Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Rapid administration of multiple cycles of high-dose myelosuppressive chemotherapy in patients with metastatic breast cancer.

PURPOSE: To determine the feasibility and safety of a rapidly cycled sequence of high-dose myelosuppressive chemotherapy courses. PATIENTS AND METHODS: Seventeen patients with metastatic breast cancer were treated with two courses of cyclophosphamide (CPA; 3.0 g/m2) supported by granulocyte colony-stimulating factor (G-CSF). Following the first CPA treatment, peripheral-blood leukaphereses commenced when the leukocyte count recovered to 1.0 x 10(9)/L. After hematologic recovery from the second dose of CPA, patients were treated with carboplatin 1,500 mg/m2, etoposide 1,200 mg/m2, and CPA 5.0 g/m2 administered over 3 days. The peripheral-blood progenitors (PBPs) were reinfused 3 days later, and G-CSF was recommenced. RESULTS: All patients received the three courses. The median interval between treatments was 14 days (range, 13 to 21). Sixteen of the 34 courses of CPA resulted in admissions for fever. Following the third course, neutrophil counts recovered to 0.5 x 10(9)/L at a median of 9 days (range, 8 to 18) after PBP reinfusion and platelets recovered to 50 x 10(9)/L at a median of 12 days (range, 9 to 102). There were no treatment-related deaths. Flow-cytometric analysis was performed on the leukapheresis collections of eight patients. Seven patients with at least 2.0 x 10(6) CD34+ CD33- cells per kilogram body weight exhibited prompt hematologic recovery. One patient with 0.03 x 10(6) CD34+ CD33- cells was still cytopenic on day 21, and required reinfusion of her back-up marrow. Among seven patients with measurable or assessable disease, there were two complete responses (CRs) and four partial responses (PRs). CONCLUSION: These preliminary results suggest that multiple, rapidly cycled courses of high-dose myelosuppressive chemotherapy can be administered. PBPs, harvested during the G-CSF-augmented rebound from CPA-induced cytopenia, produce rapid hematologic recovery in patients undergoing high-dose chemotherapy (HDC). Further follow-up will be necessary to assess the efficacy of this specific regimen in the treatment of metastatic breast cancer.

Severe toxicity limits intensification of induction therapy for acute lymphoblastic leukemia.

Fourteen adult patients with newly-diagnosed acute lymphoblastic, leukemia (ALL), and lymphoblastic lymphoma, were treated with a dose-intense induction regimen. This regimen was designed to increase the fraction of patients achieving an early complete remission, in an attempt to increase the fraction of patients who are long-term disease-free survivors. The induction regimen included vincristine, prednisone, intermediate-dose cytarabine (Ara-C), and idarubicin, all given during the first week of therapy. This combination led to significant hepatic, gastro-intestinal, infectious, and neurologic toxicity. There was unacceptable treatment-related mortality (29%). After the first eight patients, the study was modified, omitting the Ara-C from the induction phase. Gastrointestinal morbidity was less in the cohort treated without Ara-C; however, infectious morbidity persisted at unacceptable levels and this program was terminated as too toxic to administer. There were nine complete remissions, three early deaths, and two patients with resistant disease. There have been six relapses, three of which occurred in patients who, because of protracted grade III/IV toxicity, were no longer receiving chemotherapy. With a minimum follow-up of 20 months, only three patients are still alive. We conclude that this combination of vincristine, prednisone, Ara-C, and idarubicin, is too toxic to be used as induction therapy for adult patients with ALL and lymphoblastic lymphoma.

A double-blind trial of carbamazepine in negative symptom schizophrenia.

Twenty-eight residual schizophrenics hospitalized in a chronic institution with a 9 to 30 year history of disease, with predominantly negative symptoms were given carbamazepine. Carbamazepine was administered in a double-blind trial and therapeutic effects were measured by the Scale for the Assessment of Negative Symptoms (SANS). Patients were also assessed for positive symptoms using the Brief Psychiatric Rating Scale (BPRS), for depression using the Hamilton Depression Scale, for extrapyramidal symptoms by the Simpson and Angus scale, to rule out these symptoms as sources of secondary negative symptoms. The study continued for 7 weeks with therapeutic carbamazepine levels achieved during the last 5 weeks. There was no significant positive effect of carbamazepine on negative symptoms.

High-intensity chemotherapy with peripheral blood progenitor cell support.

In a series of clinical studies at Memorial Sloan-Kettering Cancer Center, we have used hematopoietic growth factors and peripheral blood-derived hematopoietic progenitor cells to facilitate delivery of multiple courses of high-dose chemotherapy at abbreviated treatment intervals. In these studies, we have demonstrated the feasibility of cross-over regimens involving induction chemotherapy with high-dose cyclophosphamide, supported by granulocyte colony-stimulating factor and followed by multiple peripheral blood leukapheresis to harvest progenitor cells. These cells are then used as rescue for the consolidation component of treatment, which, in the earlier-generation studies, consisted of a single course of high-dose carboplatin/etoposide/cyclophosphamide chemotherapy. In subsequent studies, patients received either four courses of high-dose carboplatin or carboplatin/cyclophosphamide or tandem courses of thiotepa. In all cases, the planned interval between treatments was 14 days, and the achieved median was approximately 16 days. These studies show that the administration of high-intensity regimens that deliver multiple courses of very high-dose chemotherapy at relatively brief intervals is feasible. Our current research focuses on exploiting these findings to devise disease-specific regimens for breast and ovarian cancer.

Factors affecting the mobilization of primitive and committed hematopoietic progenitors into the peripheral blood of cancer patients.

Rapid hematopoietic reconstitution following peripheral blood progenitor cell (PBPC) autotransplantation is thought to result from reinfusion of committed progenitor cells. This has raised concern that PBPC autografts might be rich in committed hematopoietic progentors responsible for early engraftment, but deficient in more primitive progenitors required for long-term hematopoietic reconstitution. The granulomonocytic colony-forming unit (CFU-GM) assay measures committed progenitors responsive to a single species of colony-stimulating activity such as granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas the pre-CFU assay identifies more primitive progenitors by measuring interleukin-3 (IL-3) and kit ligand (KL) induced generation of secondary CFU-GM from CD34+, 4-hydroperoxycyclophosphamide resistant progenitors that require multiple cytokine stimuli. Paired bone marrow (BM) and PBPC samples from 17 breast and ovarian cancer patients participating in four separate clinical trials were compared in these assay systems. In seven of nine patients, PBPC autografts mobilized with cyclophosphamide rebound and G-CSF compared favorably with paired BM autografts in both committed and primitive progenitor capacity. Failure to mobilize substantial primitive progenitor cell numbers occurred in two of nine patients undergoing this mobilization regimen and could not have been predicted by either circulating CFU-GM or CD34+ cell number. Prior myelosuppressive treatment experiences reduced peripheral progenitor yields somewhat, but still allowed for the collection of PBPC autografts which compared favorably with BM autografts in total CFU-GM and Pre-CFU. Mobilization of PBPC with G-CSF or GM-CSF alone in patients who had received prior myelosuppressive therapies produced autografts which were relatively deficient in committed progenitors, but absolutely deficient in primitive progenitors. We conclude that optimization of patient characteristics and mobilization parameters can achieve PBPC autografts rich in both the primitive and committed hematopoietic progenitor cells.

High-intensity chemotherapy with hematopoietic support in breast cancer.

Chemotherapy can produce excellent palliation for many patients with metastatic breast cancer. Survival impact is, however, limited, and permanent remission is extremely rare. There is increasing evidence that dose and dose intensity may be important determinants of outcome in the chemotherapy of breast cancer. Single courses of chemotherapy in doses requiring autologous bone marrow support produce high rates of objective response in patients with metastatic disease that was refractory to prior standard-dose therapy. When used as first chemotherapy for metastases or as consolidation in patients whose disease is responding to lower-dose therapy, high-dose chemotherapy can result in prolonged disease-free survival for some patients. The major cause of treatment failure is relapse from a chemotherapy-induced complete response. Kinetic models suggest that multiple, rapidly cycled courses of high-dose chemotherapy might be superior to single applications or to multiple treatments that are widely spaced in time. Heretofore, the substantial toxicity of high-dose chemotherapy (up to 20% mortality in some early trials) has largely precluded the consideration of timely retreatment; however, the risk appears to have been reduced through the use of hematopoietic growth factors and peripheral blood progenitor cells. Our group has used these new technologies to develop regimens consisting of multiple cycles of high-dose chemotherapy that are rapidly administered. We are currently refining these regimens in preparation for phase II and III studies.

Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12.

Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma.

Pilot study of 5-azacytidine (5-AZA) and carboplatin (CBDCA) in patients with relapsed/refractory leukemia.

5-azacytidine (5-AZA) and carboplatin (CBDCA) are two agents which have demonstrated antileukemic activity in a number of phase I-II trials. Their mechanisms of action and pharmacology related to cell resistance suggested suitability for combination therapy. The aim of this pilot was to evaluate the effects of this combination in the treatment of patients with relapsed/refractory acute leukemia. A total of 21 patients was enrolled. 5-azacytidine, at doses ranging from 50-150 mg/m2/day, was administered as a 2-hr infusion for 5 consecutive days. On day 3, patients began a 5-day course of CBDCA given as a 24-hr continuous intravenous infusion of 250 mg/m2/day. There were no complete remissions with this regimen. Although there were three partial responses, these were generally of short duration. Nonhematologic toxicities were mild. No correlation was seen between response and serum platinum levels. These results demonstrate that the 5-AZA/CBDCA combination is ineffective therapy for heavily pretreated patients with acute leukemia.

Beneficial impact of peripheral blood progenitor cells in patients with metastatic breast cancer treated with high-dose chemotherapy plus granulocyte-macrophage colony-stimulating factor. A randomized trial.

BACKGROUND: This study compared the efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with peripheral blood-derived hematopoietic progenitor cells (PBP) as support for patients receiving high-dose chemotherapy and assessed the adequacy of these strategies as alternatives to autologous bone marrow rescue. METHODS: The authors studied patients with metastatic breast carcinoma who had a major response to conventional chemotherapy or had achieved a complete remission by surgical resection of all known metastases. They were treated with carboplatin 1500 mg/m2, etoposide 1200 mg/m2, and cyclophosphamide 5.0 g/m2. Before this high-dose chemotherapy, the patients had been randomly assigned to one of two hematopoietic support strategies: GM-CSF alone (Group 1) or GM-CSF-primed PBP and GM-CSF (Group 2). Autologous bone marrow was harvested from all patients for use only in the event of persistent pancytopenia with marrow aplasia on day 15. RESULTS: A total of 18 patients were treated. Randomization was halted after the initial 10 patients because of the significant advantages for patients in Group 2 in comparison with those in Group 1 in regard to (1) the median number of days to absolute neutrophil count 0.5 x 10(9)/l (12 versus 21) and platelet count to 50 x 10(9)/l (13 versus 23), (2) platelet transfusions (3 versus 15.5), and (3) episodes of neutropenic sepsis (0 versus 4, respectively). One patient in Group 1 died from treatment-related complications. All patients in Group 1 required bone marrow reinfusion. No patient in Group 2 required bone marrow reinfusion, and no early mortality was observed in this group. Eight subsequent patients were treated with PBP and GM-CSF (Group 3). This group was more heavily pretreated than Groups 1 or 2 and had a slower hematologic recovery than Group 2. However, none of these patients required bone marrow reinfusion. The four patients in Group 1 that did not have early bone marrow rescue all had neutrophil counts of 0.0 on day 15. For Groups 2 and 3, the neutrophil counts on day 15 ranged from 0.3-2.1 x 10(9)/l (median, 1.9) and from 0.2-2.1 x 10(9)/l (median 0.6), respectively. CONCLUSIONS: The use of PBP plus GM-CSF accelerated hematologic recovery after this chemotherapeutic regimen compared with GM-CSF alone; there were reduced morbidity and platelet transfusion requirements. Recovery was sufficiently rapid that PBP were an acceptable alternative to autologous bone marrow transplantation in patients receiving high-dose carboplatin, etoposide, and cyclophosphamide.