Replacement of bortezomib with carfilzomib for multiple myeloma patients progressing from bortezomib combination therapy.

In this open-label, intra-patient phase I/II trial, bortezomib was replaced with carfilzomib (escalated from 20 to 45 mg/m(2) on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle) for multiple myeloma (MM) patients who progressed while on or within 12 weeks of receiving a bortezomib-containing combination regimen. Study objectives included determination of the maximum tolerated dose (MTD), overall response rate (ORR), clinical benefit rate (CBR), time to progression, time to response, duration of response, progression-free survival and overall survival (OS). Of 38 registered patients, 37 were treated and evaluable for efficacy and safety. Thirty-one carfilzomib-based regimens using 14 different drug combinations were tested. One regimen (carfilzomib (45 mg/m(2)), ascorbic acid (1000 mg) and cyclophosphamide (2.2 mg/kg)) reached MTD. ORR and CBR were 43.2 and 62.2%, respectively. Median progression-free survival, time to progression and OS were 8.3, 9.9 and 15.8 months, respectively. Hematologic adverse events (AEs; ⩾grade 3) included lymphopenia (35.1%), thrombocytopenia (24.3%), anemia (10.8%) and neutropenia (10.8%). Nonhematologic AEs (⩾grade 3) included fever (5.4%) and hypokalemia (5.4%). These results demonstrate that replacing bortezomib with carfilzomib is safe and can be effective for MM patients failing bortezomib-containing combination regimens. This trial was registered at http://www.clinicaltrials.gov (#NCT01365559).

A phase 2 study of pegylated liposomal doxorubicin, bortezomib, dexamethasone and lenalidomide for patients with relapsed/refractory multiple myeloma.

Our previous studies have shown that lowering the dose of pegylated liposomal doxorubicin (PLD) and bortezomib in combination with intravenous dexamethasone on a longer 4-week cycle maintained efficacy and improved tolerability in both previously untreated and relapsed/refractory (R/R) multiple myeloma (MM) patients. Lenalidomide has shown efficacy in combination with bortezomib and dexamethasone but this combination has been poorly tolerated. We conducted this phase 2 study (clinicaltrials.gov identifier: NCT01160484) to evaluate whether a longer 4-week schedule using modified doses and schedules of IV dexamethasone (40 mg), bortezomib (1.0 mg/m(2)) and PLD (4.0 mg/m(2)) administered on days 1, 4, 8, and 11 with lenalidomide 10 mg daily on days 1-14 (DVD-R) would be effective and tolerated for patients with R/R MM. A total of 40 heavily pretreated patients were enrolled and 84.6% showed clinical benefit (complete response, 20.5%; very good partial response, 10.3%; partial response, 17.9%; minimal response, 35.9%) to the combination regimen. An additional 10.3% showed stable disease and 5.1% progressed while on study. The regimen was well tolerated, with a low incidence of adverse events such as fatigue (40%), thrombocytopenia (35%), neutropenia (35%), anemia (30%), peripheral neuropathy (25%) and pneumonia (15%). Thus, the DVD-R regimen is well tolerated and produces high response rates for patients with R/R MM.

A Phase I, open label, dose ranging trial of intravenous bolus zoledronic acid, a novel bisphosphonate, in cancer patients with metastatic bone disease.

BACKGROUND: Bone metastases typically are associated with osteolytic bone destruction, resulting in bone pain, pathologic fractures, spinal cord compression, and hypercalcemia. Bisphosphonates are potent inhibitors of normal and pathologic bone resorption and represent a significant therapeutic improvement in the management of patients with lytic bone metastases. Zoledronic acid is a new-generation, highly potent, nitrogen-containing bisphosphonate that to the authors knowledge is the most potent inhibitor of bone resorption currently in clinical trials. The objectives of the current study were to assess the safety and tolerability of increasing doses of zoledronic acid and to determine its activity with respect to reducing biochemical markers of bone resorption in cancer patients with bone metastases. METHODS: Forty-four cancer patients with bone metastases or primary bone lesions were enrolled sequentially into 1 of 5 fixed ascending-dose treatment groups. Each patient received a single intravenous bolus injection of 1, 2, 4, 8, or 16 mg of zoledronic acid over 30-60 seconds. Patients were monitored for 8 weeks for the evaluation of clinical findings, adverse events, vital signs, electrocardiograms, markers of bone resorption, and urinary N-acetyl-beta-D-glucosaminidase. RESULTS: Zoledronic acid was safe and well tolerated at all dose levels tested. Commonly reported adverse events included bone pain, fever, anorexia, constipation, and nausea, which were experienced by a similar proportion of patients in each treatment group. Seven patients reported serious adverse events, none of which appeared to be related to the study drug. Zoledronic acid effectively suppressed biochemical markers of bone resorption, including the highly specific markers N-telopeptide and deoxypyridinoline, for up to 8 weeks in the 2-16-mg dose groups and for a shorter duration in the 1-mg group. CONCLUSIONS: In the current study, zoledronic acid was safe and well tolerated and demonstrated potent inhibition of bone resorption. The authors believe it may improve the treatment of metastatic bone disease.

C-type natriuretic peptide expression and pulmonary vasodilation in hypoxia-adapted rats.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are potent pulmonary vasodilators that are upregulated in hypoxia-adapted rats and may protect against hypoxic pulmonary hypertension. To test the hypothesis that C-type natriuretic peptide (CNP) also modulates pulmonary vascular responses to hypoxia, we compared the vasodilator effect of CNP with that of ANP on pulmonary arterial rings, thoracic aortic rings, and isolated perfused lungs obtained from normoxic and hypoxia-adapted rats. We also measured CNP and ANP levels in heart, lung, brain, and plasma in normoxic and hypoxia-adapted rats. Steady-state CNP mRNA levels were quantified in the same organs by relative RT-PCR. CNP was a less potent vasodilator than ANP in preconstricted thoracic aortic and pulmonary arterial rings and in isolated lungs from normoxic and hypoxia-adapted rats. Chronic hypoxia increased plasma CNP (15 +/- 2 vs. 6 +/- 1 pg/ml; P < 0.05) and decreased CNP in the right atrium (35 +/- 14 vs. 65 +/- 17 pg/mg protein; P < 0.05) and in the lung (3 +/- 1 vs. 14 +/- 3 pg/mg protein; P < 0.05) but had no effect on CNP in brain or right ventricle. Chronic hypoxia increased ANP levels fivefold in the right ventricle (49 +/- 5 vs. 11 +/- 2 pg/mg protein; P < 0.05) but had no effect on ANP in lung or brain. There was a trend toward decreased ANP levels in the right atrium (2,009 +/- 323 vs. 2,934 +/- 397 pg/mg protein; P = not significant). No differences in CNP transcript levels were observed between the two groups of rats except that the right atrial CNP mRNA levels were lower in hypoxia-adapted rats. We conclude that CNP is a less potent pulmonary vasodilator than ANP in normoxic and hypoxia-adapted rats and that hypoxia raises circulating CNP levels without increasing cardiopulmonary CNP expression. These findings suggest that CNP may be less important than ANP or BNP in protecting against hypoxic pulmonary hypertension in rats.

B-lymphoma induction by reticuloendotheliosis virus: characterization of a mutated chicken syncytial virus provirus involved in c-myc activation.

Nondefective reticuloendotheliosis virus induces chicken bursal lymphoma in a manner similar to that of avian leukosis virus. The provirus integrates in the c-myc locus and uses a promoter insertion mechanism to activate c-myc expression. We cloned a provirus involved in c-myc activation from a B lymphoma. Detailed structural characterization of this clone, including sequence determination, revealed proviral insertion at 512 base pairs preceding the second c-myc exon. The provirus has a deletion of 80% of the viral genes but retains two intact long terminal repeats (LTRs). A segment of the viral env sequence is present in an inverted orientation. Elevated expression of c-myc, apparently directed by the 3' LTR, was detected. However, despite the presence of an intact 5' LTR, no viral transcripts were detected. Thus, the internal proviral rearrangement can affect 5' LTR transcription or stability of the message or both. This finding is in consonance with the view that proviral deletion plays an important role in the induction of bursal lymphomas by nonacute retroviruses.

Insertional activation of c-myc by reticuloendotheliosis virus in chicken B lymphoma: nonrandom distribution and orientation of the proviruses.

Chicken syncytial virus, a member of the reticuloendotheliosis virus family, can induce chicken B lymphomas indistinguishable from those caused by avian leukosis virus. Previously, we have demonstrated that the chicken syncytial virus proviruses in these tumors are linked to the proto-oncogene c-myc. We have now determined the arrangement of chicken syncytial virus proviruses in 22 tumors. The results indicate that these proviruses, without exception, are integrated upstream from the second c-myc exon. At least 70% of these insertion sites are clustered in a 0.5-kilobase region immediately preceding the exon. The proviruses are all arranged in the same transcriptional orientation as c-myc. This type of provirus organization bears strong resemblance to that of the avian leukosis virus proviruses involved in c-myc activation.

In vitro transcription analysis of the viral promoter involved in c-myc activation in chicken B lymphomas: detection and mapping of two RNA initiation sites within the reticuloendotheliosis virus long terminal repeat.

Chicken syncytial virus, a member of the reticuloendotheliosis virus family, induces B-cell lymphomas in chickens that arise by transcriptional activation of the chicken c-myc gene. In vitro transcription studies on cloned tumor DNA containing a deleted chicken syncytial virus provirus integrated upstream from, and in the same transcriptional orientation as, the chicken c-myc coding region were utilized to map possible transcriptional promoters and initiation sites. In vitro transcripts extending into c-myc sequences were initiated at two sites within the downstream long terminal repeat (LTR) closest to c-myc coding sequences. Both initiation sites have been precisely mapped by S1 nuclease and DNA sequencing methods. One site (I1) lies at the U3-R junction of the LTR, and the other site (I2) lies approximately 160 nucleotides upstream. Transcriptional control signals, including TATA- and CAAT-like sequences are present at appropriate distances upstream from the initiation sites. Both initiation sites are utilized to a similar extent. The upstream chicken syncytial virus LTR was also shown to be transcriptionally active in vitro. Two strong transcriptional initiation sites were also found in the LTR of spleen necrosis virus, a related member of the reticuloendotheliosis virus family; therefore, it seems likely that the existence of two transcriptional initiation sites is a common feature of the reticuloendotheliosis virus LTR, in contrast to other previously studied retroviral LTRs that exhibit one such site. The possible implications of these findings are discussed.

Double cos site vectors: simplified cosmid cloning.

A new vector for construction of cosmid libraries is described. Cosmid c2XB contains restriction sites for use in the insertion of foreign DNA and two lambda cos sites separated by a blunt-end restriction site. The presence of two cos sites on a single plasmid eliminates the need to prepare two separate cosmid arms, and the internal blunt-end restriction site prevents cosmid concatemerization. Thus, a double restriction-enzyme digestion is sufficient to prepare the vector for subsequent ligation with DNA fragments which are dephosphorylated to prevent their self-ligation. The use of this vector system allows efficient cosmid cloning (1 X 10(5) colonies per micrograms insert DNA) and eliminates background due to vector self-ligation. Furthermore, the procedure is so rapid as to eliminate the need to amplify cosmid libraries for storage and reuse. Also described is a cosmid vector for use in construction of cosmid libraries which are to be introduced into cultured eukaryotic cells. This vector contains the Herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker and a retroviral long terminal repeat (LTR) region as an enhancer sequence.