Pioglitazone as a novel therapeutic approach in chronic granulomatous disease
Authors: Maddalena Migliavacca, Andrea Assanelli, Francesca Ferrua, Maria Pia Cicalese, Alessandra Biffi, Marta Frittoli, Paolo Silvani, Giovanna Chidini, Edoardo Calderini, Anna Mandelli, Anna Camporesi, Raffaella Milani, Giada Farinelli, Roberto Nicoletti, Fabio Ciceri, Alessandro Aiuti, Maria Ester Bernardo
Journal: Journal of Allergy and Clinical Immunology (2016)

Chronic granulomatous disease (CGD) is a rare genetic disease caused by defects in genes encoding the subunits of the nicotinamide adenine dinucleotide phosphate oxidase complex.1Holland S.M. Chronic granulomatous disease.Clin Rev Allergy Immunol. 2010; 38: 3-10Crossref PubMed Scopus (268) Google Scholar Accordingly, patients with CGD are affected by life-threatening bacterial and fungal infections, as well as extensive tissue granuloma formation. Most frequently, CGD is caused by mutations in the X-chromosomal CYBB gene, which encodes for the gp91phox subunit.2Di Matteo G. Giordani L. Finocchi A. Ventura A. Chiriaco M. Blancato J. et al.IPINET (Italian Network for Primary Immunodeficiencies)Molecular characterization of a large cohort of patients with chronic granulomatous disease and identification of novel CYBB mutations: an Italian multicenter study.Mol Immunol. 2009; 46: 1935-1941Crossref PubMed Scopus (36) Google Scholar X-chromosome–linked CGD (X-CGD) generally produces a severe phenotype, with a mortality rate of 3% to 5% per year despite state-of-the-art prophylaxis and intensive multimodal treatment.3Cole T. Pearce M.S. Cant A.J. Cale C.M. Goldblatt D. Gennery A.R. Clinical outcome in children with chronic granulomatous disease managed conservatively or with hematopoietic stem cell transplantation.J Allergy Clin Immunol. 2013; 132: 1150-1155Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for patients with X-CGD with excellent survival thanks to recent improvements in HSCT protocols.4Güngör T. Teira P. Slatter M. Stussi G. Stepensky P. Moshous D. et al.Inborn Errors Working Party of the European Society for Blood and Marrow TransplantationReduced-intensity conditioning and HLA-matched haemopoietic stemcell transplantation in patients with chronic granulomatous disease: a prospective multicentre study.Lancet. 2014; 383: 436-448Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar The treatment of patients without an HLA-matched donor and active infections/inflammatory complications remains challenging and requires novel approaches.5Gennery A.R. Slatter M.A. Grandin L. Taupin P. Cant A.J. Veys P. et al.Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation; European Society for ImmunodeficiencyTransplantation of hematopoietic stem cells and long-term survival for primary immunodeficiencies in Europe: entering a new century, do we do better?.J Allergy Clin Immunol. 2010; 126 (e1-11): 602-610Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar Recently, pioglitazone, a peroxisome proliferator–activated receptor gamma agonist approved for type 2 diabetes, was reported in this journal to induce mitochondrial (mt) reactive oxygen species (ROS) production in mice with X-CGD. Restored phagocytes demonstrated significantly enhanced killing of Staphylococcus aureus in vitro and in vivo.6Fernandez-Boyanapalli R.F. Frasch S.C. Thomas S.M. Malcolm K.C. Nicks M. Harbeck R.J. et al.Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease.J Allergy Clin Immunol. 2015; 135: 517-527.e12Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar We report, for the first time, the use of pioglitazone as a novel therapeutic approach in a 5-month-old boy with X-CGD experiencing multiple severe infections. At 1 month of life, the patient developed a cutaneous abscess, fever, and respiratory distress requiring artificial ventilation in pediatric intensive care unit. During the hospitalization, he developed 3 septic shocks, secondary to a peritonsillar abscess, Candida albicans, and Staphylococcus epidermidis dissemination, respectively. Thoracic computed tomography scan showed multiple pulmonary abscesses (Fig 1, A). Because of the susceptibility to severe infections with organ damage and failure to thrive, he was investigated for a potential congenital immunodeficiency. Dihydrorhodamine (DHR) fluorescence showed impaired response in his peripheral blood and a mutation of the CYBB gene was identified (c.483+1G>T). This mutation, in literature, is associated with a X-linked CGD form with undetectable level of gp91phox protein.7Roos D. Kuhns D.B. Maddalena A. Bustamante J. Kannengiesser C. de Boer M. et al.Hematologically important mutations: the autosomal recessive forms of chronic granulomatous disease (second update).Blood Cells Mol Dis. 2010; 44: 291-299Crossref PubMed Scopus (132) Google Scholar Because of the underlying general conditions of the patient with persistent pulmonary distress requiring protracted noninvasive ventilation, severe delay in neuromuscular development, and failure to thrive, HSCT was postponed despite availability of an HLA-identical sibling. Encouraged by the innovative findings on the effect of pioglitazone on ROS production and consequent protection from infection in vitro in human cells and in vivo in an animal model,6Fernandez-Boyanapalli R.F. Frasch S.C. Thomas S.M. Malcolm K.C. Nicks M. Harbeck R.J. et al.Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease.J Allergy Clin Immunol. 2015; 135: 517-527.e12Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar we sought to investigate, for the first time to our knowledge in children, the potential beneficial effect of oral pioglitazone. After parent informed consent for off-label use, pioglitazone was administered at a starting dose of 1 mg/kg and given the absence of adverse effects was progressively increased up to 3 mg/kg (15 mg/daily). After 10 days of pioglitazone at target dose (3 mg/kg), the DHR test showed a percentage of granulocytes with increased DHR fluorescence (bright fluorescent cells = 12.90%; value before treatment 0.06%; Fig 2, A and B), albeit at a lower mean fluorescence intensity than normal donor cells. This effect was maintained in the following weeks until withdrawal (day+30), although at a lower level (day+17, 7.55%; day+25, 4.86%). Moreover, an overall shift in DHR of the whole population of patient's granulocytes was noted after pioglitazone treatment (for details, see Table E1 in this article's Online Repository at www.jacionline.org). These findings paralleled well with clinical, radiological, and blood parameters. The patient experienced a progressive improvement in general clinical condition, respiratory parameters, and function with stable oxygen saturation under neurally adjusted ventilatory assist-noninvasive ventilation. Lung computed tomography scan performed on the day of pioglitazone withdrawal showed clear improvement (Fig 1, B). White blood cell (WBC) counts, as well as neutrophil counts, progressively decreased to normal levels (WBC, 13.9 × 109/L, and neutrophil, 8.2 × 109/L before pioglitazone; WBC, 6.8 × 109/L, and neutrophil, 2 × 109/L at pioglitazone withdrawal, respectively). C-Reactive protein level decreased from 24.4 mg/L to 13.1 mg/L before and after pioglitazone, respectively. The clinical and radiologic amelioration allowed for HSCT; pioglitazone was withdrawn and after 1 week, conditioning regimen based on treosulfan/fludarabine/Thiotepa was administered to the patient.8Bernardo M.E. Piras E. Vacca A. Giorgiani G. Zecca M. Bertaina A. et al.Allogeneic hematopoietic stem cell transplantation in thalassemia major: results of a reduced-toxicity conditioning regimen based on the use of treosulfan.Blood. 2012; 120: 473-476Crossref PubMed Scopus (145) Google Scholar Graft versus host disease prophylaxis was conducted through administration of cyclosporin-A and short-term methotrexate. Neutrophil and platelet engraftment took place at day+17 and +32, respectively. The patient did not develop signs of graft versus host disease. He is currently at day+150 after HSCT in good clinical condition with donor chimerism above 90% on CD14+, CD15+, and CD3+ cells in peripheral blood. He does not require any respiratory support, he has a satisfactory oral food intake with weight gain, and he shows neuromuscular development improvement. Although observed in a single child, pioglitazone might have played a role in the protection of the patient from further infections and in the improvement of his pulmonary situation, by reducing the inflammatory status and by partial restoration of host defense through induction of mtROS production in his granulocytes. This is also supported by the fact that concomitant medications were not increased, but rather reduced in intensity during pioglitazone treatment (for details, see Table E2 in this article's Online Repository at www.jacionline.org). Moreover, corticosteroids were never administered to the patient, neither before nor during pioglitazone treatment. Moreover, the patient did not develop any adverse effects related to pioglitazone administration, confirming the safety of this therapeutic approach. Considering data in female carriers, patients with HSCT, and improvement after gene therapy,7Roos D. Kuhns D.B. Maddalena A. Bustamante J. Kannengiesser C. de Boer M. et al.Hematologically important mutations: the autosomal recessive forms of chronic granulomatous disease (second update).Blood Cells Mol Dis. 2010; 44: 291-299Crossref PubMed Scopus (132) Google Scholar, 9Ott M.G. Schmidt M. Schwarzwaelder K. Stein S. Siler U. Koehl U. et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (984) Google Scholar we speculate that even an enhancement in DHR fluorescence in a relatively small population of leukocytes might be the expression of a protective ROS production. Accordingly, Fernandez-Boyanapalli et al6Fernandez-Boyanapalli R.F. Frasch S.C. Thomas S.M. Malcolm K.C. Nicks M. Harbeck R.J. et al.Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease.J Allergy Clin Immunol. 2015; 135: 517-527.e12Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar suggest that the mechanism responsible for peroxisome proliferator–activated receptor gamma agonist effect in mice with CGD is related to increased ROS production by mitochondria in a subpopulation of neutrophils, monocytes, and inflammatory macrophages. It remains to be determined which population is targeted by pioglitazone in vivo. Presently our observations cannot provide information regarding long-term effects on mtROS production, but suggest that pioglitazone might be used as bridge treatment for these fragile chronic patients. Fernandez-Boyanapalli et al's findings and our experience on the use of pioglitazone provide relevant insights into the treatment of this rare disease especially for those patients without a prompt suitable matched donor or for whom the critical disease conditions force to postpone HSCT. Further studies conducted through well-designed clinical trials are warranted to investigate the drug's precise mechanism of action, as well as to define the role of pioglitazone in the therapeutic armamentarium of patients with X-CGD. We thank all the clinical and nurse staff of the Pediatric Immunohematology Unit. We thank Dr Gigliola Di Matteo for genetic analyses, Dr Benedetta Mazzi for chimerism analyses, and Dr Raisa Jofra Hernandez and Dr Federica Salerio for technical support. Table E1Percentage of DHR bright and DHR positive granulocytes according to pioglitazone dose∗The fold increase in MFI is also depicted and is calculated as the ratio between the MFI on total granulocytes after stimulation with PMA and the MFI on unstimulated total granulocytes.Time pointPioglitazone doseDHR bright fluorescent cells(%)DHR fluorescent cells(%)MFI on total granulocytes (fold increase)Before treatment/0.060.070.905 d of pioglitazone1 mg/kg0.200.250.9210 d of pioglitazone2 mg/kg0.540.650.9725 d of pioglitazone (d+10)3 mg/kg12.9076.402.8732 d of pioglitazone (d+17)3 mg/kg7.5521.001.3940 d of pioglitazone (d+25)3 mg/kg4.866.481.29After HSCT/94.1098.3020.54MFI, Mean fluorescent intensity.∗ The fold increase in MFI is also depicted and is calculated as the ratio between the MFI on total granulocytes after stimulation with PMA and the MFI on unstimulated total granulocytes. Open table in a new tab Table E2Concomitant medications received by the patient during pioglitazone treatment∗Days are expressed in relation to the first day of pioglitazone administration.Concomitant medicationsDose per dayState at pioglitazone startState at pioglitazone stopLinezolid IV30 mg/kgOngoingStopped 7 d after pioglitazone startPiperacillin-tazobactam IV300 mg/kgOngoingStopped 10 d after pioglitazone startAmoxicillin-clavulanic acid PO50 mg/kgStarted 10 d after pioglitazone initiationOngoingTrimethoprim-sulphamethoxazole PO5-25 mg/kgOngoingOngoingVoriconazole IV8 mg/kgOngoingStopped 6 d after pioglitazone startItraconazole PO10 mg/kgStarted 6 d after pioglitazone initiationOngoingNoninvasive ventilationNAOngoingOngoingIV, Intravenous; NA, not applicable; PO, per os (by mouth).∗ Days are expressed in relation to the first day of pioglitazone administration. Open table in a new tab MFI, Mean fluorescent intensity. IV, Intravenous; NA, not applicable; PO, per os (by mouth).