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1.
Mol Immunol ; 105: 150-164, 2019 01.
Article in English | MEDLINE | ID: mdl-30513451

ABSTRACT

Complement activation in human diseases is characterized by the local covalent deposition of the long-lived C3 fragments iC3b/C3dg/C3d. Previously, TT30, a complement alternative pathway (AP)-selective inhibitor, was designed as a fusion protein linking the first four short consensus repeats (SCRs) of human complement receptor type 2 (CR2) with the first five SCRs of human factor H (fH). TT30 acts by utilizing CR2 SCR1-4 to bind the initially formed iC3b/C3dg/C3d fragments and delivering surface-targeted inhibition of AP C3 and C5 convertases through fH SCR 1-5. In order to combine classical (CP) and lectin (LP) pathway inhibitory abilities employing CR2-mediated targeting, TT32 was developed. TT32 is a CR2-CR1 fusion protein using the first ten SCRs of CR1, chosen because they contain both C3 and C5 convertase inhibitory activity through utilization of decay-acceleration and cofactor activity for both AP and CP. In Wieslab assays, TT32 showed potent inhibition of the CP and AP with IC50 of 11 and 46 nM, respectively. The TT32 inhibitory activity is partially blocked with a molar excess of a competing anti-CR2 mAb, thus demonstrating the importance of the CR2 targeting. TT32 was studied in the type II (CII) collagen-induced arthritis (CIA), an active immunization model, and the CII antibody-induced arthritis (CAIA) passive transfer model. In CIA, injection of 2.0 mg TT32 at day 21 and 28 post disease induction, but not untargeted CR1 alone, resulted in a 51.5% decrease in clinical disease activity (CDA). In CAIA, treatment with TT32 resulted in a 47.4% decrease in CDA. Therefore, a complement inhibitor that targets both the AP and CP/LP C3/C5 convertases was shown to limit complement-mediated tissue damage and inflammation in disease models in which all three complement activation pathways are implicated.


Subject(s)
Arthritis, Experimental/therapy , Complement C3 Convertase, Alternative Pathway/immunology , Immunization, Passive , Receptors, Complement 3d/immunology , Receptors, Complement/immunology , Recombinant Fusion Proteins/immunology , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Complement C3 Convertase, Alternative Pathway/genetics , Humans , Male , Mice , Rabbits , Receptors, Complement/genetics , Recombinant Fusion Proteins/genetics , Sheep
2.
Blood ; 131(17): 1960-1973, 2018 04 26.
Article in English | MEDLINE | ID: mdl-29519807

ABSTRACT

Naturally occurring, large deletions in the ß-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and ß-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and ß-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the ß-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of ß-hemoglobinopathies.


Subject(s)
Anemia, Sickle Cell , CRISPR-Cas Systems , Fetal Hemoglobin , Gene Editing , Genetic Loci , Hematopoietic Stem Cells/metabolism , beta-Globins/genetics , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/metabolism , Anemia, Sickle Cell/pathology , Anemia, Sickle Cell/therapy , Cell Line , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/genetics , Hematopoietic Stem Cells/pathology , Humans , beta-Globins/metabolism
4.
J Clin Oncol ; 33(11): 1252-7, 2015 Apr 10.
Article in English | MEDLINE | ID: mdl-25732165

ABSTRACT

PURPOSE: Secondary acute myeloid leukemia (sAML), defined as AML arising after a prior myelodysplastic syndrome or after antineoplastic therapy, responds poorly to current therapies. It is often associated with adverse karyotypic abnormalities and overexpression of proteins that mediate drug resistance. We performed a phase III trial to determine whether induction therapy with cytarabine and amonafide L-malate, a DNA intercalator and non-ATP-dependent topoisomerase II inhibitor that evades drug resistance mechanisms, yielded a superior complete remission rate than standard therapy with cytarabine and daunorubicin in sAML. PATIENTS AND METHODS: Patients with previously untreated sAML were randomly assigned at a one-to-one ratio to cytarabine 200 mg/m(2) continuous intravenous (IV) infusion once per day on days 1 to 7 plus either amonafide 600 mg/m(2) IV over 4 hours on days 1 to 5 (A + C arm) or daunorubicin 45 mg/m(2) IV over 30 minutes once per day on days 1 to 3 (D + C arm). RESULTS: The complete remission (CR) rate was 46% (99 of 216 patients) in A + C arm and 45% (97 of 217 patients) in D + C arm (P = .81). The 30- and 60-day mortality rates were 19% and 28% in A + C arm and 13% and 21% in D + C arm, respectively. CONCLUSION: Induction treatment with A + C did not improve the CR rate compared with D + C in patients with sAML.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Induction Chemotherapy , Leukemia, Myeloid, Acute/drug therapy , Adenine , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cytarabine/administration & dosage , Daunorubicin/administration & dosage , Drug Administration Schedule , Female , Humans , Infusions, Intravenous , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Naphthalimides/administration & dosage , Organophosphonates , Prospective Studies , Remission Induction , Time Factors , Treatment Outcome , Young Adult
5.
Blood ; 118(17): 4705-13, 2011 Oct 27.
Article in English | MEDLINE | ID: mdl-21860027

ABSTRACT

To selectively modulate human complement alternative pathway (CAP) activity implicated in a wide range of acute and chronic inflammatory conditions and to provide local cell surface and tissue-based inhibition of complement-induced damage, we developed TT30, a novel therapeutic fusion protein linking the human complement receptor type 2 (CR2/CD21) C3 fragment (C3frag = iC3b, C3dg, C3d)-binding domain with the CAP inhibitory domain of human factor H (fH). TT30 efficiently blocks ex vivo CAP-dependent C3frag accumulation on activated surfaces, membrane attack complex (MAC) formation and hemolysis of RBCs in a CR2-dependent manner, and with a ∼ 150-fold potency gain over fH, without interference of C3 activation or MAC formation through the classic and lectin pathways. TT30 protects RBCs from hemolysis and remains bound and detectable for at least 24 hours. TT30 selectively inhibits CAP in cynomolgus monkeys and is bioavailable after subcutaneous injection. Using a unique combination of targeting and effector domains, TT30 controls cell surface CAP activation and has substantial potential utility for the treatment of human CAP-mediated diseases.


Subject(s)
Complement C3-C5 Convertases/antagonists & inhibitors , Complement C3d/metabolism , Complement Factor H/therapeutic use , Complement Pathway, Alternative/immunology , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Immune System Diseases/drug therapy , Immune System Diseases/immunology , Receptors, Complement 3d/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Animals , Complement C3-C5 Convertases/metabolism , Complement Factor H/administration & dosage , Drug Design , Drug Evaluation, Preclinical , Female , Humans , Immune System Diseases/metabolism , Macaca fascicularis , Male , Models, Immunological , Molecular Targeted Therapy/methods , Rabbits , Receptors, Complement 3d/administration & dosage , Recombinant Fusion Proteins/administration & dosage
6.
Expert Opin Investig Drugs ; 20(7): 995-1003, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21591994

ABSTRACT

INTRODUCTION: Amonafide is a novel topoisomerase II (Topo II) inhibitor and DNA intercalator that induces apoptotic signaling by blocking the binding of Topo II to DNA. Amonafide retains cytotoxic activity even in the presence of P-glycoprotein (Pgp)-mediated multi-drug resistance (MDR), a major contributor to clinical treatment failure. AREAS COVERED: In vitro, Pgp-mediated transport (efflux) of amonafide from myeloblasts obtained from patients with secondary acute myeloid leukemia (sAML) was significantly less than efflux of daunorubicin. Amonafide has shown efficacy in patients with sAML, as well as in patients with poor prognostic characteristics such as older age and unfavorable cytogenetics, all associated with MDR. Improved antileukemic activity is observed when amonafide is given together with cytarabine, rather than as monotherapy, with a complete remission rate of ∼ 40% in a recent Phase II trial in sAML. The efficacy of amonafide was maintained among poor-risk subsets of patients, including older patients and patients who had previous myelodysplastic syndrome or previous leukemogenic therapy. The safety profile was acceptable and manageable. EXPERT OPINION: Amonafide plus cytarabine may have clinical utility in patients with sAML and in other poor-risk subgroups of acute myeloid leukemia (AML). Ongoing trials will help define the role for amonafide in the treatment of poor-risk AML.


Subject(s)
Antineoplastic Agents/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Naphthalimides/therapeutic use , Adenine , Animals , Antineoplastic Agents/pharmacokinetics , Clinical Trials as Topic/methods , DNA Topoisomerases, Type II/metabolism , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/mortality , Naphthalimides/pharmacokinetics , Organophosphonates , Survival Rate/trends , Treatment Outcome
7.
Biopharm Drug Dispos ; 31(1): 72-81, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19862681

ABSTRACT

The pharmacokinetics, bioavailability and effects on electrocardiographic (ECG) parameters of fludarabine phosphate (2F-ara-AMP) were evaluated in adult patients with B-cell chronic lymphocytic leukemia. Patients received single doses of intravenous (IV) (25 mg/m(2), n=14) or oral (40 mg/m(2), n=42) 2F-ara-AMP. Plasma concentrations of drug and metabolites and digital 12-lead ECGs were monitored for 23 h after dosing. The dephosphorylated product fludarabine (2F-ara-A) was the principal metabolite present in the systemic circulation. Mean (+/-SD) elimination half-life did not differ significantly between IV and oral dosage groups (11.3+/-4.0 vs 9.7+/-2.0 h, p=0.053). Renal excretion was a major clearance pathway, along with transformation to a hypoxanthine metabolite 2F-ara-Hx. Estimated mean oral bioavailability of 2F-ara-A was 58%. Compared to the time-matched drug-free baseline Fridericia correction of the QT interval (QTcF), the mean QTcF change following 2F-ara-AMP did not differ from zero, and a treatment effect of >+10 and >+15 ms could be excluded following oral and IV 2F-ara-AMP, respectively. Similarly, heart rate, PR interval and QRS duration did not change following 2F-ara-AMP treatment. Thus the 25 mg/m(2) IV and 40 mg/m(2) oral doses of 2F-ara-AMP produce similar systemic exposure, and do not prolong QTcF, indicating low risk of drug induced Torsades de Pointes.


Subject(s)
Metabolic Clearance Rate , Vidarabine Phosphate/analogs & derivatives , Vidarabine/analogs & derivatives , Administration, Oral , Adult , Biological Availability , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Synergism , Female , Half-Life , Heart Rate/drug effects , Heart Rate/physiology , Humans , Male , Middle Aged , Models, Chemical , Vidarabine/pharmacokinetics , Vidarabine Phosphate/metabolism , Vidarabine Phosphate/pharmacokinetics
8.
Leuk Res ; 34(4): 487-91, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19748672

ABSTRACT

Amonafide-l-malate (amonafide) is a unique DNA intercalator that maintains activity in the presence of MDR mechanisms, a frequent cause of treatment-failure in secondary AML. 43 patients with relapsed/refractory or secondary AML or CML blast crisis were enrolled into two phase I dose-escalation studies investigating amonafide as monotherapy or in combination with cytarabine. 3/17 patients in the monotherapy trial and 10/26 patients in the combination trial achieved a complete remission. Between both trials responses occurred in 9/20 patients with secondary AML. Both trials demonstrated an acceptable safety profile and significant antileukemic activity in patients with poor-risk AML, especially those with secondary AML.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cytarabine/administration & dosage , Leukemia, Myeloid, Acute/drug therapy , Naphthalimides/administration & dosage , Adenine , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cytarabine/adverse effects , Female , Humans , Male , Middle Aged , Naphthalimides/adverse effects , Organophosphonates , Remission Induction , Risk Factors , Treatment Outcome , Young Adult
9.
J Oncol Pharm Pract ; 11(3): 127-30, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16390601

ABSTRACT

OBJECTIVE: To report a case of who a patient developed clinical and radiographical evidence of interstitial lung disease (ILD) on erlotinib after having tolerated gefitinib therapy. CASE SUMMARY: A 58-year-old man with stage IV non-small-cell lung cancer (NSCLC) failed first and second line chemotherapy. He then received gefitinib, a small molecule epidermal growth factor receptor (EGFR) inhibitor, a therapy which was well tolerated, but did cause a grade 1 rash. On gefitinib, the patient's disease remained stable for seven months. Subsequent disease progression was treated with the newer EGFR inhibitor, erlotinib. After 5 days of erlotinib therapy, the patient presented with a sore throat and dyspnea, followed by a grade 2 rash and significant hemoptysis. Erlotinib was discontinued for three days, during which time his symptoms abated. Erlotinib was restarted and the patient again developed sore throat, dyspnea and severe hemotpysis, with progression of the rash to grade 3. Erlotinib therapy was discontinued and the patient recevied prednisone and supplemental oxygen. A CT scan of the chest demonstrated new areas of patchy ground glass opacity bilaterally and increased interstitial markings consistent with ILD. DISCUSSION: The case demonstrates that clinical ILD can occur following erlotinib therapy, even in patients who previously tolerated gefitinib. ILD has not been reported to occur more frequently with erlotinib than with gefitinib. However, the dose of erlotinib employed clinically is the maximum tolerated dose identified in phase 1 trials, and is associated with an increased incidence of grade 3-4 rash and diarrhea, as compared to gefitinib. Thus, the observation of clinical ILD following erlotinib, but not gefitinib, may be the consequence of increased potency of erlotinib 150 mg/day compared to gefitinib 250 mg/day. CONCLUSION: Clinical ILD can occur following erlotinib even in patients who previously tolerated gefitinib. IT is important to carefully monitor pulmonary symptoms in all patients who are receiving erlotinib, as early diagnosis and timely intervention are critical in managing drug-induced ILD.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Diseases, Interstitial/chemically induced , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/adverse effects , Quinazolines/adverse effects , Erlotinib Hydrochloride , Gefitinib , Humans , Lung Diseases, Interstitial/diagnostic imaging , Male , Middle Aged , Quinazolines/therapeutic use , Tomography, X-Ray Computed
10.
Proc Natl Acad Sci U S A ; 99(20): 12606-11, 2002 Oct 01.
Article in English | MEDLINE | ID: mdl-12193655

ABSTRACT

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras. Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.


Subject(s)
Cell Transformation, Neoplastic , Telomerase/physiology , Blotting, Western , Catalysis , Cell Division , DNA-Binding Proteins , Electrophoresis, Polyacrylamide Gel , Glucose/metabolism , Green Fluorescent Proteins , Humans , Karyotyping , Kinetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Oxygen/metabolism , Retroviridae/genetics , Telomere/enzymology , Telomere/physiology , Time Factors , Tumor Cells, Cultured
11.
Oncogene ; 21(29): 4577-86, 2002 Jul 04.
Article in English | MEDLINE | ID: mdl-12085236

ABSTRACT

One critical step in the development of a cancerous cell is its acquisition of an unlimited replicative lifespan, the process termed immortalization. Experimental model systems designed to study cellular transformation ex vivo have relied to date on the in vitro selection of a subpopulation of cells that have become immortalized through treatment with chemical or physical mutagens and the selection of rare clonal variants. In this study, we describe the direct immortalization of primary human airway epithelial cells through the successive introduction of the Simian Virus 40 Early Region and the telomerase catalytic subunit hTERT. Cells immortalized in this way are now responsive to malignant transformation by an introduced H-ras or K-ras oncogene. These immortalized human airway epithelial cells, which have been created through the stepwise introduction of genetic alterations, provide a novel experimental model system with which to study further the biology of the airway epithelial cell and to dissect the molecular basis of lung cancer pathogenesis.


Subject(s)
Bronchi/metabolism , Bronchi/pathology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Transduction, Genetic , Blotting, Western , Cell Differentiation , Cell Division , Cell Line, Transformed , Cells, Cultured , DNA-Binding Proteins , Genes, ras/genetics , Genetic Vectors , Humans , Simian virus 40/genetics , Telomerase/genetics , Telomerase/metabolism , Time Factors
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