., Cauwels, A. &Poeze, M. (2015) Citrulline supplementationimproves organ perfusion and arginine availabil-ity under conditions with enhanced arginaseactivity. Nutrients, 7, 5217–5238.Correspondence636 ª 2018 British Society for Haematology and John Wiley & Sons LtdBritish Journal of Haematology, 2019, 184, 634–696

A phase 1 dose-finding study of intravenous L-citrulline in sickle cell disease: a potential novel therapy for sickle cell pain crisis

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A phase 1 dose-finding study of intravenous L-citrulline in sickle cell disease: a potential novel therapy for sickle cell pain crisis

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