The biodistribution and radiation dosimetry of the Arg-Gly-Asp peptide 18F-AH111585 in healthy volunteers.

UNLABELLED: We report the safety, biodistribution, and internal radiation dosimetry of a new PET tracer, (18)F-AH111585, a peptide with a high affinity for the alpha(v)beta(3) integrin receptor involved in angiogenesis. METHODS: PET scans of 8 healthy volunteers were acquired at time points up to 4 h after a bolus injection of (18)F-AH111585. (18)F activity in whole blood and plasma and excreted urine were measured up to 4 h after injection. In vivo (18)F activities in up to 12 source regions were determined from quantitative analysis of the images. The cumulated activities subsequently calculated were then used to determine the internal radiation dosimetry, including the effective dose. RESULTS: Injection of (18)F-AH111585 was well tolerated in all subjects, with no serious or drug-related adverse events reported. The main route of (18)F excretion was renal (37%), and the 3 highest initial uptakes were by liver (15%); combined walls of the small, upper large, and lower large intestines (11%); and kidneys (9%). The 3 highest absorbed doses were received by the urinary bladder wall (124 microGy/MBq), kidneys (102 microGy/MBq), and cardiac wall (59 microGy/MBq). The effective dose was 26 microGy/MBq. CONCLUSION: (18)F-AH111585 is a safe PET tracer with a dosimetry profile comparable to other common (18)F PET tracers.

Phase I trial of the positron-emitting Arg-Gly-Asp (RGD) peptide radioligand 18F-AH111585 in breast cancer patients.

UNLABELLED: The integrin alpha v beta3 receptor is upregulated on tumor cells and endothelium and plays important roles in angiogenesis and metastasis. Arg-Gly-Asp (RGD) peptide ligands have high affinity for these integrins and can be radiolabeled for PET imaging of angiogenesis or tumor development. We have assessed the safety, stability, and tumor distribution kinetics of a novel radiolabeled RGD-based integrin peptide-polymer conjugate, 18F-AH111585, and its feasibility to detect tumors in metastatic breast cancer patients using PET. METHODS: The biodistribution of 18F-AH111585 was assessed in 18 tumor lesions from 7 patients with metastatic breast cancer by PET, and the PET data were compared with CT results. The metabolic stability of 18F-AH111585 was assessed by chromatography of plasma samples. Regions of interest (ROIs) defined over tumor and normal tissues of the PET images were used to determine the kinetics of radioligand binding in tissues. RESULTS: The radiopharmaceutical and PET procedures were well tolerated in all patients. All 18 tumors detected by CT were visible on the 18F-AH111585 PET images, either as distinct increases in uptake compared with the surrounding normal tissue or, in the case of liver metastases, as regions of deficit uptake because of the high background activity in normal liver tissue. 18F-AH111585 was either homogeneously distributed in the tumors or appeared within the tumor rim, consistent with the pattern of viable peripheral tumor and central necrosis often seen in association with angiogenesis. Increased uptake compared with background (P = 0.002) was demonstrated in metastases in lung, pleura, bone, lymph node, and primary tumor. CONCLUSION: 18F-AH111585 designed to bind the alpha v beta3 integrin is safe, metabolically stable, and retained in tumor tissues and detects breast cancer lesions by PET in most anatomic sites.

Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain.

Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an index AML patient, an algorithm of analyses was applied using clinical material. In vivo and in vitro investigation of primary blasts at relapse revealed persistent TK phosphorylation of FLT3 despite sufficient PKC412 serum levels. Through additional molecular analyses, we identified a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in this patient. Reconstitution experiments expressing the N676K mutant in 32D cells demonstrated that FLT3-ITD-N676K was sufficient to confer an intermediate level of resistance to PKC412 in vitro. These studies point out that a genetically complex malignancy such as AML may retain dependence on a single oncogenic signal.

Activity of the tyrosine kinase inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT mutation.

The majority of patients with systemic mast cell disease express the imatinib-resistant Asp816Val (D816V) mutation in the KIT receptor tyrosine kinase. Limited treatment options exist for aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). We evaluated whether PKC412, a small-molecule inhibitor of KIT with a different chemical structure from imatinib, may have therapeutic use in advanced SM with the D816V KIT mutation. We treated a patient with MCL (with an associated myelodysplastic syndrome (MDS)/myeloproliferative disorder [MPD]) based on in vitro studies demonstrating that PKC412 could inhibit D816V KIT-transformed Ba/F3 cell growth with a 50% inhibitory concentration (IC50) of 30 nM to 40 nM. The patient exhibited a partial response with significant resolution of liver function abnormalities. In addition, PKC412 treatment resulted in a significant decline in the percentage of peripheral blood mast cells and serum histamine level and was associated with a decrease in KIT phosphorylation and D816V KIT mutation frequency. The patient died after 3 months of therapy due to progression of her MDS/MPD to acute myeloid leukemia (AML). This case indicates that KIT tyrosine kinase inhibition is a feasible approach in SM, but single-agent clinical efficacy may be limited by clonal evolution in the advanced leukemic phase of this disease.

Identification of a novel activating mutation (Y842C) within the activation loop of FLT3 in patients with acute myeloid leukemia (AML).

Fms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated AML. Mutation screening of FLT3 exon 20 in AML patients (n = 110) revealed 2 patients with a novel mutation (Y842C) within the highly conserved activation loop of FLT3. FLT3-Y842C-transfected 32D cells showed constitutive FLT3 tyrosine phosphorylation and interleukin 3 (IL-3)-independent growth. Treatment with PKC412 led to inhibition of proliferation and apoptotic cell death. Primary AML blasts bearing FLT3-Y842C mutations showed constitutive FLT3 and signal transducer and activator of transcription 5 (STAT-5) tyrosine phosphorylation. Ex vivo PKC412 treatment of primary blasts resulted in suppression of constitutive FLT3 and STAT-5 activation and apoptotic cell death. Inspection of the FLT3 structure revealed that Y842 is the key residue in regulating the switch from the closed to the open (= active) conformation of the FLT3 activation loop. Overall, our data suggest that mutations at Y842 represent a significant new activating mutation in AML blasts. Since FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412 are currently being investigated in clinical trials in AML, extended sequence analysis of FLT3 may be helpful in defining the spectrum of TKI-sensitive FLT3 mutations in AML.

PKC412 inhibits the zinc finger 198-fibroblast growth factor receptor 1 fusion tyrosine kinase and is active in treatment of stem cell myeloproliferative disorder.

Human stem cell leukemia-lymphoma syndrome usually presents itself as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-fibroblast growth factor receptor (FGFR) 1 fusion tyrosine kinase. Current empirically derived cytotoxic chemotherapy is inadequate for treatment of this disease. We hypothesized that small-molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo. Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLC-gamma, STAT5, and phosphatidylinositol 3-kinase/AKT, required the proline-rich domains, but not the ZNF domains, of ZNF198. A small-molecule tyrosine kinase inhibitor, PKC412 (N-benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1-induced MPD. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disorder with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.