A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

BACKGROUND: Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). METHODS: We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. RESULTS: Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event. CONCLUSIONS: Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440 .).

The impact of comorbidity on health-related quality of life in elderly patients with chronic myeloid leukemia.

The primary objective of this study was to investigate whether the presence of comorbidities was associated with a lower health-related quality of life (HRQOL) in elderly patients with chronic myeloid leukemia (CML). A sample of 174 CML patients aged 60 years or above was analyzed. HRQOL was assessed with the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). A number of pre-selected sociodemographic and disease-related factors were considered as potential confounding factors for the association between comorbidity and HRQOL. Mean age of the 174 patients analyzed was 70 years (range 60-87 years) and 55 % were male. Overall, 111 patients (64 %) reported at least one comorbidity. Analysis stratified by age group category showed a greater proportion of patients with comorbidities in the older sub-group population (≥70 years) compared to younger patients (60 to 69 years). Differences in HRQOL outcomes between patients with no comorbidity at all and those with two or more comorbid conditions were at least twice the magnitude of a clinically meaningful difference in all the physical and mental health scales of the SF-36. In multivariate analysis, after adjusting for key confounding factors, the following scales were significantly lower in those with comorbidity: general health (p < 0.001), bodily pain (p < 0.001), physical functioning (p = 0.002), and vitality (p = 0.002). Assessing comorbidity in elderly patients with CML is important to facilitate identification of those most in need of HRQOL improvements.

Differences among young adults, adults and elderly chronic myeloid leukemia patients.

BACKGROUND: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. PATIENTS AND METHODS: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CML WP over a 40-year period. RESULTS: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P < 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P < 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P < 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). CONCLUSIONS: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome. CLINICAL TRIAL NUMBERS: NCT00510926, NCT00514488, NCT00769327, NCT00481052.

Profiling of drug-metabolizing enzymes/transporters in CD33+ acute myeloid leukemia patients treated with Gemtuzumab-Ozogamicin and Fludarabine, Cytarabine and Idarubicin.

Genetic heterogeneity in drug-metabolizing enzyme/transporter (DMET) genes affects specific drug-related cancer phenotypes. To investigate the relationships between genetic variation and response to treatment in acute myeloid leukemia (AML), we genotyped 1931 variants on DMET genes in 94 CD33-positive AML patients enrolled in a phase III multicenter clinical trial combining Gemtuzumab-Ozogamicin (GO) with Fludarabine-Cytarabine-Idarubicin (FLAI) regimen, with the DMET Plus platform. Two ADH1A variants showed statistically significant differences (odds ratio (OR)=5.68, P=0.0006; OR=5.35, P=0.0009) in allele frequencies between patients in complete/partial remission and patients without response, two substitutions on CYP2E1 (OR=0.13, P=0.001; OR=0.09, P=0.003) and one on SLCO1B1 (OR=4.68, P=0.002) were found to differently influence liver toxicity, and two nucleotide changes on SULTB1 and SLC22A12 genes correlated with response to GO (OR=0.24, P=0.0009; OR=2.75, P=0.0029). Genetic variants were thus found for the first time to be potentially associated with differential response and toxicity in AML patients treated with a combination of GO-FLAI regimen.

Investigating factors associated with adherence behaviour in patients with chronic myeloid leukemia: an observational patient-centered outcome study.

BACKGROUND: Optimal adherence to imatinib therapy is of paramount importance to maximise treatment effectiveness in patients with chronic myeloid leukaemia (CML). The main objective of this study was to investigate patient-reported personal factors associated with adherence behaviour. METHODS: Analysis was conducted on 413 CML patients receiving long-term therapy with imatinib. Adherence behaviour was measured with the Morisky Medication Adherence Scale and personal factors investigated included: quality of life, perceived social support, fatigue, symptom burden, psychological wellbeing and desire for additional information. Key socio-demographic and treatment-related factors were also taken into account. Univariate and multivariate logistic regression analyses were used to investigate factors associated with optimal adherence to therapy. RESULTS: In all, 53% of patients reported an optimal adherence behaviour. The final multivariate model retained the following variables as independent predictors of optimal adherence to therapy: desire for more information (ref. no), odds ratio (OR)=0.43 (95% confidence interval (CI), 0.29-0.66; P<0.001), social support (higher score representing greater support), OR=1.29 (95% CI, 1.11-1.49; P<0.001) and concomitant drug burden (ref. no), OR=1.82 (95% CI, 1.18-2.80; P=0.006). CONCLUSION: This study suggests that a higher level of social support, satisfaction with information received and concomitant drug burden are the main factors associated with greater adherence to long-term imatinib therapy.

A phase II trial of short course fludarabine, mitoxantrone, rituximab followed by 9°Y-ibritumomab tiuxetan in untreated intermediate/high-risk follicular lymphoma.

BACKGROUND: A prospective, single-arm, open-label, multicenter, nonrandomised phase II trial to evaluate efficacy and safety of short fludarabine, mitoxantrone, and rituximab (FMR) induction followed by radioimmunotherapy, in untreated, intermediate/high-risk follicular non-Hodgkin's lymphoma (NHL) patients. PATIENTS AND METHODS: Fifty-five patients were treated using a sequential treatment schedule of four induction cycles of FMR chemoimmunotherapy, and a subsequent consolidating single administration of (90)Y-ibritumomab tiuxetan ((90)Y-IT), 8-14 weeks later. Patients were eligible for radioimmunotherapy if at least in partial response (PR) after induction, with normal platelet and granulocyte counts and a bone marrow infiltration ≤ 25%. Primary study end points were response rate and hematologic toxic effects; secondary end points were overall survival (OS) and progression-free survival (PFS). RESULTS: All the 55 patients received four induction cycles with an overall response rate of 96% (38 complete responses [CR] and 15 PR). Fifty-one patients (38 in CR and 13 in PR) received (90)Y-IT. By the end of the treatment, 49/55 patients achieved a CR. With a median follow-up of 21 months, the estimated 3-year PFS was 81% and the 3-year OS 100%. CONCLUSIONS: This study has established feasibility, tolerability, and efficacy of a regimen composed by short FMR induction with (90)Y-IT consolidation in untreated intermediate/high-risk follicular NHL patients.

Gemcitabine as single agent in pretreated T-cell lymphoma patients: evaluation of the long-term outcome.

BACKGROUND: Peripheral T-cell lymphoma unspecified (PTCLU) and mycosis fungoides (MF) often show resistance to conventional chemotherapy. Gemcitabine should be considered a suitable option. We report the long-term update of 39 pretreated T-cell lymphoma patients treated with gemcitabine. PATIENTS AND METHODS: From May 1997 to September 2007, 39 pretreated MF and PTCLU patients received gemcitabine. Inclusion criteria were as follows: histologic diagnosis of MF or PTCLU; relapsed/refractory disease; age > or =18 years; and World Health Organization performance status of two or less. Nineteen patients had MF and 20 PTCLU. All patients with MF had a T3-T4, N0, and M0 disease and patients with PTCLU had stage III-IV disease. Gemcitabine was given on days 1, 8, and 15 on a 28-day schedule (1200 mg/m(2)/day) for a total of three to six cycles. RESULTS: Overall response rate was 51% (20 of 39 patients); complete response (CR) and partial response (PR) rates were 23% (9 of 39 patients) and 28% (11 of 39 patients), respectively. Patients with MF had a CR rate of 16% and a PR rate of 32% compared with a CR rate of 30% and a PR rate of 25% of PTCLU patients. Among the CR patients, 7 of 9 are in continuous complete response with a variable disease-free interval (15-120 months). CONCLUSION: In our experience, gemcitabine proved to be effective in pretreated MF and PTCLU patients, even in the long term.

MACOP-B regimen in the treatment of adult Langerhans cell histiocytosis: experience on seven patients.

BACKGROUND: Adult Langerhans cell histiocytosis (LCH) is a rare disease. The combination of vinblastine and prednisone, given in a 6-month course, is the standard of care but prospective randomized trials are lacking. PATIENTS AND METHODS: We report our monocentric experience in the treatment of seven adult patients with multisystem (MS) LCH (n = 3) or single-system multifocal (SS-m) LCH (n = 4) with the short-course intensive chemotherapy regimen methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone and bleomicin (MACOP-B). RESULTS: The overall response rate was 100% [five complete response (CR), two partial response (PR)]. After a median follow-up of 6.5 years, four patients are in first continuous CR and three patients relapsed after 5, 8 and 62 months, respectively. Four patients were evaluated with positron emission tomography (PET) scan: all three PET-negative patients at the end of treatment had a long-lasting response with only one patient relapsing after 5 years. PET scan detected additional bone lesions at diagnosis in two of four patients, changing the treatment program in one of them. CONCLUSIONS: MACOP-B regimen seems to be very active in the treatment of adult MS or SS-m LCH, with long-lasting responses in five of seven patients. PET scan merits further evaluation in the initial staging and in the evaluation of the response to chemotherapy.

European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia.

The therapeutic landscape of chronic myeloid leukemia (CML) has profoundly changed over the past 7 years. Most patients with chronic phase (CP) now have a normal life expectancy. Another goal is achieving a stable deep molecular response (DMR) and discontinuing medication for treatment-free remission (TFR). The European LeukemiaNet convened an expert panel to critically evaluate and update the evidence to achieve these goals since its previous recommendations. First-line treatment is a tyrosine kinase inhibitor (TKI; imatinib brand or generic, dasatinib, nilotinib, and bosutinib are available first-line). Generic imatinib is the cost-effective initial treatment in CP. Various contraindications and side-effects of all TKIs should be considered. Patient risk status at diagnosis should be assessed with the new EUTOS long-term survival (ELTS)-score. Monitoring of response should be done by quantitative polymerase chain reaction whenever possible. A change of treatment is recommended when intolerance cannot be ameliorated or when molecular milestones are not reached. Greater than 10% BCR-ABL1 at 3 months indicates treatment failure when confirmed. Allogeneic transplantation continues to be a therapeutic option particularly for advanced phase CML. TKI treatment should be withheld during pregnancy. Treatment discontinuation may be considered in patients with durable DMR with the goal of achieving TFR.

A phase II trial of CHOP chemotherapy followed by yttrium 90 ibritumomab tiuxetan (Zevalin) for previously untreated elderly diffuse large B-cell lymphoma patients.

BACKGROUND: A prospective, single-arm, open-label, nonrandomized phase II combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus radioimmunotherapy trial was conducted to evaluate the efficacy and safety in untreated elderly diffuse large B-cell lymphoma (DLBCL) patients. PATIENTS AND METHODS: From February 2005 to April 2006, in our institute we treated 20 eligible elderly (age > or =60 years) patients with previously untreated DLBCL using a novel regimen consisting of six cycles of CHOP chemotherapy followed 6-10 weeks later by (90)Y ibritumomab tiuxetan. RESULTS: The overall response rate to the entire treatment regimen was 100%, including 95% complete remission (CR) and 5% partial remission. Four (80%) of the five patients who achieved less than a CR with CHOP improved their remission status after radioimmunotherapy. With a median follow-up of 15 months, the 2-year progression-free survival was estimated to be 75%, with a 2-year overall survival of 95%. The (90)Y ibritumomab tiuxetan toxicity included grade > or =3 hematologic toxicity in 12 of 20 patients; the most common grade > or =3 toxic effects were neutropenia (12 patients) and thrombocytopenia (7 patients). Transfusions of red blood cells and/or platelets were given to one patient. CONCLUSION: This study has established the feasibility, tolerability, and efficacy of this regimen for elderly patients with DLBCL.

WT1 transcript amount discriminates secondary or reactive eosinophilia from idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia.

Idiopathic hypereosinophilic syndromes (HES) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to eosinophilia, it can be present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The hybrid gene FIP1L1-PDGRFalpha was identified in a subset of patients presenting with HES or chronic eosinophilic leukemia (CEL). In spite of this, the majority of HES patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. In this study we explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on WT1 transcript amount. For this purpose, 312 patients with hypereosinophilia were characterized at the molecular and cytogenetic level and analyzed for WT1 expression at diagnosis and during follow-up. This study clearly demonstrates that WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients.

Myeloid sarcoma (MS) is a rare neoplasm whose knowledge is largely based on case reports and/or technically dated contributions. Ninety-two MSs in adulthood with clinical data available were evaluated both morphologically and immunohistochemically. Seventy-four cases were also studied by fluorescent in situ hybridization on tissue sections and/or conventional karyotyping on bone marrow or peripheral blood. Histologically, 50% of the tumors were of the blastic type, 43.5% either monoblastic or myelomonocytic and 6.5% corresponded to different histotypes. CD68/KP1 was the most commonly expressed marker (100%), followed by myeloperoxidase (83.6%), CD117 (80.4%), CD99 (54.3%), CD68/PG-M1 (51%), CD34 (43.4%), terminal-deoxy-nucleotidyl-transferase (31.5%), CD56 (13%), CD61/linker for activation of T cells (2.2%), CD30 (2.2%) and CD4 (1.1%). Foci of plasmacytoid monocyte differentiation were observed in intestinal cases carrying inv16. Chromosomal aberrations were detected in about 54% of cases: monosomy 7(10.8%), trisomy 8(10.4%) and mixed lineage leukemia-splitting (8.5%) were the commonest abnormalities, whereas t(8;21) was rare (2.2%). The behavior was dramatic irrespective of presentation, age, sex, phenotype and cytogenetics. Most if not all, long survivors received bone-marrow transplantation. The present report expands the spectrum of our knowledge showing that MS has frequent monoblastic/myelomonocytic differentiation, displays distinctive phenotypic profile, carries chromosomal aberrations other than t(8;21), and requires supra-maximal therapy.

Cryotherapy in the prevention of oral mucositis in patients receiving low-dose methotrexate following myeloablative allogeneic stem cell transplantation: a prospective randomized study of the Gruppo Italiano Trapianto di Midollo Osseo nurses group.

Severe oral mucositis is a major cause of morbidity following allogeneic hematopoietic stem cell transplantation (AHSCT). Cryotherapy, that is, the application of ice chips on the mucosa of the oral cavity during the administration of antineoplastic agents, may reduce the incidence and severity of chemotherapy-related oral mucositis. In this multicenter randomized study, we addressed whether cryotherapy during MTX administration is effective in the prevention of severe oral mucositis in patients undergoing myeloablative AHSCT. One hundred and thirty patients undergoing myeloablative AHSCT and MTX-containing GVHD prophylaxis were enrolled and randomized to receive or not receive cryotherapy during MTX administration. The incidence of severe (grade 3-4) oral mucositis, the primary end point of the study, was comparable in patients receiving or not cryotherapy. Moreover, no difference was observed in the incidence of oral mucositis grade 2-4 and the duration of oral mucositis grade 3-4 or 2-4, or in the kinetics of mucositis over time. In univariate and multivariate analysis, severe oral mucositis correlated with TBI in the conditioning regimen and lack of folinic acid rescue following MTX administration. Thus, cryotherapy during MTX administration does not reduce severe oral mucositis in patients undergoing myeloablative allogeneic HSCT. Future studies will assess cryotherapy before allogeneic HSCT.

Effects of granulocyte colony stimulating-factor in a rat model of acute liver injury.

BACKGROUND/AIM: Controversial experimental observations suggest that granulocyte colony stimulating-factor may promote hepatic regeneration after hepatectomy and chemical injury either by directly stimulating adult liver cells or facilitating the mobilization of bone marrow cells and their homing to the liver. We investigated whether different schedules of granulocyte colony stimulating-factor administration protect against experimental acute liver injury. METHODS: Acute liver injury was induced in Sprague-Dawley fed rats by injecting a single intraperitoneal dose of carbon tetrachloride. Recombinant human granulocyte colony stimulating-factor or vehicle was given daily after intoxication (4 days) or before (7 days) and after carbon tetrachloride administration. Liver injury and regeneration were assessed 2 and 4 days after damage. Bone marrow cells mobilization was evaluated by the white blood cell count and the assessment of circulating clonogenic haematopoietic progenitors (colony forming unit-cells). RESULTS: In this experimental model, although granulocyte colony stimulating-factor induced the significant mobilization of colony forming unit-cells, the study cytokine had no effect on liver injury (serum alanine amino transaminase level and necrotic index) and liver regeneration (mitotic index and bromodeoxyuridine incorporation), regardless of the administration schedule. CONCLUSIONS: This study does not support the conclusion that: (1) granulocyte colony stimulating-factor exerts a protective effect against toxic-induced, non-lethal acute liver injury and (2) promotes hepatocyte regeneration.

The NF-kappaB pathway blockade by the IKK inhibitor PS1145 can overcome imatinib resistance.

Imatinib represents at present the most attractive therapy for BCR-ABL positive leukemias, even though a percentage of CML patients develop resistance to this compound. For these resistant patients a therapeutic approach based on a combination of drugs is more likely to be effective. In the last years, constitutive NF-kappaB/Rel activity has been demonstrated in several hematological malignancies. As a result, NFkB/Rel-blocking approaches have been proposed as antineoplastic strategies. Furthermore, the identification of specific kinases within the NF-kappaB activation pathway offers a selective target to address tailored therapies. In the current study, we show that the IKK inhibitor PS1145 is able to inhibit the proliferation of CML cell lines and primary BM cells. Moreover, the addition of Imatinib increases the effects of PS1145 in resistant cell lines and BM cells from resistant patients, with a further increase of apoptosis and inhibition of proliferation and colony growth. Our data provide the rational for a new therapeutic approach, which combines Imatinib and the IKK inhibitor PS1145 in CML resistant patients.

Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia.

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

Treatment and outcome of 2904 CML patients from the EUTOS population-based registry.

The European Treatment and Outcome Study (EUTOS) population-based registry includes data of all adult patients newly diagnosed with Philadelphia chromosome-positive and/or BCR-ABL1+ chronic myeloid leukemia (CML) in 20 predefined countries and regions of Europe. Registration time ranged from 12 to 60 months between January 2008 and December 2013. Median age was 55 years and median observation time was 29 months. Eighty percent of patients were treated first line with imatinib, and 17% with a second-generation tyrosine kinase inhibitor, mostly according to European LeukemiaNet recommendations. After 12 months, complete cytogenetic remission (CCyR) and major molecular response (MMR) were achieved in 57% and 41% of patients, respectively. Patients with high EUTOS risk scores achieved CCyR and MMR significantly later than patients with low EUTOS risk. Probabilities of overall survival (OS) and progression-free survival for all patients at 12, 24 and 30 months was 97%, 94% and 92%, and 95%, 92% and 90%, respectively. The new EUTOS long-term survival score was validated: the OS of patients differed significantly between the three risk groups. The probability of dying in remission was 1% after 24 months. The current management of patients with tyrosine kinase inhibitors resulted in responses and outcomes in the range reported from clinical trials. These data from a large population-based, patient sample provide a solid benchmark for the evaluation of new treatment policies.

Dual tyrosine kinase inhibitors in chronic myeloid leukemia.

The Bcr-Abl inhibitor imatinib mesylate induces complete hematologic and cytogenetic remissions in most newly diagnosed chronic myeloid leukemia (CML) patients, but relatively few of them achieve molecular remission. In addition, imatinib is much less effective in advanced phase-CML as well as in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), mainly due to the development of drug resistance. The challenge for the future is to improve current clinical results with kinase inhibitors in CML, developing strategies that can eradicate residual disease and overcome or prevent resistance. 'Dual' Src and Abl kinase inhibitors are an attractive class of compounds, since (a) these molecules are able to bind Bcr-Abl with less stringent conformational requirements with respect to imatinib, therefore allowing for efficient inhibition of several, resistance-associated mutant forms of Bcr-Abl; (b) Src kinases have been shown to be involved in Bcr-Abl-mediated leukemogenesis as well as upregulated in some patients resistant to imatinib. Here, we review the development, the mode of action and the preclinical or early clinical evaluation of several novel dual Src and Abl kinase inhibitors.