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 .).

Phase I/IIa trial of the mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) administered orally in patients with refractory or advanced malignancies and sarcoma.

BACKGROUND: Ridaforolimus is an inhibitor of mTOR with evidence of antitumor activity in an I.V. formulation. This multicenter, open-label, 3 + 3 design nonrandomized, dose-escalation, phase I/IIa trial was conducted to determine the safety, pharmacokinetic (PK) and pharmacodynamic parameters, maximum tolerated dose, and antitumor activity of oral ridaforolimus. PATIENTS AND METHODS: Patients with metastatic or unresectable solid tumors refractory to therapy were eligible. Seven different continuous and intermittent dosing regimens were examined. RESULTS: One hundred and forty-seven patients were enrolled in this study among which 85 were patients with sarcoma. Stomatitis was the most common DLT observed. The dosing regimen, 40 mg QD × 5 days/week, provided the best combination of cumulative dose, dose density, and cumulative exposure, and was the recommended dosing regimen for subsequent clinical development. PK was nonlinear, with less than proportional increases in day-1 blood AUC0-∞ and Cmax, particularly with doses >40 mg. The terminal half-life estimate of ridaforolimus (QD × 5 40 mg) was 42.0 h, and the mean half-life ∼30-60 h. The clinical benefit rate, (complete response, partial response, or stable disease for ≥4 months was 24.5% for all patients and 27.1% for patients with sarcoma. CONCLUSION: Oral ridaforolimus had an acceptable safety profile and exhibited antitumor activity in patients with sarcoma and other malignancies. ClinicalTrials.gov Identifier NCT00112372.

A humanized system for pharmacologic control of gene expression.

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.

Phase Ib study of weekly mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) with weekly paclitaxel.

BACKGROUND: The additive cytotoxicity in vitro prompted a clinical study evaluating the non-prodrug rapamycin analogue ridaforolimus (AP23573; MK-8669; formerly deforolimus) administered i.v. combined with paclitaxel (PTX; Taxol). MATERIALS AND METHODS: Patients with taxane-sensitive solid tumors were eligible. The main dose escalation foresaw 50% ridaforolimus increments from 25 mg with a fixed PTX dose of 80 mg/m(2), both given weekly 3 weeks in a 4-week cycle. Collateral levels with a lower dose of either drug were planned upon achievement of the maximum tolerated dose in the main escalation. Pharmacodynamic studies in plasma, peripheral blood mononuclear cells (PBMCs) and skin biopsies and pharmacokinetic (PK) interaction studies at cycles 1 and 2 were carried out. RESULTS: Two recommended doses were determined: 37.5 mg ridaforolimus/60 mg/m(2) PTX and 12.5 mg/80 mg/m(2). Most frequent toxic effects were mouth sores (79%), anemia (79%), fatigue (59%), neutropenia (55%) and dermatitis (48%). Two partial responses were observed in pharyngeal squamous cell and pancreatic carcinoma. Eight patients achieved stable disease > or =4 months. No drug interaction emerged from PK studies. Decrease of eukaryotic initiation factor 4E-binding protein1 (4E-BP1) phosphorylation was shown in PBMCs. Similar inhibition of phosphorylation of 4E-BP1 and mitogen-activated protein kinase was present in reparative epidermis and vascular tissues, respectively. CONCLUSION: Potential antiangiogenic effects and encouraging antitumor activity justify further development of the combination.

Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands.

OBJECTIVES: Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands. METHODS: A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval. RESULTS: AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal. CONCLUSION: Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

Pharmacologic control of a humanized gene therapy system implanted into nude mice.

Systemic delivery of specific therapeutic proteins by a parenteral route of administration is a recognized practice in the management of several gene defects and acquired diseases. As an alternative to repetitive parenteral administration, gene therapy may provide a novel means for systemic delivery of therapeutic proteins while improving patient compliance and therapeutic efficacy. However, for gene therapy to be an efficacious and safe approach to the clinical management of such diseases, gene expression must be tightly regulated. These investigations demonstrate precise in vivo control of protein expression from cells that are engineered to secrete human growth hormone (hGH) in response to stimulation by rapamycin. The cells were implanted intramuscularly into nu/nu mice and stimulated by intravenous or oral administration of rapamycin. In vivo experiments demonstrate that the activity and pharmacokinetics of rapamycin determine the level of serum hGH that result from the engineered cells. In addition, responsiveness of the cells to rapamycin, number of cells implanted, hGH expression kinetics, and the pharmacokinetics of hGH itself, also influence the circulating levels of hGH after rapamycin stimulation. Controlled manipulation of several of these parameters, either independently or in combination, allows for precise regulation of circulating hGH concentration in vivo.

Clustering of Syk is sufficient to induce tyrosine phosphorylation and release of allergic mediators from rat basophilic leukemia cells.

In mast cells, antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, stimulates tyrosine phosphorylation and activation of multiple signaling pathways leading to the release of several classes of mediators of the allergic response. Early events induced upon cross-linking of Fc epsilon RI include tyrosine phosphorylation of Fc epsilon RI subunits and activation of the tyrosine kinase p72syk (Syk), which binds to tyrosine-phosphorylated Fc epsilon RI. Clustering of Syk, as a result of its interaction with aggregated Fc epsilon RI, may play a role in activating one or more of the signaling pathways leading to mediator release. To test this possibility, Syk was introduced into a model mast cell line (rat basophilic leukemia cells) as part of a chimeric transmembrane protein containing the extracellular and transmembrane domains of CD16 and CD7, respectively. Clustering of the Syk chimera, using antibodies against CD16, was found to be sufficient to stimulate early and late events normally induced by clustering of Fc epsilon RI. Specifically, aggregation of Syk induced degranulation, leukotriene synthesis, and expression of cytokine genes. Induction of mediator release was dependent on the kinase activity of Syk. Consistent with this finding, clustering of Syk also induced the tyrosine phosphorylation of a profile of proteins, including phospholipase C-gamma 1 and mitogen-activated protein kinase, similar to that induced upon clustering of Fc epsilon RI. These results strongly suggest that Syk is an early and critical mediator of multiple signaling pathways that emanate from the Fc epsilon RI receptor and give rise to the allergic response.

The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts.

Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.

A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

Modified snake venom in amyotrophic lateral sclerosis. Lack of clinical effectiveness.

Diverse observations including neurological examination, manual muscle testing, quantitative electromyography, videotape esophagrams, and pulmonary function tests were used in a double-blind study evaluating the use of detoxified snake venoms as modified neurotoxin in 64 patients with amyotrophic lateral sclerosis (ALS) treated for six months. Transient periods of improvement were more common in the patients who received placebo. This study did not demonstrate any benefit from administration of modified snake venom to patients with ALS.

Immunoregulation and blocking antibodies induced by interferon beta treatment in MS.

OBJECTIVE: To examine the in vivo immunoregulatory properties of interferon beta-1a (IFN beta-1a) on the T cell responses to myelin basic protein (MBP) and to evaluate the occurrence of the blocking antibodies characterized by the ability to reverse the effects of IFN beta on T cells in MS patients treated with IFN beta. METHODS: The precursor frequency of T cells recognizing MBP and control antigens was estimated in a microwell culture system. The cytokine profile of T cell lines was measured in ELISA. The binding antibodies were determined in ELISA and Western blot. Cytopathic test and the T cell functional assays were used to determine the blocking effects of the binding antibodies. RESULTS: Treatment with IFN beta resulted in a substantial reduction in the precursor frequency of MBP-reactive T cells in MS patients. The cytokine profile of MBP-reactive T cells that sustained the treatment was altered toward an increased production of interleukin (IL)-10 and decreased production of tumor necrosis factor (TNF)alpha and IFN-gamma. The immunoregulatory properties of IFN beta on T cells could be blocked by the binding antibodies derived from a proportion of patients treated with IFN beta (4 of 64, 6.25%). The blocking antibodies also neutralized anti-viral activity of IFN beta in cytopathic assays, corresponding to previously described neutralizing antibodies. CONCLUSIONS: Treatment with IFN beta alters the cytokine profile by enhancing the production of IL-10 and downregulating Th1 cytokines, which may contribute to clinical benefit in MS. The treatment also induces blocking antibodies that impair the immunoregulatory properties of IFN beta in some individuals.

Interferon beta induces T-helper 2 immune deviation in MS.

OBJECTIVE: To define the in vitro effects of interferon beta la (IFN-beta1a) on myelin basic protein (MBP)-reactive T cells and to determine its regulatory mechanism on cytokine networks in patients with MS. METHODS: The proliferation and cytokine production of MBP-reactive T-cell clones were measured in thymidine uptake assays and ELISA respectively. The precursor frequency of MBP-reactive T cells was estimated in a microwell culture system. RESULTS: IFN-beta inhibited the proliferation of established MBP-reactive T-cell clones, which correlated with enhanced production of anti-inflammatory interleukin (IL)-4 and IL-10, and a decrease in tumor necrosis factor alpha (TNF-alpha) and IFN-gamma. When examined with peripheral blood mononuclear cells (PBMCs), IFN-beta was found to reduce the in vitro T-cell responses to MBP, as indicated by the significantly decreased frequency of MBP-reactive T cells. The decreased frequency of MBP-reactive T cells corresponded to an augmented production of IL-4 and IL-10. Although the level of TNF-alpha and IFN-gamma was generally unaltered or decreased, IFN-beta appeared to enhance the production of IFN-gamma in PBMCs derived from some individuals with MS. CONCLUSION: Interferon beta la (IFN-beta) suppresses myelin basic protein (MBP)-reactive T cells and induces immune deviation toward the production of T-helper 2 cytokines, which may contribute to its therapeutic benefit in MS. The study also suggests some heterogeneity in MBP-reactive T-cell responses to IFN-beta in different individuals with MS.

Regulation of chemokine receptor CCR5 and production of RANTES and MIP-1alpha by interferon-beta.

Trafficking of inflammatory T cells into the brain is associated with interactions of certain chemokines with their receptors, which plays an important role in the pathogenesis of multiple sclerosis (MS). We examined whether interferon-beta (IFN-beta) had the ability to regulate the production of chemokines and the expression of their receptors in T cells derived from patients with MS. It was demonstrated for the first time that in vitro exposure of T cells to IFN-beta-1a selectively inhibited mRNA expression for RANTES and MIP-1alpha and their receptor CCR5. T cell surface expression of CCR5 was significantly reduced in MS patients treated with IFN-beta, correlating with decreased T cell transmigration toward RANTES and MIP-1alpha. The study provides new evidence suggesting that IFN-beta treatment impairs chemokine-induced T cell trafficking by reducing the production of RANTES and MIP-1alpha and the expression of their receptors CCR5.

T cell responses to myelin basic protein in patients with spinal cord injury and multiple sclerosis.

Autoimmune inflammation secondary to myelin destruction may play an inhibitory role in restoration of nerve functions in spinal cord injury (SCI). In this study, we demonstrated that T cells recognizing myelin basic protein (MBP) occurred at a high precursor frequency in patients with SCI, which was compatible to that in patients with multiple sclerosis (MS), a disease of presumed autoimmune pathology. The findings suggest of hyperactivity of MBP-reactive T cells in patients with SCI. MBP-reactive T cell lines derived from patients with SCI exhibited a preferential recognition pattern toward the 81-99 and the 151-169 regions of MBP. There were functional differences in the epitope recognition and cytokine profile between two panels of MBP-reactive T cell lines derived from patients with SCI and patients with MS. The study provides new evidence important for further investigation of the role of the inflammatory component in SCI.

Neuromyopathy in malignant hyperthermia.

A 31-year old male developed malignant hyperthermia (MH) during the initial minutes of Halothane induction. CPK rose to 6120 U/ml and decreased to normal levels as the patient became afebrile over a 10 day period of cooling measures and metabolic management. Muscle weakness, predominantly proximal and depressed deep tendon reflexes were found upon examination during convalescence. Muscle biopsy showed neurogenic changes characterized by fiber type grouping and targetoid fibers. CPK was elevated in one of the patient's children. This case supports the view of underlying hereditary neuromyopathy in MH.

Precordialgia as a manifestation of vertebrobasilar insufficiency.

In 6 patients with vertebrobasilar insufficiency (VBI), precordialgia and autonomic-nervous-system manifestation such as diaphoresis and tachycardia were associated with the symptoms of VBI. Investigation ruled out cardiac and gastrointestinal causes. Possible mechanisms are discussed on the basis of intermitent ischemia of the central autonomic centers supplied by the posterior circulation.